Female Gametophyte Development in Maize: Microtubular Organization and Embryo Sac Polarity

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1 The Plant Cell, Vol. 6, , June 1994 O 1994 American Society of Plant Physiologists Female Gametophyte Development in Maize: Microtubular Organization and Embryo Sac Polarity Bing-Quan Huang and William F. Sheridan Department of Biology, University of North Dakota, Grand Forks, North Dakota The developmental stages of the maize embryo sac were correlated with the corresponding silk lengths of ear florets in the female inflorescence. The development of embryo sacs in the ovules of spikes occurs in a gradient pattern with the initiation of the embryo sac beginning at the base of the ear and progressing to the top. At the beginning of meiosis, the presence of conspicuous cortical microtubules coincides with the extensive elongation of the megasporocyte. The spindles at metaphase I and II align along the long axis of the megasporocyte leading to the linear alignment of the dyad and tetrad of megaspores. During megagametogenesis, micropylar and chalazal nuclei of the embryo sac undergo synchronized divisions and migration at the second and third mitosis. Radiate perinuclear microtubules are present during the interphase of the second and third mitosis, and inter-sister nuclear microtubules occur at the late four-nucleate embryo sac. The configuration and orientation of the spindles, phragmoplasts, and pairs of nuclei result in precise positioning of the nuclei. The fusion of the polar nuclei and the formation of a microtubule organizing center-like structure in the filiform apparatus occur right after the first division of the antipodal cells. The different patterns of organization of microtubules in the cells of the mature embryo sac reflect their structural adaptations for their future function. INTRODUCTION The maize ear is a spike composed of spikelet pairs on the rachis of the cob. As the pistil develops, each functional ear floret has a single ovary that ends in an elongated silk. Each ovary contains an ovule in which a single megasporocyte or megagametophyte is located beneath one or severa1 layers of nucellus cells toward the micropylar pole of the ovule (Kiesselbach, 1949). The ear has a range of florets with variable length of silks and provides a range of developmental stages of megasporocytes and megagametophytes that are well suited for studying embryo sac development. In the study of maize pollen development, the externa1 morphological features were used to estimate different developmental stages of the microspores and the male gametophyte (Chang and Neuffer, 1989). However, no such developmental index for the female counterpart has been established. The basic events of embryo sac development in maize have been extensively studied using classical paraffin and plastic resin techniques in light microscopy and electron microscopy (for review, see Randolph, 1936; Kiesselbach, 1949; Russell, 1979, 1993; Bedinger and Russell, 1994). Embryo sac development in maize is monosporic and conforms to the fblygonum type (Kiesselbach, 1949; Maheshwari, 1950; Russell, 1979). In each ovule, the single megasporocyte (megaspore mother cell) displays the micropylar-chalazal polarity manifested by its asymmetrical organelle distribution, callose deposition, and To whom correspondence should be addressed. the chalazal location of plasmodesmata (Russell, 1979; Willemse, 1981; Haig, 1990; Huang and Russell, 1992). Only the chalazal-most megaspore survives, and it then goes through three mitotic divisions and cellularization giving rise to the seven-celled embryo sac, which consists of two synergids, one egg cell, and one central cell (Randolph, 1936; Kiesselbach, 1949). Two synergid cells locate at the micropylar end of the embryo sac, each with a strongly thickened wall ingrowth called the filiform apparatus at the extreme micropylar end of the cell. The egg cell lies below the apices of the synergids and extends somewhat further toward the center of the embryo sac than do the two synergids. The central cell is the largest cell occupying?he center of the embryo sac, and avariable number of antipodal cells are located at the chalazal pole (Diboll and Larson, 1966). Despite the recognized importance of the microtubular cytoskeleton in establishing and maintaining specific polarities and spindle orientation in plant cells (Baskin and Cande, 1990), little information is available concerning the involvement of the microtubular cytoskeleton in nuclear positioning and anchoring, nuclear division, and cellularization during the development of the embryo sac as a result of the inaccessibility of megasporocytes and the megagametophytes (Bednara et al., 1988; Wiliemse and van Lammeren, 1988; Webb and Gunning, 1990,1991). The characterization of embryo sac development has become more feasible with the development of a technique for the isolation of individual embryo sacs (Huang et al., 1990). This technique has been used to

2 846 The Plant Cell Figure 1. The Patterns of Embryo Sac Development in the Different Stages of Ears.

3 Embryo Sac Development in Maize 847 investigate the polarity of nuclear and organellar DNA during the development of the embryo sac (Huang and Russell, 1993) and the cytoskeletal changes during fertilization (Huang et al., 1993). A similar technique involving enzymatic digestion of ovules followed by squashing was used to study the microtubular cytoskeleton during megasporogenesis and early embryogenesis in Arabidopsis (Webb and Gunning, 1990, 1991). In this study, we used differential interference contrast (DIC) and fluorescent light microscopy to investigate embryo sac development in the different stages of ear development and correlated the events of embryo sac formation with the external morphological feature of silk length to obtain a developmental index for embryo sac development along the length of the ear. In addition, we characterized the changes of organellar and nuclear DNA and the role of the microtubular cytoskeleton during megasporogenesis and megagametogenesis by examining the cellular changes from the premeiotic megasporocyte stage to the mature female gametophyte, the mature embryo sac. RESULTS Developmental Pattern of Ear Florets and lndex of Embryo Sac Development The silk length was used as an external morphological feature to correlate the extent of embryo sac development at different developmental stages of the ear, namely, from the very early stage when silks are initiated to the mature ear in which silks emerge from the husk. The development of embryo sacs in the ovules corresponds with silk development in the ear in which a gradient of developmental pattern reveals that ovule development progresses from the base to the top of the ear. On the basis of analysis of immature ears differing in their extent of silk lengths, we observed five overlapping phases of ear development (Figure 1A). In the first phase of ear development, the range of silk length in the florets was from O to 0.4 cm, and the ovules contained either megasporocytes in meiosis or megaspores (Figure la, ear 1; Figures 1B to 1G). In the second phase, the range of silk length was from O to 0.8 cm, and the ovules contained stages from megasporocytes to immature megagametophytes (Figure la, ear 2; Figures 1B to 1K). In the third phase, the silk lengths on the ear ranged up to 1.2 cm, and most ovules of the ear contained megagametophytes with eight nuclei (Figure la, ear 3; Figures 1L and 1M). In the fourth phase, the silk lengths on the ear ranged up to 1.5 cm, and the most advanced ovules contained embryo sacs with more than three antipodal cells (Figure la, ear 4; Figures 1N and 10). In the fifth phase, with silk lengths up to 12 cm on the mature ear, most of the ovules contained embryo sacs with 40 or more antipodal cells (Figure la, ear 5). A more detailed analysis was performed on a second group of four ears with overlapping phases of development. Each immature ear was divided transversely into three equal sections, and in each section the silk lengths were measured for groups of 17 to 78 florets before manual dissection of their ovules. The liberated reproductive cells isolated from a total of 262 ovules were stained (dye #33258, Hoechst, St. Louis, MO) and analyzed microscopically to ascertain their stage of Figure 1. (continued). (A) Five overlapping phases of ear development. The first ear is the smallest ear in which florets have a silk length ranging from O to 0.4 cm; the megasporocytes are at premeiosis and meiosis stages. The second ear is the transitional ear in which the embryo sac development is from megasporocytes to immature megagametophytes with the silk length ranging from O to O8 cm. The young ear is third; the sevencelled megagamete phytes form at the bottom part with silk length up to 1.2 cm. The fourth ear is premature; most of the florets have an embryo sac with variable antipodal cells and silk length more than 1.5 cm. The fifth ear is mature; the embryo sac in most of the florets has more than 40 antipodal cells with silk length up to 12 cm. A correlation between reproductive cell development and silk length is shown in (E) to (O). (8) Megasporocyte in the premeiotic stage. (C) Silk lengths corresponding to the megasporocyte shown in (E); up to 0.2 cm. (D) Megasporocyte at prophase I of meiosis. (E) Silk lengths of 0.1 to 0.4 cm long. (F) Functional megaspore. (G) Silk lengths of 0.3 to 0.6 cm. (H) A two-nucleate immature embryo sac. (I) Silk lengths of 0.4 to 0.7 cm. (J) A four-nucleate immature embryo sac. (K) Silk lengths of 0.4 to 0.8 cm. (L) An eight-nucleate embryo sac. (M) Silk lengths of 0.5 to 1.0 cm. (N) Mature embryo sac with 10 (or more) antipodal cells. (O) Silk lengths of more than 1.2 cm. E, egg; S, synergid; PN, polar nucleus; AN, antipodal cell nuclei.

4 848 The Plant Cell m Figure 2. Microtubules and Organellar DMA in the Isolated Megasporocytes at Meiosis I. (A) The megaspore mother cell (MMC) surrounded by nucellus tissue in the ovule. Bar = 10 nm. (B) An isolated megaspore mother cell at premeiosis. Bar = 1 im. (C) Anti-tubulin immunofluorescence staining of isolated megaspore mother cell showing short microtubule bundles in the cytoplasm and cortical region (arrowhead). N, nucleus. Bar = 1 urn. (D) DMA staining of cytoplasmic organelles at the chalazal end of the cell (arrow). Bar = 1 urn. (E) An isolated megaspore mother cell elongates at prophase I. Bar = 1 urn. (F) Longitudinally aligned microtubules in both poles of megaspore mother cell (arrowheads) visualized by anti-tubulin immunofluorescence staining. N, nucleus. Bar = 1 urn. (G) Hoechst fluorescence staining of the cell in (E) showing the zygotene chromosomes. Bar = 1 urn.

5 Embryo Sac Development in Maize 849 development. The results of these analyses and the comparisons of silk length with developmental stage of the reproductive cells (megasporocytes, megaspores, and embryo sacs) for each of the three sections of the four ears are presented in Table 1. The ears are listed from youngest at the top to oldest at the bottom in Table 1. The results of these detailed observations agree well with those presented in Figure 1. Florets with silk lengths ranging from O to -0.2 cm usually contained megasporocytes in the premeiotic stage, while those bearing silks between 0.2 and 0.3 cm contained megasporocytes in meiotic stages. As silk lengths approached 0.5 cm, the megaspore stage became more frequent (see Table 1, bottom section of ear 1 and middle section of ear 2). In florets with silk lengths between 0.4 and 0.5 cm, it appears that megasporogenesis was completed (see the overlap in stages of the middle and basal sections of ear 2 in Table 1). Development of the megagametophyte follows soon thereafter, so that florets with silk lengths no longer than 0.65 cm contained two-nucleate embryo sacs. Florets with silks up to 1.O cm in length contained two-, four-, and eight-nucleate embryo sacs. All florets with silks of 1.0 cm or greater contained eight-nucleate embryo sacs. These data confirmed the gradient of ovule development in florets from the base of the ears upward to the top. These results also provided a rather detailed correlation of the relationship of silk length to the stage of formation and development of the female reproductive cells. Megasporogenesis A single hypodermal cell, the archesporium, significantly increases its size and develops directly into the megasporocyte (megaspore mother cell) that is surrounded by numerous nucellus cells (Figure 2A). The early megasporocyte is initially cup shaped and characterized by a large nucleus located toward the micropylar end of the cell (Figures 26 and 2D). Numerous short cortical microtubules and cytoplasmic microtubules are aligned predominantly toward the micropylar pole and in the perinuclear region (Figure 2C). The staining with Hoechst revealed the clustering of DNA-containing cytoplasmic organelles at the chalazal end of the cell distant from the nucleus (Figure 2D). As the megasporocyte proceeds in prophase I of meiosis, the cell elongates and the nucleus moves to the center of the cell (Figures 2E and 2G). Distinct parallel microtubules align principally at both poles, while reticulate cytoplasmic microtubules distribute to a lesser extent in the perinuclear region (Figure 2F). At the early pachytene stage, the nucleus, with a distinct nucleolus (Figure 2H), is localized in the central region, and dense microtubules become obviously localized around the nuclear envelope and the cortical region of the micropylar pole (Figure 21). The chromosomes become more condensed while the organellar DNA is distributed throughout both polar regions of the cell (Figure 2J). During the late pachytene stage (Figures 2K and 2M) and diplotene stage (Figure 20), the megasporocyte contains a cytoplasmic reticulate array of microtubules, and the perinuclear microtubules become even more conspicuous (Figures 2L and 2N). At the late prophase I stage (Figures 3A and 3C), the cortical microtubule bundles are obvious at both poles, and the perinuclear microtubules retain the same configuration (Figure 36). At the metaphase I stage, the chromosomes line up at the equator of the cell while numerous DNA-containing organelles are localized at the opposite poles of the cell (Figures 3D and 3F). The metaphase spindle is characterized by focused poles aligned along the longitudinal ais of the cell (Figure 3E). During anaphase I, the two sets of chromosomes migrate to opposite poles (Figures 3G and 3H). At the end of meiosis I, the DNAcontaining organelles have relocated between the two nuclei (Figures 31 and 3K). The microtubules are more densely arranged at the midzone of the cell where the cross wall will form (Figure 3J). At prophase II (Figures 3L and 3N), the microtubules are arranged in prominent bundles and aligned at the perinuclear region (Figure 3M). Metaphase II is characterized by highly focused spindle poles (Figure 30) and the alignment of the chromosomes at the equator (Figure 3P). By the completion of meiosis, the megasporocyte has produced four megaspores, among which only the chalazal-most megaspore survives while the three other megaspores degenerate (Figure 4A). Sometimes degeneration even occurs in the micropylar cell of the dyad but more commonly in the micropylar tetrad megaspores before completion of the cytokinesis in meiosis II. The surviving megaspore shows normal morpho-.logical characters, while the degenerating megaspore has a distorted nucleus (Figures 48 and 4C). Figure 2. (continued). (H) An isolated megasporocyte at early pachytene. Bar = 1 pm. (I) Anti-tubulin immunofluorescence staining of the cell in (H) showing the perinuclear microtubules (arrowhead) and cortical microtubules (arrow). N, nucleus. Bar = 1 pm. (J) The pachytene chromosomes and DNA-containing organelles at both ends of the cell shown in (I) (arrowheads). Bar = 1 pm. (K) An isolated megasporocyte at late pachytene. Bar = 1 pm. (L) Anti-tubulin immunofluorescence staining of the cell in (K) reveals the reticulate microtubule array in the cytoplasm, microtubules surrounding the nuclear envelope (arrowhead), and short longitudinally aligned microtubules at the micropylar cortical region (arrow). N, nucleus. Bar = 1 pm. (M) Hoechst fluorescence staining of the cell shown in (L). Bar = 1 pm. (N) A diplotene megasporocyte with reticulate cytoplasmic microtubules (arrowhead). N, nucleus. Bar = 1 pm. (O) Hoechst fluorescence staining of the cell shown in (N). Bar = 1 pm.

6 850 The Plant Cell Table 1. The Pattern of Embrvo Sac Development alona the Lenath of the DeveloDina Ear Total Number Number Number Silk Number of MMC of of Number Number Number of Position - Length of MMC at Pre- MMC at Mega- of of 8N or of Ear X f SD (cm) Range (cm) or ES meiosis Meiosis spore 2N ES 4N ES More ES Ear 1 TOP Middle Base Total Ear 2 TOP Middle Base Total Ear 3 TOP Middle Base Total Ear 4 TOP Middle Base Total 0.05 f 0.03 n = * 0.06 n = f 0.11 n = f 0.03 n = n = f 0.95 n = f 0.06 n = f 0.12 n = n = f 0.14 n = f 1.48 n = n = O O oo ~ ~ MMC, megaspore rnother cell; ES, embryo sac; 2N, two-nucleate; 4N, four-nucleate; 8N, eight-nucleate Megagametogenesis Formation of the Female Gametophyte Following the completion of meiosis, the surviving megaspore comprises the initial cell of the female gametophyte generation. It expands somewhat by formation of a large vacuole at each pole of the cell with the nucleus localized at its center (Figures 4D and 40. The microtubules are mostly perinuclear, and two longitudinal microtubule bundles extend from the megaspore nucleus to the opposite cell poles (Figure 4E). The wall of the enlarged megaspore will eventually enclose the embryo sac, but first its nucleus undergoes three successive nuclear divisions (the free nuclear phase) followed by cytokinesis and cell wall formation. The first mitotic division of the megaspore gives rise to the two-nucleate embryo sac (immature female gametophyte), which undergoes a significant degree of vacuolization during its elongation and enlargement. The two nuclei migrate to the opposite poles of the developing embryo sac, and a large vacuole occupies its center (Figures 4G and 41). The microtubules are localized at the perinuclear region, and some of the microtubule bundles extend to the cortex of the embryo sac (Figure 4H). The size of the micropylar end of the embryo sac expands to a large extent (Figure a), and the micropylar end is easily distinguishable

7 Figure 3. The Organization of Microtubules and Organellar DNA in Isolated Megasporocytes at Late Meiosis I and Meiosis II. (A) A megasporocyte at diakinesis. Bar = 1 urn. (B) Reticulate microtubules in the cytoplasm that parallel microtubules at cortical regions of both poles (arrowhead) and perinuclear microtubules (arrow) visualized by anti-tubulin immunofluorescence staining. N, nucleus. Bar = 1 urn. (C) The chromosomes at diakinesis. Bar = 1 urn. (D) A megasporocyte at metaphase I. Bar = 1 urn. (E) Anti-tubulin immunofluorescence staining of the cell in (D) showing the spindle at the equator of the cell (arrowheads). Bar = 1 urn. (F) Organellar DNA distributes at both poles of the same cell in (E) (arrowheads). CH, chromosomes. Bar = 1 urn. (G) A megasporocyte at anaphase I. Bar = 1 urn. (H) Staining of the chromosomes at anaphase I with Hoechst Bar = 1 \im. (I) An isolated dyad. Bar = 1 urn. (J) Short parallel microtubules align at the midzone of the dyad shown in (I) (arrowhead). N, nucleus. Bar = 1 urn. (K) The organellar DNA (arrows) relocated to the midzone between the two nuclei of the dyad. Bar = 1 urn. (L) A megasporocyte at prophase II. Bar = 1 urn. (M) The nucleus of the prophase II cell surrounded by cage-like microtubule bundles (arrowhead). Bar = 1 urn. (N) Hoechst fluorescence staining of the cell shown in (M). Bar = 1 urn. (0) Spindle microtubules (arrowheads) at both poles of the cell. Bar = 1 urn. (P) The chromosomes of the cell shown in (O). Bar = 1 im.

8 852 The Plant Cell Figure 4. The Localization of Microtubules and Organellar DMA in the Megaspore and Embryo Sac.

9 Embryo Sac Development in Maize 853 after isolation (Figure 4K). Microtubule bundles in the perinuclear region of the micropylar end form an anastomosing network extending from the nucleus to the cell cortex (Figure 4L). The polarization of the nucleus and organellar DNA becomes conspicuous; the micropylar nucleus is surrounded by a dense accumulation of organellar DNA (Figure 4M) and perinuclear microtubules, while those in the chalazal end are present to a lesser extent (Figure 4L). The second mitotic division of the developing embryo sac synchronously occurs at both the micropylar and chalazal nuclei. Anaphase is distinguished by a focused spindle at opposite poles, interzone microtubules between kinetochores, and migrating chromosomes (Figures 4N, 40, and 4P). The late-telophase stage is characterized by the simultaneous formation of phragmoplasts between the two sister nuclei at the micropylar and at the chalazal poles (Figures 4Q and 5A). The phragmoplast at the micropylar end is oriented parallel to the long axis of the embryo sac and is nearly perpendicular to the transverse orientation of the chalazal phragmoplast (Figure 5A). Consequently, the plane of alignment of the pair of sister nuclei at the micropylar pole constitutes nearly a right angle to the plane of alignment of the pair of sister nuclei at the chalazal pole (Figure 58). No cell plates normally form after the second mitosis. Initially, diffuse and patchy microtubules organize at the perinuclear region around each pair of sister nuclei in the four-nucleate embryo sac (Figure 5C). The sister nuclei initially lie close together with the two micropylar nuclei aligned side-by-side on a line perpendicular to the long axis of the embryo sac, while the two chalazal nuclei are aligned side-by-side on a line parallel and nearly coincident with the long axis of the embryo sac (Figure 5D). However, at the late stage of the four-nucleate embryo sac, one of each of the pairs of sister nuclei migrates away from its sister nucleus and moves, respectively, from the micropylar and the chalazal poles toward the center of the embryo sac (Figures 5E and 5H). Numerous microtubule bundles are localized between the sister nuclei, and the bundle ends obviously associate with the envelopes of the sister nuclei (Figure 5F). One of the sister nuclei migrates toward the center of the embryo sac where some cortical microtubules are localized (Figure 5G). At the third mitotic division, the pair of nuclei at both the micropylar and chalazal poles all simultaneously divide. At the anaphase stage, the sister chromatids separate and the daughter chromosomes migrate in opposite directions, but at each cellular pole the two pairs of sister nuclei are aligned nearly at right angles to each other. Consequently, the orientation of the resulting pairs of sister nuclei is nearly perpendicular to each other both at the micropylar pole and at the chalazal pole (Figures 51 and 5J). At the micropylar end of the embryo sac, the resulting pair of micropylar sister nuclei oriented ata right angle to the axis of the embryo sac (indicated by topmost pair of arrows in Figure 5J) presumably develop as the synergid nuclei, while in the axial-orientated pair of sister nuclei (indicated by the pair of arrows in Figure 54, one will develop as the egg nucleus and the other as a polar nucleus. A similar pattern occurs at the chalazal region; one of the axial-oriented sister nuclei (indicated by middle pair of arrows in Figure 5J) will become a polar nucleus, and all the rest of the nuclei will develop as antipodal cells (Figures 5J and 5K). At cytokinesis in the eight-nucleate embryo sac, the phragmoplasts are simultaneously organized at both the micropylar and the chalazal poles, indicating the locations of the future cell plates between Figure 4. (continued). (A) The surviving megaspore (MP) is located at the chalazal pole while three other megaspores degenerate (arrowhead). Bar = 10 pm. (B) Two isolated megaspores: One is a degenerating megaspore (arrowhead), and the other is a surviving megaspore (arrow). Bar = 2 pm. (C) Staining with Hoechst shows the shrinkage of nuclear DNA in the degenerating megaspore (arrowhead). Bar = 2 pm. (D) The megaspore elongates and large vacuoles are visible. Bar = 2 pm. (E) The perinuclear microtubules and two longitudinally aligned microtubule bundles connect the nucleus to both poles (arrows). Bar = 2 pm. (F) The nucleus of the megaspore shown in (E). Bar = 2 pm. (O) Two-nucleate embryo sac. Bar = 2 pm. (H) The perinuclear microtubule bundles connect to the periphery region of the embryo sac (arrowhead). Bar = 2 pm. (I) Two nuclei at both poles of the embryo sac shown in (H) visualized by labeling with Hoechst Bar = 2 pm. (J) The location of the two-nucleate embryo sac as seen in a plastic thick section. ES, embryo sac. Bar = 10 pm. (K) The polarity of the two-nucleate embryo sac is evident, reflected by the larger size of its micropylar pole compared to its chalazal pole. Bar = 2 pm. (L) The perinuclear microtubule bundles connect to the peripheral region of the embryo sac (arrowhead). Bar = 2 pm. (M) The polarity of the embryo sac shown in (L) is manifested by more organellar DNA located at the micropylar pole (arrowhead) than that at the chalazal pole. Bar = 2 pm. (N) An embryo sac during the second mitotic division. Bar = 2 pm. (O) The distinct spindles (arrow) at opposite poles and interzonal spindles (arrowhead) at late anaphase of the second mitotic division of the embryo sac shown in (N). Note that the two nuclei at opposite ends are dividing simultaneously. The alignment of the spindles was distorted during preparation of the slide. A typical orientation of spindles is shown in Figure 58. Bar = 2 pm. (P) Chromosomes stained with Hoechst in the embryo sac shown in (O). CH, chromosome. Bar = 2 pm. (a) The embryo sac at telophase of the second mitotic division. Bar = 2 pm.

10 Figure 5. The Organization of Microtubules and Organellar DNA in the Four- and Eight-Nucleate Embryo Sac. (A) The phragmoplast microtubules extend between the two pairs of daughter nuclei (arrowheads) at telophase. Note the nearly perpendicular orientation of the two phragmoplasts at the opposite poles. Bar = 2 urn. (B) The telophase chromosomes at the end of the second mitotic division in the embryo sac shown in (A). Bar = 2 urn. (C) Numerous perinuclear microtubules (arrowhead) surround the pairs of nuclei at both ends of an early four-nucleate embryo sac. Bar = 2 urn. (D) The four-nucleate embryo sac shown in (C) visualized by labeling of Hoechst Note the perpendicular orientation of two pairs of nuclei. Bar = 2 jim. (E) Late four-nucleate embryo sac. Bar = 2 urn. (F) Numerous microtubule bundles are aligned between the two pairs of sister nuclei (arrowheads), and some cortical microtubule bundles (arrow) are localized on the peripheral region of the embryo sac. Bar = 2 urn. (G) A different focal plane of the embryo sac shown in (F) reveals the microtubule bundles that are adjacent to the internal nuclei of the two pairs of sister nuclei (arrows). Bar = 2 urn. (H) Four nuclei of the embryo sac shown in (E) visualized by labeling of Hoechst Bar = 2 urn. (I) An embryo sac at the third mitotic division. Bar = 2 urn. (J) Anaphase of the third mitotic division. Note that three pairs of groups of chromosomes (arrows) are conspicuous at the same focal plane. Bar = 2 urn. (K) Different focal plane of the cell shown in (J). Note the fourth pair of groups of chromosomes (arrows) at the micropylar pole of the embryo sac. Bar = 2 urn.

11 Embryo Sac Development in Maize 855 these nuclei (Figures 6A, 6B, 6C, and 6D). After formation of the cell walls, the two synergids and the egg cell are at the micropylar pole, the three antipodal cells are at the chalazal pole, and the central cell occupies the center of the embryo sac. At this stage, the two polar nuclei are still at the opposite poles of the large vacuole that is located in the center of the central cell (Figures 6E, 6F, and 6G). Numerous longitudinally aligned microtubule bundles are localized at the synergids and the antipodal cells in the micropylar and chalazal ends of the embryo sac, while sparse transverse microtubule arrays are seen in the central cell, mainly at the cortical region (Figures 6H and 61). The polar nuclei fuse (Figures 7A, 7C, and 7E), and the microtubule organizing center-like structure forms in the synergid cells right after the first division of the antipodal cells (Figures 78 and 7D). The organization of the microtubules in the synergids is characterized by dense longitudinal microtubules that radiate from the microtubule organizing center-like structure at the filiform apparatus (Figure 78). The microtubules transversely align at the peripheral region of the central cell and randomly organize in the antipodal cells (Figures 78 and 7D). Numerous DNA-containing organelles are localized in the young egg cell (Figure 7F), and the antipodal cells continue to divide and produce variable numbers of cells at the chalazal end (up to more than 40 cells); some of them extend into the chalazal area of the ovule (Figure 7F). At maturity, the embryo sac is ready for entry of the pollen tube and the events of double fertilization. DISCUSSION Developmental lndex of Embryo Sac Development In the study of Chang and Neuffer (1989), the relative length of the glume and anther in the floret was identified as a reliable morphological indicator to identify the microspore and pollen developmental stages. However, such an easily measured morphological indicator has not been reported for the development of the female gametophyte. Although the number of nodes and internodes, leaf number, and the length of the ear can be used to roughly estimate the extent of development of the ear, these parameters cannot be used to precisely determine the stages of embryo sac development. Our investigation revealed that embryo sac development in the ovules of spikes occurs in a gradient pattern, with embryo sac initiation beginning at the base of the ear and progressing toward the top. The development of the embryo sac correlates with the elongation of the silk. The ovules contain megasporocytes at premeiosis and meiosis in pistils with silks ranging from O to 0.4 cm in length. The functional megaspore undergoes three successive mitotic divisions as the silk length progresses up to 1.0 cm. The number of antipodal cells is up to 40 or more when the silks emerge from the husk. Selection of an ear with silk lengths ranging from O to 4.5 cm will provide an investigator with all stages of reproductive cells from premeiotic megasporocytes to eight-celled embryo sacs. A developmental index of embryo sac development and silk lengths similar to those described above was found for two inbred lines, Mo17 and ND101; however, the developmental index for the early flowering inbred line A344 has shorter silk lengths for corresponding developmental stages of embryo sac (B.4. Huang and W.F. Sheridan, manuscript in preparation). Kranz et al. (1992) have shown that egg cells become capable of successful in vitro fertilization when isolated from the embryo sacs of pistils with silks one-sixth as long as those required for in vivo fertilization following pollination. This indicates that the silk length may be used to determine the receptivity and functional capacity of the female gamete (egg cell) as previously suggested by Dupuis and Dumas (1989, 1990). Polarity of Nuclei and DNAContllining Organelles during Megasporogenesis and Megagametogenesis The polarity of the nuclei and DNAcontaining organelles is mainly manifested by the regular pattern of positioning of the nuclei and the asymmetrical distribution of the DNA-containing organelles during embryo sac development. The determination of the functional megaspore is probably related to the polarity of cellular features, including the disposition of the nuclei, plastid, mitochondria, and callose (Willemse, 1981). We observed that the DNA-containing organelles are predominantly localized at the chalazal end of the megaspore mother cell before meiosis and confirmed the premeiotic establishment of megasporocyte polarity (Russell, 1979). After meiosis I, a DNA-containing organelle zone is present at the future division site between the two nuclei of the dyad. A similar organelle band has been observed during microsporogenesis in orchids, and this band appears to facilitate equal apportionment of membrane-bound organelles (Brown and Lemmon, 1991). After meiosis II, the chalazal-most megaspore has received most of the organelles from the megaspore mother cell, and it is capable of further megagametogenesis (Russell, 1979). The three other micropylar megaspores, however, degenerate as manifested by their distorted nuclei. Our observations confirm the widely accepted view that in the hlygonum type of embryo sac development, the two synergids are derived from the division of one of the nuclei in the micropylar pair of the four-nucleate embryo sac, whereas the egg and one polar nucleus are the mitotic products of the other micropylar nucleus; the other polar nucleus and the three antipodal nuclei are derived from the division of the two nuclei of the chalazal pair (Reiser and Fischer, 1993; Russell, 1993). But what is intriguing and possibly most significant about our results are the alignments of the spindles and phragmoplasts during the nuclear divisions of the two- and four-nuclear stages. When the nuclei of the two-nucleate embryo sac undergo mitosis, one spindle (micropylar) is aligned in a transverse plane while the other spindle (chalazal) is oriented longitudinally,

12 856 The Plant Cell Figure 6. The Organization of Microtubule and Nuclear DMA in an Eight-Nucleate Embryo Sac and the Cells of the Megagametophyte. (A) An isolated embryo sac at cytokinesis of the third mitotic division. Bar = 2 urn. (B) Phragmoplasts are simultaneously formed at the opposite poles of the embryo sac. Note the ring-like phragmoplast (arrowheads) indicating the location of the cell plates. Bar = 2 urn.

13 Embryo Sac Development in Maize 857 relative to the micropylar-chalazal axis. This pattern is repeated again at both ends of the embryo sac in a mirror image fashion at the third mitotic division. The polarity of DNA-containing organelles is apparently established in the late two-nucleate embryo sac with predominant DNA-containing organelles surrounding the micropylar nucleus. Because the young egg contains a significant number of the DNA-containing organelles after cellularization of the embryo sac, it appears that this early event in the determination of the reproductive cells occurs in the two-nucleate embryo sac, although cellular differentiation is delayed until cytokinesis. The pattern of cell partition in the embryo sac appears to be determined by cytoplasmic determinants (Haig, 1990) or cytoplasmic domains (Brown and Lemmon, 1991). The disturbance of the positioning of the nuclei and their spatial relationship during cell formation and differentiation in the maize indeterminate gametophyte (ig) mutation results in formation of unanticipated cell types (Lin, 1978,1981; B.-Q. Huang and W.F. Sheridan, manuscript in preparation). On the basis of our observations as well as those of others, it appears that nuclear positioning in the embryo sac is an important factor in cell determination. The Role of Microtubules in Megasporogenesis Sequential morphological changes are accompanied by the modification of microtubule configuration during megasporogenesis (Bednara et al., 1988; Willemse and van Lammeren, 1988; Webb and Gunning, 1990). First, the cell shape of the megasporocyte elongates extensively along its long axis at the beginning of meiosis. The expansion of the megasporocyte in Gasteria verrucosa and Arabidopsis was believed to be controlled by the surrounding cells of the nucellus and integument because cortical microtubules were found less frequently at the edge of the cell (Willemse and van Lammeren, 1988; Webb and Gunning, 1990). In contrast, we observed that in maize dense cortical microtubules were localized at the cortex of the megasporocyte, especially at the micropylar end during prophase I. The major function of cortical microtubules is probably to aid in the establishment of cell shape (Gunning and Hardham, 1982). The presence of cortical microtubules coincides with elongation of the megasporocyte and suggests that cell expansion may also be internally controlled by cortical microtubules in this species. The nucleus of the megasporocyte expands simultaneously with cell elongation during prophase I and is characterized by dense microtubules associated with the nuclear envelope. The rapid change of concentrated perinuclear microtubules to the highly focused metaphase spindles occurs during the transition from prophase I to metaphase I and prophase I1 to metaphase 11. According to Staiger and Cande (1990), a similar change of the microtubule configuration was found throughout most of the cell cycle in maize microsporocytes and is probably controlled by a nuclear envelope-associated microtubule organization center. Second, the formation of the linear dyad and tetrad of megaspores occurs after cytokinesis of meiosis I and meiosis II. Although the preprophase band is absent during meiosis in the megasporocyte (Willemse and van Lammeren, 1988; Webb and Gunning, 1990), the future division site can be predicted by spindle orientation, the phragmoplast, and nuclear position. During metaphase I and 11, the longitudinally aligned spindle assures the formation of the linear arrangement of dyad and tetrad megaspores. These features deviate from those previously reported in Arabidopsis, where the asymmetrical metaphase I spindle and absence of an intermediate dyad stage result in the formation of a multiplanar tetrad of spores followed by simultaneous cytokinesis (Webb and Gunning, 1990). These results suggest that the microtubular cytoskeleton plays an important role in determining the pattern of tetrads of spores. The Role of Microtubules in Megagametogenesis As previously described, the regular pattern of positioning of the nuclei of the embryo sac and polarized distribution of the DNA-containing organelles are two distinct morphological features during megagametogenesis. Three distinct types of Figure 6. (continued). (C) A different focal plane of the embryo sac shown in (e). Note an oblique view of the ring-like phragmoplast at the micropylar and chalazal poles (arrowheads). Bar = 2 pm. (D) Eight nuclei in the same embryo sac shown in (B) and (C) are visible. Bar = 2 pm. (E) An isolated eight-nucleate embryo sac containing seven cells; the egg cell is not in focus. S, synergid; CC, central cell; A, antipodal cell (arrow). Bar = 2 vm. (F) Corresponding nuclei in the cells of the embryo sac shown in (E) visualized by staining with Hoechst SN, synergid nucleus; PN, polar nucleus; AN, antipodal cell nucleus. Bar = 2 pm. (G) Localization of the embryo sac in the ovule in plastic section. Bar = 10 pm. (H) An isolated eight-nucleate embryo sac. There are parallel microtubule bundles in the synergid cells, transverse microtubule arrays in the central cell, and random microtubules in the antipodal cells. S, synergid; E, egg cell; CC, central cell; A, antipodal cells. Bar = 2 pm. (I) Corresponding nuclei in the embryo sac shown in (H). SN, synergid nucleus; EN, egg nucleus; PN, polar nucleus; AN, antipodal cell nucleus. Bar = 2.

14 858 The Plant Cell Figure 7. The Organization of Microtubules and Organellar DNA in the Premature Embryo Sacs and "Female Germ Unit." (A) An isolated female germ unit including two synergid cells (S), one egg cell (not in focus), and one central cell (CC). Bar = 2 urn. (B) An array of dense longitudinally aligned microtubules is distinct in the synergid cells (S) and radiates out from the microtubule organizing center-like structure at the filiform apparatus (arrowhead). Transverse microtubules (arrow) are localized in the peripheral region of the central cell (CC). Bar = 2 urn. (C) Two synergid nuclei (SN) and one central cell nucleus (CCN) are seen in the isolated female germ unit. Bar = 2 urn. (D) An isolated embryo sac. The facial view of a microtubule organizing center-like structure at the filiform apparatus (arrowhead) shows that the microtubule bundles radiate from this structure. Transverse microtubules are conspicuous in the central cell (CC), and random microtubules are visible in the antipodal cells (A). S, synergid; E, egg cell. Bar = 2 urn. (E) Fusion of polar nuclei (PN) in the central cell and the presence of six antipodal cells. AN, antipodal nuclei; SN, synergid nuclei; EN, egg nuclei. Bar = 2 urn. (F) An isolated embryo sac. The embryo sac has 18 antipodal cells (A) and abundant organellar DNA in the egg cell (E) (arrowhead). CC, central cell; S, synergid. Bar = 2 urn. microtubules are probably involved in establishing these nuclear patterns and organellar polarity. The first type of microtubules is the perinuclear microtubules, which are mainly present in the interphase stages of the embryo sac. The perinuclear microtubules are closely associated with the free nuclei and mostly radiate from the nuclear envelope. Some of the microtubule bundles extend to the circumferential fringe of the embryo sac at the two- and fournucleate embryo sac. A similar pattern of microtubules has been reported in the development of endosperm in wheat and was believed to contribute to the precise position of the nuclei in the coenocytic phase (van Lammeren, 1988; Webb and Gunning, 1991). Its role may be especially important in the freenucleate stages, because these nuclei need a physical means of support so that they do not randomly move about in the developing embryo sac. The perinuclear network of microtubules is organized predominantly at the micropylar pole of the two-nucleate embryo sac and coincides with the polarized distribution

15 Embryo Sac Development in Maize 859 of the DNA-containing organelles at this region. Although the precise function of these perinuclear microtubules with regard to the abundant micropylar DNA-containing organelles is unclear, it is likely that each pole of microtubules claims a region of cytoplasm in which the microtubules extend and maintain one pattern of polarity (Russell, 1993). The second type of microtubules is the inter-sister nuclear microtubules. This microtubule array occurs at the late fournucleate embryo sac and is characterized by numerous parallel microtubule bundles connecting the two sister nuclear envelopes at each pole of the embryo sac. These inter-nuclear microtubules probably maintain the spatial positioning of the sister nuclei and allow or cause one member of each pair of the sister nuclei to change position in preparation for the third mitotic division. During this period, one of the pair of micropylar nuclei migrates chalazally, while one of the chalazal pair shifts micropylarly. The third type of microtubules are those of the spindles and phragmoplasts of lhe three mitotic divisions of megagametogenesis. The nearly perpendicular orientation of the sister spindles and phragmoplasts (one transverse and the other longitudinal) during the second and third mitosis is the feature that determines future nuclear positions. But the prior orientation of the spindles (and the resulting phragmoplasts) is likely to result from the interaction of the perinuclear microtubules with the inter-sister nuclear microtubules. After the third mitotic division, the position of the eight nuclei delimits the location of the seven cells of the embryo sac, where two synergid cells and an egg cell occupy the micropylar pole, the antipodal cells locate at the chalazal pole, and the central cell occupies the middle of the embryo sac. This suggests that the positioning of the nuclei by control of mitotic spindle orientation and by control of nuclear migration is fundamentally related to the process of cell differentiation and the determination of cell fate in the mature embryo sac. Cellularization Phragmoplasts accompany cell plate formation during cellularization after the third mitosis, and three models were proposed to interpret the pattern of cell plate organization (Russell, 1993). All the models agree that cellularization includes the formation of one common cell wall between the two synergids and the formation of a second cell plate between the central cell and egg apparatus. The difference between the above models is how many cell wall plates partition the four nuclei of the micropylar half of the embryo sac. According to the two cell plates model (Cass et al., 1985,1986; Folsom and Cass, 1990), only two cell plates partition the four nuclei of the micropylar half of the embryo sac, one of which, the chalazal-most egg apparatus wall, bifurcates and encloses the egg cytoplasm, separating it from the synergid nuclei. Bhandari and Chitralekha (1989), however, proposed that in Ranunculus an additional curved cell plate partitions the egg nucleus from the two synergid nuclei to form a third cell plate in the cellularization of the micropylar half of the embryo sac. According to the four cell plates model (Battaglia, 1991), two additional cell plates are organized between the egg nucleus and two adjacent synergid nuclei. In our investigation, we observed that three phragmoplasts are organized between the four nuclei in the micropylar half of the embryo sac, one of which encloses the egg cytoplasm. This result supports the model that three cell plates may partition the nuclei of the egg apparatus and central cell nucleus in maize. However, the mechanism of establishing the phragmoplast between the non-sister nuclei is still obscure. The occurrence of similar patterns of phragmoplast appearance without accompanying cell plate formation in the first and second mitotic divisions during megagametogenesis and in the free nuclei stage of endosperm development raises the issue of a possible general mechanism that controls cell plate formation (van Lammeren, 1988; Huang et al., 1990). Organization of Microtubules in the Cells of the Mature Embryo Sac After cellularization, the organization of microtubules differs significantly among the synergids, the egg cell, the central cell, and the antipodal cells. These cytoskeletal differences most likely represent both the consequences of past developmental events and structural adaptations for future function. The synergid is a highly polarized cell characterized by dense microtubules at the filiform apparatus and longitudinal arrays at the micropylar pole. The microtubules at the filiform apparatus are organized with a microtubule organizing center-like structure, and it is likely to be a common feature in the mature embryo sac of angiosperms because a similar organization of microtubules was found at the filiform apparatus in flumbago and Nícotiana (Huang et al., 1990; Huang and Russell, 1992). The organization of microtubules in the synergid may be related to its highly active metabolism reflected by rapid organelle movement in the living isolated synergids (Huang et al., 1992) and highly polarized distribution of organelles (Diboll and Larson, 1966). The micropylar distribution of microtubules suggests more rapid physiological activity at the micropylar pole in association with the filiform apparatus and supports the potential for secretory activity and transfer function in this cell (Jensen, 1974; Huang and Russell, 1992). The prominent feature of microtubules in the central cell is the transverse alignment of microtubules localized at the peripheral region of the cell. The principally cortical distribution of microtubules may aid in the expansion of the volume of the cell. In the antipodal cells, the interphase microtubules appear to be random in distribution. These rapidly divided cells extend into the nucellus tissue in the chalazal region of the ovule. The antipodal cells have a high rate of metabolic activity and may function as transfer cells between the sporophytic cells and the embryo sac (Gunning and Pate, 1969; Willemse and van Went, 1984).

16 860 The Plant Cell METHODS Analysls of Developmental lndex of Embryo Sac Development Different developmental stages of ears were collected from a standard genetic stock planted in the greenhouse of the Biology Department, University of North Dakota. The ear was dissected transversely as three equal sections. The silk lengths of 20 to 78 florets were measured individually, and their ovarias were dissected and immediately fixed in the fixative consisting Of 4% paraformaldehyde in Pipes buffer, ph 6.9, for 1 to 1.5 hr. Embryo sacs were isolated by enzyme digestion followed by manipulation and stained with Hoechst according to the protocols described below and reported previously (Huang and Russell, 1989). The embryo sacs isolated from different sections of theear werecorrelated with the silk length of corresponding sections. Chemlcal Fixation and Plastic Thlck ing Ovaries were dissected and fixed in a solution of 3% glutaraldehyde in 0.1 M sodium cacodylate buffer, ph 7.2, for 5 min under vacuum and further fixed in glutaraldehyde for 4 hr at room temperature and 1% osmium tetroxide at 4% overnight. Tissue was embedded in Spurr's resin and then sectioned at 4pm thickness. The observation of the thick sections was conducted using a microscope (Laborlux S; Leitz, Germany) equipped for differential interference contrast (DIC). Double Labellng of Microtubules and DNA Materials were fixed in the mixture of fixative consisting of 4% paraformaldehyde, 10% DMSO, 0.1% Triton X-100 in PEMG buffer (50 mm Pipes, 5 mm EGTA, 2 mm MgSO., and 4% glycerol, ph 6.8) for 30 minto 1 hr at room temperature. After rinsing with PEMG buffer three to four times, materials were incubated in the enzyme solution (2% cellulysin, 2% pectinase, 0.3% pectolyase, and protease inhibitors in PEMG buffer) at 37% for 1 hr. The protein inhibitors consisted of 2 mm phenylmethylsulfonyl fluoride, 16 pm leupeptin, and 25 pm pepstatin. The megaspore mother cells, megaspores, and embryo sacs were isolated by hand dissection with glass needles using inverted phase contrast microscopy. The isolated embryo sacs were collected in a small via1 or on a poly-l-leucine-coated cover slip. After being extracted with 1% Triton X-I00 (in PEMG buffer) for 1 hr and rinsed with PEMG buffer two to three times, the specimens were incubated in blocking solution consisting of 1% egg albumin in PBS buffer for 10 min and then were labeled with anti-a-tubulin (N356; Amersham International) diluted t200 with blocking solution for 1 to 2 hr at 37% and subsequently incubated in anti-mouse IgG diluted 1:800 with blocking solution and 0.2 to 0.4 pglml Hoechst (1 mglml in stock solution) at the same temperature for 1 to 2 hr. 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