Writer:Roy C. Brown etc Reporter:Zhaowei-Xie Instructor:Professor Wang

1. Development of endosperm in Arabidopsis thaliana Writer:Roy C. Brown etc Reporter:Zhaowei-Xie Instructor:Professor Wang

2. Abstract :The process of endosperm development in Arabidopsis was studied using immunohistochemistry of tubulin/microtubules coupled with light and confocal laser scanning microscopy. Arabidopsis undergoes the nuclear type of development in which the primendosperm nucleus resulting from double fertilization divides repeatedly without cytokinesis resulting in a syncytium lining the central cell. Development occurs as waves originating in the micropylar chamber and moving through the central chamber toward the chalazal tip.

3. Priorto cellularization, the syncytium is organized into nuclear cytoplasmic domains (NCDs) defined by nuclear based radial systems of microtubules. The NCDs become polarized in axes perpendicular to the central cell wall, and anticlinal walls deposited among adjacent NCDs compartmentalize the syncytium into open-ended alveoli overtopped by a crown of syncytial cytoplasm. Continued centripetal growth of the anticlinal walls is guided by adventitious phragmoplasts that form at interfaces of microtubules emanating from adjacent interphase nuclei.

4. Polarity of the elongating alveoli is reflected in a subsequent wave of periclinal divisions that cuts off a peripheral layer of cells and displaces the alveoli centripetally into the central vacuole. This pattern of development via alveolation appears to be highly conserved; it is characteristic of nuclear endosperm development in angiosperms and is similar to ancient patterns of gametophyte development in gymnosperms.

5. Introduction • Seed development in flowering plants is initiated by the unique process of double fertilization in which the two sperms resulting from division of the single generative cell of the pollen grain unite with cells of the megagametophyte, one sperm nucleus fusing with the nucleus of the egg cell and the other with one or more polar nuclei of the central cell. Even though the zygote and primary endosperm nucleus have like genomes, they develop along totally different pathways to give rise to the embryo and accompanying nutritive endosperm.

6. Differences in the developmental patterns of the diploid embryo and the usually triploid endosperm cannot be attributed solely to differences in the level of ploidy. The number of polar nuclei varies depending upon the pattern of embryo sac development. In the Oenothera pattern of development, both the zygote and primary endosperm nucleus are diploid yet subsequent embryo and endosperm development follow different programs.

7. The genetic and physiological balance among endosperm, embryo and maternal tissues of the ovule have long been known to be important in regulation of seed development, but the specific controls responsible for the different patterns of development in the same ovule have remained enigmatic. As part of an overall goal to understand the basic cellular mechanisms involved in plant development, we have studied the microtubule cycle as related to the pattern of nuclear endosperm development in the cereals, barley, rice and wheat.

8. These studies show that the microtubule cycle in endosperm development is characterized by abrupt changes reflecting distinct developmental stages which are correlated with the cell cycle, cell polarity, control of the division plane, and wall deposition. The typical microtubule arrays of dividing plant cells which characterize early embryo development are compared to the unique microtubule arrays associated with early nuclear endosperm development in Fig. 1.

9. Fig. 1A–E, A’–E’ Diagrammatic comparison of microtubule systems associated with initial divisions in the zygote and nuclear endosperm

10. During the initial period of syncytial development in the cereals, numerous mitoses occur without cytokinesis. Ephemeral phragmoplasts are initiated between telophase nuclei but do not expand beyond the interzone and no cell plates are laid down. This uncoupling of mitosis and cytokinesis results in a peripheral layer of multinucleate cytoplasm surrounding a large central vacuole. The syncytial stage is followed by a mitotic hiatus during which time the cytoplasm is organized into nuclear cytoplasmic domains(NCDs) that are defined by nuclear-based radial microtubule systems.

11. This is followed by alveolation, a curious form of wall deposition that is not related directly to nuclear division. Growth of anticlinal walls between adjacent NCDs forms a peripheral layer of openended alveoli overtopped by the remaining syncytial cytoplasm. The anticlinal walls continue to elongate unidirectionally into the central vacuole in association with adventitious phragmoplasts that form in the common cytoplasm adjacent to the central vacuole. These unusual phragmoplasts are organized at the interface of microtubule systems emanating from apical tips of nuclei in adjacent alveoli.

12. Periclinal division in the alveoli occurs as a coordinated wave resulting in a peripheral layer of cells and an inner layer of alveoli. The periclinal divisions are more typical of higher plant development in that mitosis is followed by cytokinesis via the classic interzonal phragmoplast/cell plate that joins to parental (alveolar) walls. Periclinal division differs, however, in the lack of a predictive preprophase band (PPB) of microtubules and the resulting interphase cells do not develop a hoop-like cortical system of microtubules (see Fig. 1).

13. The inner layer of alveoli elongate as do the anticlinal walls again in association with adventitious phragmoplasts, all of which form between non-sister nuclei. Repeated cycles of periclinal cell division and renewed anticlinal wall growth completes cellularization of the central cell. In the later development of a multilayered aleurone in barley, the microtubule cycle includes PPBs before mitosis and hoop-like cortical microtubules in interphase cells

14. An interesting hypothesis drawn from these data is that while the endosperm is capable of typical cell division with involvement of the four microtubule arrays (cortical, PPB, spindle and phragmoplast), and these components appear to be added as the endosperm becomes cellular and more typically plant like, morphogenesis can proceed at a more basic level in lieu of the microtubule arrays that characterize typical plant cells.

15. Thus, endosperm provides the opportunity to learn more of the hierarchy of controls operative in plant morphogenesis. The model dicotyledon, Arabidopsis thaliana, is ideal for extension of these studies as its embryology is well known and it undergoes the nuclear type of endosperm development as do the cereals.

16. The purpose of this investigation was to characterize the process of endosperm development in Arabidopsis, to assess commonalities among Arabidopsis and other species of the Brassicaeae and the cereals, to more fully understand the general pattern of nuclear endosperm development and, in particular, to analyze changes in microtubule arrays associated with the basic processes of endosperm development.

17. Materials and methods • Plant material: • Developing endosperm of Arabidopsis thaliana (L.) Heyn h. ecotype Columbia was studied in situ using material embedded in plastic for light microscopy and unembedded material sectioned with a vibratome for immunofluorescence. To optimize fixation, ovules were removed from siliques and the micropylar/stalk region was sliced or nicked to facilitate penetration by fixatives and other fluids used in processing. Ovules were sectioned longitudinally in the plane of the micropyle and stalk. The stage of embryo development is used in referring to development of ovule and endosperm.

18. Light microscopy (LM): • Ovules were fixed in 4% glutaraldehyde in 0.1 M phosphate buffer (pH 6.9), postfixed in osmium ferricyanide (Hepler 1981), dehydrated in a graded acetone series, and infiltrated with Spurr’s resin (all at room temperatures). Thin sections (ca. 0.5 mm) were stained with methylene-blue borax (Postek and Tucker 1976) or polychrome stain (Fox 1997).

19. Immunofluorescence of microtubules: • Ovules were stripped from a developmental series of siliques and fixed in 4% formaldehyde freshly prepared from paraformaldehyde in PHEM-DMSO microtubule-stabilizing buffer for 1 h Following a thorough wash in PHEM-DMSO, ovules were mounted to specimen holders with cyanoacrylate cement and sectioned at 50 μm with an OTS microtome Sections were adhered to coverslips with Mayer’s egg albumen histological adhesive and covered by a thin agarose-gelatin film.

20. A rapid improved protocol for immunostaining modified from Harper et al. and Brown and Lemmon (1995) was used for immunohistochemistry of tubulin/microtubules. Reagents were exchanged throughthe agarose-gelatin film preventing loss of sections and minimizing damage to the delicate endosperm tissues. Sections were permeabilized in a cocktail of enzymes and Triton X-100 Harper. and incubated in a monoclonal antibody against yeasttubulin for 1 h at 37°C, rinsed in PHEM-DMSO buffer, and incubated for 3 h with secondary antibody conjugated to fluorescein.

21. Following several washes in water, nuclei acids were stained by a 0.1% aqueous solution of propidium iodide. Coverslips with sections were mounted to a microscope slide in Mowiol 4/88 containing phenylenediamine as a antifade reagent. Fluorescence was examined with a Bio-Rad MRC 600 confocal laser scanning microscope.

22. Results • The primary endosperm nucleus undergoes mitosis without cytokinesis to give rise to a row of evenly spaced nuclei in the narrow post-fertilization embryo sac (Fig. 2A). The ovule becomes circinotropous as it grows, bending like a horseshoe with the chalazal region adjacent to the micropylar region. The abaxial surface of the large central chamber bends gradually but the adaxial surface is sharply bent and the chalazal and micropylar chambers are separated by the adaxial ridge (Fig. 2B).

23. The central vacuole enlarges and the multinucleate endosperm becomes peripheral (Fig. 2B). The embryo sac wall enclosing the endosperm is appressed to inner integument cells of the ovule which develop massive electrondense deposits (Fig. 2C,D) identified as tannins (Schulz and Jensen 1974).

24. In the early ovule containing an embryo of a few cells, the enlarged central cell is lined with a thin peripheral layer of syncytial endosperm surrounding a large central vacuole (Fig. 2C). At the globular embryo stage (Figs. 2D–3B), syncytial cytoplasm surrounds the developing embryo in the narrow micropylar chamber. In the central chamber, the multinucleate syncytium is a thin peripheral layer.

25. Fig. 2A–D Syncytial stage of endosperm development in longitudinal sections of ovules. All figures are oriented with the micropyle on the left. Bar = 21 mm

26. Microtubules in the micropylar endosperm appear as a delicate network throughout the cytoplasm (Fig. 3A). At this stage, cells of the embryo (Fig. 3B) are actively dividing and exhibit the characteristic microtubule arrays of meristematic division (cortical, PPB, spindle and phragmoplast. In the central chamber, the syncytium has a distinctive beaded appearance (Fig. 2D).

28. Waves of division result in proliferation of the syncytium (Fig. 3C). The nuclei in the syncytium are evenly spaced in a single layer, each surrounded by a sphere of cytoplasm (Fig. 3C–E) comprising the nuclear cytoplasmic domain (NCD) and interconnected to surrounding NCDs by microtubules that radiate in the interconnecting cytoplasmic strands among the lateral vacuoles that accumulate around the NCDs (Fig. 3D).

30. Prior to cellularization, the late syncytial endosperm is organized into widely spaced NCDs protruding into the central vacuole with a thin layer of cytoplasm closely appressed to the central cell wall interconnecting them (Figs. 2D, 3E). Microtubules radiating from nuclei delimit the NCDs which have the appearance of “cells” before anticlinal walls are formed (Fig. 3E).

31. Fig. 2D Ovule with late globular (transition) embryo. In the central chamber the peripheral syncytium consists of widely spaced nuclei (arrowheads) with a small mass in the chalazal portion. Fig. 3E Organization of NCDs. Microtubules radiating from nuclei (one labelled N) in the syncytial endosperm lining the central chamber define islands of cytoplasm that bulge into the central vacuole.

32. Cellularization of the endosperm begins in the micropylar chamber and coincides with initiation of cotyledons in the embryo. In the transition to cellular endosperm, the NCDs become polarized, elongating in an axis perpendicular to the embryo sac wall (Fig. 3F). This is accompanied by rearrangement of microtubules from a nearly symmetrical radial arrangement to one in which microtubules extend in columns of cytoplasm connecting the NCDs to the embryo sac wall (Fig. 3F).

33. Fig. 3F Polarization of NCDs. Just prior to deposition of anticlinal walls, NCDs elongate. Nuclei are apical in the cytoplasmic columns and microtubules (two bundles marked by arrowheads) are arranged in the axis of elongation.

34. Cellularization proceeds as a wave into the central chamber. This course of development is evident in transverse sections at the two-chamber level (Fig. 4A). Pre-heart stage ovules contain syncytial endosperm in both chambers, whereas in heart stage ovules the endosperm in the micropylar chamber is cellularized throughout.

35. Cellularization events proceed rapidly in a wave from the micropylar chamber to the chalazal chamber and a succession of developmental stages is present in ovules with early heart stage embryos. Along the gently curved abaxial surface of the central cell, the transition is gradual and all stages of polarization and alveolar formation can be seen in the large central chamber (Fig. 4B).

36. The alveoli are most advanced (greatly elongated) adjacent to the embryo and gradually grade into syncytial endosperm in the central chamber. At the sharply curved adaxial surface, however, alveolar formation ceases abruptly at the ridge separating the micropylar and chalazal chambers (Fig. 4B,C).

37. Fig. 4A–C Initial cellularization.

38. Whereas endosperm quickly becomes cellular around the embryo in the micropylar chamber, it remains syncytial in the chalazal chamber until late stages of seed maturation (Figs. 4C, 7B). Nodules of multinucleate endosperm line the abaxial wall of the chalazal chamber, and a large coenocytic cyst of multinucleate cytoplasm is positioned in the tip of the chalazal chamber atop the nucellar proliferating tissue (Figs. 4C, 7B).

39. Fig.4C Longitudinal section showing transition from endospermic nodules (arrowheads) to cyst (asterisk) in the chalazal chamber. Fig.7B Longitudinal section of an ovule with torpedo-stage embryo showing cellularized endosperm except in the chalazal chamber where a pronounced ceonocytic cyst (asterisk) persists.

40. Deposition of anticlinal walls is not directly associated with nuclear division and occurs in the same fashion between sister and non-sister nuclei. This unusual form of wall deposition results in anticlinal walls in axes perpendicular to the central cell wall. The walls between adjacent NCDs join with the central cell wall only at the periphery and compartmentalize the syncytium into open-ended cylinders or alveoli (Figs. 4B, 5A).

41. Fig. 4B Longitudinal section. Alveolation proceeds as a wave beginning in the micropylar chamber near the embryo (EM). A periclinal wall proximate to the embryo is indicated by the arrowhead. Alveoli and first periclinal division. Bars = 9.4 mm. Fig. 5A Single layer of alveoli during radial growth of anticlinal walls (one labelled AW) prior to periclinal cell division.

42. The leading edges of the anticlinal walls adjacent to the tonoplast grow in association with adventitious phragmoplasts (Fig. 6A) that form at the interface of arrays of microtubules emanating from interphase nuclei. When a face view of the alveoli is obtained, as in a tangential section, it can be seen that interaction of microtubules radiating from the nuclei control phragmoplast placement/wall deposition resulting in a honeycomb of polygonal chambers (Fig. 6B).

43. Fig. 6A In alveoli, microtubules emanate from tips of nuclei (N) which are restricted to a column of cytoplasm by lateral vacuoles. Phragmoplasts which guide continued centripetal growth of alveolar walls (arrowheads) develop at the interface of microtubules radiating from adjacent nuclei. Fig.6B A tangential section showing microtubules radiating from adjacent nuclei (N) in polygonal chambers of a honeycomb system of alveolar walls. Phragmoplasts, two marked by asterisks, are associated with the leading edges of the anticlinal walls.

44. Before the peripheral cytoplasm in the central chamber has been completely compartmentalized into alveoli, a wave of cell division begins in the oldest alveoli near the embryo (Fig. 4B). The majority of these divisions are oriented with the spindles at right angles to the embryo sac wall and parallel to the anticlinal walls (Figs. 4B, 5B).

45. Fig. 4B Longitudinal section. Alveolation proceeds as a wave beginning in the micropylar chamber near the embryo (EM). A periclinal wall proximate to the embryo is indicated by the arrowhead. Fig. 5B Mitosis in the alveolate layer is synchronous. Most division planes are periclinal (P) but occasional anticlinal (A) divisions are seen.

46. This round of division differs from previous nuclear divisions of the endosperm in that karyokinesis is followed immediately by phragmoplast/cell plate formation resulting in cytokinesis, but no PPBs predict the division plane. The phragmoplast begins in the interzone of the spindle (Fig. 6C) expanding in a thin raft of cytoplasm suspended among numerous large vacuoles (Fig. 5B).

47. Fig. 6B Mitosis in the alveolate layer is synchronous. Most division planes are periclinal (P) but occasional anticlinal (A) divisions are seen. This round of karyokinesis is followed immediately by cell plates. Fig. 6C Phragmoplasts originating in the interzone following chromosome separation guide deposition of periclinal cell plates.

48. Periclinal divisions in the alveoli result in a peripheral layer of completely walled cells and an inner layer of displaced alveoli (Figs. 4B, 6D). The inner alveoli become organized similarly to those of the initial alveolar layer. The alveoli elongate with each containing a large vacuole with the nucleus suspended in the advancing front and associated with adventitious phragmoplasts that guide anticlinal wall growth (Figs. 6D, 7A).

50. The front of phragmoplast-filled cytoplasm clearly defines the leading edge of the centripetally advancing cytoplasm (Fig. 6E). The inner alveoli continue to divide periclinally to produce successive layers of endosperm that fill the central cell except near the cyst in the chalazal chamber (Fig. 7B). Cells of the early cellular endosperm are highly vacuolate (Fig. 7B); endoplasmic microtubules radiate from nuclei and traverse strands of cytoplasm which suspend nuclei among the large vacuoles (Fig. 6F).