Calcium changes during megasporogenesis and megaspore degeneration in lettuce (Lactuca sativa L.)

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1 Sex Plant Reprod (2008) 21: DOI /s ORIGINAL ARTICLE Calcium changes during megasporogenesis and megaspore degeneration in lettuce (Lactuca sativa L.) Yi Lan Qiu Æ Ru Shi Liu Æ Chao Tian Xie Æ Scott D. Russell Æ Hui Qiao Tian Received: 15 October 2007 / Accepted: 23 May 2008 / Published online: 14 June 2008 Ó Springer-Verlag 2008 Abstract Potassium pyroantimonate was used to localize loosely-bound calcium in young ovules of lettuce (Lactuca sativa L.) during megasporogenesis to investigate the relationship between ionically available calcium and megaspore degeneration. At the megasporocyte (megaspore mother cell) stage, few calcium precipitates were located in the ovule. Following meiosis in the megasporocyte, a linear tetrad of four megaspores is formed, with three of the four megaspores degenerating from the micropylar end inward. Only the chalazal-most megaspore continues to develop, becoming the functional megaspore. A decrease in amount of calcium precipitates in the megaspore, particularly in the nucleus, precedes the breakdown of the micropylar megaspores, which subsequently undergo structural disintegration and loss of recognizable cellular features. A partial recovery of calcium precipitates occurs during later degeneration. The functional megaspore retains a consistently higher concentration of calcium precipitates during development, which is retained in the developing embryo sac. This, to our knowledge, is the first report related to calcium dynamics during megaspore degeneration, and may Communicated by Mauro Cresti. C. T. Xie H. Q. Tian (&) School of Life Sciences, Xiamen University, Xiamen, China hqtian@xmu.edu.cn Y. L. Qiu R. S. Liu School of Life Sciences, Hunan Normal University, Changsha, China S. D. Russell Department of Botany and Microbiology, University of Oklahoma, Norman, OK 73019, USA facilitate future research aimed at elucidating the mechanisms of megasporogenesis. Keywords Calcium Embryo sac development Lettuce (Lactuca sativa L.) Megaspore Megasporogenesis Introduction During sexual reproduction of higher plants, meiosis in the megasporocyte (megaspore mother cell) results in the production of four haploid megaspores. In over 80% of angiosperms, only one of the four megaspores continues to develop to form an embryo sac, whereas the other three degenerate during ovule development. This predetermined megaspore abortion is a mechanism that resolves megaspore competition in monosporic angiosperms (Haig 1990). Despite an extensive classical literature on megasporogenesis, the mechanisms controlling this critical phase of embryo sac establishment and functional megaspore determination have not been fully elucidated, particularly given the difficult task of investigating megasporogenesis, which occurs very early in plant reproduction and takes place deep inside the young ovule. Current information rests largely on ultrastructural, rather than cytochemical or molecular studies (Russell 1979; Schulz and Jensen 1986; Webb and Gunning 1990), and thus the role of calcium, which is well known during plant reproduction in general (Ge et al. 2007) is incompletely known at this stage. One labile form of calcium that is readily available for metabolic activation as free Ca 2+ is in the form of looselybound calcium. This particular type of calcium may be made available by changing anionic binding sites to contribute to developmental processes that require dynamic

2 198 Sex Plant Reprod (2008) 21: activation by free cytoplasmic calcium (Ge et al. 2007). Thus, study of this particular form of calcium, and particularly its distribution in the ovule before and after fertilization, and during in vivo pollen tube passage in the pistil has been investigated in multiple species using potassium pyroantimonate. In the presence of looselybound calcium, the presence of antimonates causes the formation of calcium antimonate precipitates that can be visualized using electron microscopy (Chaubal and Reger 1994; Tian and Russell 1997; Tian et al. 2000). In these studies, the association of calcium with male fertility as well as female fertility, appears to be a sensitive marker. Significant redistribution of calcium has also been used to judge fertility in genic male sterile anthers of rice, which failed to accumulate calcium precipitates in sterile males (Tian et al. 1998). These data suggest that calcium plays crucial roles during sexual reproduction in tissues of both sexes, as well as serving as a harbinger of successful postfertilization development in situ and in vitro (Wang et al. 2006). We undertook this study to examine the relationship between calcium localization and megaspore degeneration in the Polygonum-type embryo sac of lettuce. This is, to the best of our knowledge, the first such study of calcium redistribution during megasporogenesis that has been reported, and indicates that a significant redistribution of calcium antimonate during megaspore degeneration occurs contributing to a suspected instance of programmed cell death in non-functional megaspores. Materials and methods Young ovules of lettuce (Lactuca sativa L.) were dissected from fresh flowers and fixed (3 h, room temperature) in 2% glutaraldehyde (v/v) in 0.1 mol/l KH 2 PO 4 buffer (ph 7.8) containing 1% potassium pyroantimonate (K 2 H 2 Sb 2 O 7 4H 2 O). Ovules were washed (three 30-min changes in buffered 1% potassium pyroantimonate), post fixed in 1% (w/v) buffered OsO 4 containing 1% potassium pyroantimonate (16 h, 4 C), washed in buffer (three 30-min changes), dehydrated in a graded acetone series and embedded in Spurr s resin. Over 100 ovules were embedded and sectioned; ovules were thin sectioned (80 nm), and stained with saturated aqueous uranyl acetate in 50% methanol (v/v). Stained sections were observed and photographed with a JEM-100CX II transmission electron microscope. For statistical treatment of calcium precipitates in the cells, electron micrographs were scanned and saved as digital images in a computer. The abundance of calcium precipitation in the cells was examined using imaging software (Simple PCI Version 5.3.1) from Compix, Inc. (USA). Using this software, the quantity of calcium precipitates in a cell may be automatically calculated by circumscribing a desired area for analysis. Using this method, we calculated the quantity of precipitates in the cytoplasm and nucleus of each cell examined, and then cytoplasmic precipitates were calculated by subtraction. Five or more ovules were examined at each reported stage of megasporogenesis. Results Calcium distribution in the megasporocyte The megasporocyte is an elongated cell with a stellate cross-sectional profile. Prior to meiosis, it may exceed the length of surrounding nucellar cells by 4 to 5 times and has a centrally-located nucleus (Fig. 1a). Despite the presence of conspicuous calcium precipitate accumulation in surrounding nucellar cells, calcium precipitates are rarely detected in the cytoplasm and nucleus of the megasporocyte. A few precipitates, however, are not uncommon in small vacuoles (Fig. 1b). Calcium distribution in the megaspore tetrad stage Following meiosis, four haploid megaspores form axially within the ovule, giving rise to a linear tetrad (Fig. 1c). Each of these megaspores displays similar cellular organization, with a large nucleus located in the center of the cell and numerous small organelles in each cell (Fig. 1d). There was an increase in the number of calcium precipitates in the megaspore cells within the tetrad compared to the earlier megasporocyte stage, but no conspicuous differences were noted in precipitate distribution between the four megaspores at this stage. Calcium distribution during megaspore degeneration Megaspore degeneration occurs first in the micropylar-most megaspore in the lettuce ovules. The first evidence of degeneration is a reduction in the size of the megaspore and an increase in the electron density of the cytoplasm (Fig. 2a), which permits the identification of the degenerating megaspores. As the first megaspore degenerates, it develops a significant difference in calcium distribution when compared to the other three megaspores. The micropylar-most megaspore displays tiny calcium precipitates in the cytoplasm, but only a few in its nucleus (Fig. 2b). At this stage, more calcium precipitates accumulate in the third megaspore, especially in its nucleolar vacuole (Fig. 2c), whereas the second megaspore (Fig. 2d) contains fewer calcium precipitates. The changes noted in the second megaspore seem to foreshadow imminent degeneration, despite that this

3 Sex Plant Reprod (2008) 21: Fig. 1 Transmission electron micrographs (TEM) of pyroantimonate-labeled lettuce ovules in the megasporocyte and the megaspore tetrad. a A megasporocyte (MMC) in a young ovule. 91,700; bar = 2.9 lm b No calcium precipitates were noted in the nucleus (n) of MMC, with few observed in the small vacuoles. 96,000; bar = 1 lm c A tetrad array, À` ˆgive the order of four megaspores from the micropylar toward the chalazal end. 91,700; bar = 2.9 lm d Enlargement of c. The second megaspore which would degenerate afterward displayed normal structure. 96,000; bar = 1 lm megaspore displays normal organelle structure and cellular appearance. The electron density of the cytoplasm and nucleus is greater in degenerated megaspores compared to other megaspores and surrounding nucellar cells, and calcium precipitates are highly dispersed and small in the cytoplasm (Fig. 2e). Some degenerated megaspores have fewer cytoplasmic calcium precipitates and display strong nuclear condensation, but numerous calcium precipitates appear in surrounding nucellar cells (Fig. 2f). There was a decrease in volume of the first and second megaspores following degeneration, and the shape of both cells rapidly becomes anomalous as the cells collapse (Fig. 3a). In the fourth and chalazal-most megaspore, calcium precipitates increase significantly, especially in the nucleus (Fig. 3b); this fourth megaspore represents the future functional megaspore and leads the other megaspores in calcium precipitate abundance. During the initial stages of degeneration of the third megaspore, its ultrastructure also

4 200 Sex Plant Reprod (2008) 21: Fig. 2 TEM of pyroantimonate-labeled lettuce ovules following onset of megaspore degeneration. a The first megaspore (À) in the tetrad in the micropylar end degenerated. 91,800; bar = 2.8 lm b Some calcium precipitates accumulated in the cytoplasm of the fourth megaspore (ˆ) but few in its nucleus (n). 99,600; bar = 1 lm c The third megaspore ( ) displayed a greater number of calcium precipitates in its cytoplasm and nucleus (n) compared to the second megaspore (`). 99,600; bar = 1 lm d The second megaspore (`) was noted with a decrease in calcium precipitates in its cytoplasm and nucleus (n). 99,600; bar = 1 lm e High electron density of cytoplasm and nucleus (n) in the degenerated megaspore À. Arrow represents a condensed chromatin. 99,600; bar = 1 lm f A few calcium precipitates in degenerated megaspore with condensed chromatin (arrows). 99,600; bar = 1 lm appears normal initially and various organelles displayed well developed profiles, but it also displays a decrease in calcium precipitates (Fig. 3c), both compared to the fourth megaspore (Fig. 3b) and to prior megaspores (Fig. 2c). The cytoplasmic electron density of the degenerated megaspores was high, and cell organelles were not clearly identifiable. Many calcium precipitates were noted once again in cells that had just undergone cellular degeneration (Fig. 3d). Numerous calcium precipitates appear to accumulate at the same time in the surrounding nucellar cells. Degeneration of the third megaspore resulted in increased electron density in the cytoplasm (Fig. 4a). The form of the nucleus in degenerated megaspores is still identifiable, with strongly pycnotic characteristics, including nuclear shrinkage, chromosome condensation, clumping of chromatin masses and alteration of the nuclear envelope. Cellular organelles, however, could not be readily identified. Some calcium precipitates appeared again in the degenerated megaspores (Fig. 4b). The fourth megaspore, located at the chalazal end of the tetrad, continued to develop into a functional megaspore, containing many organelles and a large nucleus located in the central region of the cell. The micropylar end wall of the functional megaspore was more electron-dense and thicker (Fig. 4c) than the chalazal end wall (Fig. 4d), displaying a strong polarity in the structure of the wall (Fig. 4a). There are numerous calcium precipitates noted in the cytoplasm, as well as some larger precipitates in the nucleus of the functional megaspore (Fig. 4e). When the nucleus of the functional megaspore divides and forms two nuclei, forming the binucleate embryo sac, a

5 Sex Plant Reprod (2008) 21: Fig. 3 TEM of pyroantimonatelabeled lettuce ovules following degeneration of the second megaspore (`). a Two megaspores (À and `) had degenerated, with intact megaspores ˆobserved in the chalazal end of tetrad. 91,800; bar = 2.7 lm b An increase in calcium precipitates is observed in the fourth megaspore (ˆ), especially in its nucleus (n). 99,600; bar = 1 lm c A significant decrease in calcium precipitates was noted in the cytoplasm and nucleus (n) of the third megaspore ( ), especially in its nucleus (n). 99,600; bar = 1 lm d Some calcium precipitates moved into the first megaspore (À) again, and many precipitates were noted in surrounding nucellar cell. 99,600; bar = 1 lm large vacuole soon forms in the central region of the cell, relegating the two nuclei toward the micropylar and chalazal ends of the cell. Some calcium precipitates were also observed in the binuclear embryo sac and in surrounding nucellar cells (Fig. 4f). Statistical analysis of calcium precipitates during megasporogenesis Following meiosis of the megasporocyte, the number of calcium precipitates in the four megaspores was about 0.1/lm 2 in the nucleus and about 0.3/lm 2 in the cytoplasm (Fig. 5). After degeneration of the first megaspore, there was a tenfold decrease in calcium precipitates in the nucleus (to 0.01/lm 2 ) and cytoplasm (to 0.03/lm 2 ) in the second megaspore prior to its degeneration. An increase in precipitates is noted in the nucleus (0.4/lm 2 ) and cytoplasm (1.5/lm 2 ) of the third and forth megaspores at this stage. We also observed a decrease in calcium precipitates in the nucleus (0.02/lm 2 ) and cytoplasm (0.06/lm 2 ) of the third megaspore following the degeneration of the second megaspore. With an increase in abundance of calcium precipitates to 0.4/lm 2 in the nucleus and 1.9/lm 2 in the cytoplasm of the fourth megaspore, no further changes

6 202 Sex Plant Reprod (2008) 21: Fig. 4 TEM of pyroantimonatelabeled lettuce ovules following degeneration of the third megaspore ( ). a Three megaspores ( ) degenerated and only one (ˆ) in the chalazal end of the tetrad developed into a functional megaspore. 91,800; bar = 2.7 lm b In a degenerated megaspore ( ), the nuclear membrane was broken, and the electron density of cytoplasm increased, with some calcium precipitates in small vacuoles. 99,600; bar = 1 lm c In the micropylar end of functional megaspore, its wall (arrow) was thicker than that in chalazal end. Numerous small calcium precipitates accumulated in its cytoplasm. 914,400; bar = 1 lm. d In the chalazal end of the functional megaspore, the wall (arrowheads) was thinner than that in micropylar end. 914,400; bar = 1 lm e The nucleus (n) of functional megaspore contained fewer, but bigger calcium precipitates than in the cytoplasm. 99,600; bar = 1 lm f The nucleus of functional megaspore divided to form a two-nucleate (n) embryo sac and a large vacuole was forming between the two nuclei. 91,800; bar = 2.5 lm were noted in the quantity of calcium precipitates in the cytoplasm of the functional megaspore until the binucleated embryo sac stage (Fig. 5). Discussion In over 80% of angiosperms, embryo sacs are of the Polygonum type, producing four megaspores, of which only the chalazal-most megaspore functions to form the mature embryo sac, with the remaining three micropylar megaspores degenerating in situ (Haig 1990). Highly correlated with megaspore degeneration, Rodkiewicz (1970) observed that in the megasporocyte, callose was detected in the micropylar walls of that cell, but that wall-related callose later disappeared or became severely depleted at the chalazal end of the ovule where the functional megaspore eventually developed. This was presumed to be related to changes in the permeability of the functional chalazal megaspore, whereas the three nonfunctional megaspores remained completely surrounded by a thick layer of callose-rich walls from their inception to their ultimate degeneration (Rodkiewicz 1975). In the present study, spatial temporal changes were observed in calcium distribution during megasporogenesis. Relatively few calcium precipitates accumulated in the megasporocyte. Following meiosis, however, calcium precipitates appeared to accumulate in the four megaspores. Initially, the localization of these calcium precipitates appeared identical in the megaspores, but preceding degeneration there was a severe and statistically significant depletion of loosely-bound calcium. As megaspore

7 Sex Plant Reprod (2008) 21: Fig. 5 Relative abundance of calcium precipitates in the megaspore mother cell (MMC), three stages during tetrads in which a gradient of abortion sweeps from the micropylar megaspores (M1, M2, M3) toward the central-most megaspore in the ovule (M4), functional megaspore (FM) and the two-nucleate embryo sac (2N-ES). Calcium concentrations are given in precipitates per lm 2 based on sectional profiles degeneration reached completion and cellular integrity was lost, loosely-bound calcium levels returned to most of their prior level, to *1.5 ppt/lm 2 (Fig. 5). The functional and adjacent megaspores, however, seem to accumulate calcium precipitates during the course of megasporogenesis and thus, appear to bind more loosely-bound calcium until just before their degeneration. Since microspore degeneration consistently follows a dramatic decrease in calcium precipitates, a pre-degeneration calcium flux (evident in the quantity of calcium precipitates localized in abortive megaspores) appears to be functionally associated with megasporogenesis. Such an association between rapid, short-term depletion of loosely-bound calcium stores and imminent degeneration is indicated according to incubation with potassium pyroantimonate. Since this is first observation of such a phenomenon in flowering plants, confirmation may be needed to verify if this occurs generally during megasporogenesis in Polygonum-type embryo sacs and whether this may occur in other embryo sac types as well. During flower development, programmed cell death (PCD) has been reported at various developmental stages and specific sites. Within the ovule, degeneration is evident in surrounding nucellar cells, which permits expansion of the female gametophyte, in addition to megaspores, and these appear to represent different pathways of degeneration (Russell 1979). Megaspore degeneration is believed to be associated with PCD (Bell 1996; Wu and Cheung 2000) and precedes the encroachment of other surrounding cells rather than crushing them, which is certainly suggestive of PCD rather than senescence. To date, however, the most complete study of PCD within the female gametophyte is the synergid, not the abortive megaspores. According to An and You (2004) degeneration of the synergid of wheat during PCD displayed chromatin condensation, nuclear deformation and distinct shrinkage, which is consistent with PCD in animals. In the present study, degenerating megaspores of lettuce also displayed chromatin condensation indicative of pycnosis, which is a PCD process, increases in electron density, loss of membrane integrity, loss of compartmentalization in organelles and ultimately many of the changes associated with PCD in animals. Programmed cell death in animals involves a number of characteristic changes in cellular organization involving activation of signal transduction pathways, new protein expression, DNA breakdown and cellular disintegration, known as apoptosis. Since changes in free Ca 2+ often serve as secondary messengers in apoptotic signaling pathways, changes in dynamic calcium concentrations have been implicated in PCD of plant cells (Kuo et al. 1996; Xuand Heath 1998; Yamaguchi et al. 1999), with transient increases in calcium suggesting a signaling mechanism that may induce cell death (Nicotera and Orrenius 1998; Ning et al.1999; Wang et al. 2001). Increases in soluble calcium concentrations have been implicated in the activation of calcium-dependent DNA restriction endonucleases, resulting in DNA-regulated decomposition (Fukuda 1996; Heath 1998; Xu and Chye 1999; Xu and Hanson 2000). In some animal studies of PCD, however, instances of decreased cellular calcium concentration along with lower cytosolic free Ca 2+ were reported [e.g., hematopoietic cells, (Baffy et al. 1993); neurons, (Eichler et al. 1994; Babcock et al. 1999)]. While the association between decreased calcium levels and PCD has not been fully elucidated, to our knowledge, no such association has been observed in higher plants. Although the current study examined loosely-bound Ca 2+ concentration independently of changes in free calcium, whether significant alterations occur in the concentration of cytosolic free Ca 2+ is not elucidated by our current study. The depletion of loosely-bound calcium in a degenerating cell, however, suggests that active recruitment of Ca 2+ in triggering responses may be associated with cell death and with enhanced cytosolic Ca 2+ availability. Whether such changes are enough to cause degeneration by themselves, whether calcium serves primarily as a secondary messenger, or whether calcium is involved solely as an incidental trigger will need to be determined by studies using other more directly informative probes. Acknowledgment This study was supported by the National Natural Science Foundation of China ( ). References An LH, You RL (2004) Studies on nuclear degeneration during programmed cell death of synergid and antipodal cells in Triticum aestivum. Sex Plant Reprod 17:

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