Cytochemical investigation of genic male-sterility in Chinese cabbage

Transcription

1 Sex Plant Reprod (2005) 18: DOI /s ORIGINAL ARTICLE CT Xie Æ YH Yang Æ YL Qiu Æ XY Zhu Æ HQ Tian Cytochemical investigation of genic male-sterility in Chinese cabbage Received: 13 September 2004 / Revised: 3 January 2005 / Accepted: 17 May 2005 / Published online: 22 July 2005 Ó Springer-Verlag 2005 Abstract A genic male sterile Chinese cabbage, Brassica campestris L. ssp. chinensis Makino, was examined using cytological and cytochemical methods to characterize the process of pollen abortion in this plant. Thick sections of both fertile and sterile anthers at different developmental stages were stained using Toluidine Blue O, Periodic Acid-Schiff s (PAS) reaction and Sudan Black B to detect cytochemical changes that may occur in the distribution of insoluble polysaccharide and lipid storage bodies. Pollen abortion in sterile anthers occurs at an early stage of microspore development. During early microspore development, reductions in the number of starch grains in the connective tissue of fertile anthers coincide with the accumulation of starch grains in cells of the anther wall. In the late microspore stage, a large vacuole forms in the microspore, and tapetal cells synthesize and accumulate lipid droplets. The cellular organization of tapetal cells in sterile anthers appears similar to that in fertile anthers, except for the absence of lipid droplets in cells of sterile anthers and diffusely labeled tapetal polysaccharides, suggesting defects in nutrient storage. Keywords Brassica campestris Æ Cytochemistry Æ Genic male-sterility Æ Microsporogenesis Supported by National Natural Science Foundation of CHINA ( ) CT Xie Æ YH Yang Æ YL Qiu Æ XY Zhu Æ HQ Tian (&) Key Laboratory of Cell Biology and Tumor Cell Engineering, Ministry of Education, Xiamen University, , Xiamen, CHINA hqtian@jingxian.xmu.edu.cn Tel.: Fax: Introduction Male sterility in higher plants has been used to facilitate cross-breeding to avoid labor-intensive emasculation in the field. Classical work has included Laser and Lersten s article in 1972 summarizing patterns and stages of pollen abortion in angiosperms and Li s paper in 1978 on anther development in male sterile plants of maize, wheat, rice and sorghum. Further research on a photoperiod sensitive genic male sterile rice showed that a special barrier between the tapetum and middle layer in the anther wall inhibited calcium influx into loculi of anther causing pollen abortion (Tian et al. 1998). Disorders of calcium distribution in a photoperiod-sensitive cytoplasmic male sterile wheat have also been shown to correlate with the failure of pollen development (Meng et al. 2000). Chinese cabbage (Brassica campestris L. ssp. chinensis Makino) is a popular native vegetable crop that is widely cultivated in China. The known pattern of male sterility in Chinese cabbage is genic and is controlled by a pair of recessive genes. Some molecular biological characterization has been done to isolate genes controlling male sterility in Chinese cabbage (Miao et al. 2000; Cao et al. 2001), but structural events involved with pollen abortion in this male sterile plant are still largely unknown. In the current study, the development of fertile and sterile anthers was compared to determine the onset and structural manifestations of pollen abortion, with particular attention given to the distribution of polysaccharides and lipids during anther development in fertile and male sterile Chinese cabbage. These studies explore the relationship between nutrient metabolism and pollen abortion and will be used as a basis for further complementary molecular studies. Materials and methods Plants of Chinese cabbage (B. campestris L. ssp. chinensis Makino) were field grown at the Xiamen

2 76 Agricultural Institution. After cabbage seeds from fertile plants are planted, the progeny yield approx. 1/4 male sterile plants (72) and 3/4 fertile plants (232), and the seeds from out-crossed sterile plants will yield approx 1/ 2 sterile (147) and 1/2 fertile plants (156). These observations support that male sterility of this cabbage is controlled by a pair of sporophytically-expressed recessive genes. Before flowering, sterile and fertile plants cannot be discriminated. An early visual marker, however, is petal color, which is somewhat whiter in sterile flowers than in fertile flowers. Anthers from both fertile and sterile plants were collected at different stages of development. At least ten anthers from different flowers were fixed and at least five anthers from each treatment were examined. Anthers fixed in 2.5% glutaraldehyde in 0.1 M KH 2 PO 4 buffer (ph 7.2) for 4 h at room temperature, washed in buffer three times (20 min each) and postfixed in 1% OsO 4 for 15 h at 4 in 0.1 M KH 2 PO 4 buffer (ph 7.2) were then washed in three changes of the buffer, dehydrated in a graded acetone series and embedded in Epon 812 resin. The anthers were sectioned at 1 lm thickness, sections floated on water on a clean slide and dried to mount the sections on a slide. The sections were stained with 2% Toluidine Blue O (Aldrich, Milwaukee, USA) solution for cytological observation. For detecting insoluble polysaccharides, sections were oxidized for 10 min in 0.5% periodic acid in 0.3% nitric acid, rinsed in running water for 1 2 min with final rinse in distilled water, stained 30 min in Schiff s reagent, washed three times in 0.5% sodium metabisulfite for 2 min each, rinsed 5 min in running water and transferred to distilled water. For detecting lipids, sections were rinsed for 1 2 min in 70% ethanol, stained in fresh 1% Sudan Black B in 70% ethanol for min at C, rinsed 1 min in 70% ethanol and transferred to distilled water (Hu and Xu 1990). Sections were observed and photographed using a Leica DMR research photomicroscope. Results Both fertile and sterile anthers were observed from the microspore mother cell stage to near anther maturity. There were no differences in structure and distribution of polysaccharides and lipids before the microspore mother cell stage in fertile and sterile anthers (data not shown). We emphasized four developmental stages: (1) microspore mother cell, (2) early microspore, (3) late microspore, and (4) bicellular pollen. Development of fertile anthers Before meiosis, microspore mother cells (MMC) in the anther loculi are round and large, with prominent, centrally located nuclei (Fig. 1a). Dark wall material around the MMCs indicates initiation of the characteristic callose wall. At this stage, the anther wall differentiates into four layers: epidermis, endothecium, middle layer and tapetum, each differing in characteristic morphology. The innermost layer of the anther wall is the tapetum, containing cells that are as large in size and thickness as the three other cell layers of the anther wall combined. There are some large vacuoles in tapetal cells and each cell generally contains one or two nuclei. Parenchyma cells of the connective tissue contain abundant granules confirmed to be starch. After MMC meiosis, tetrad cells are surrounded by a densely labeled polysaccharide wall, representing a callose wall (Fig. 2a). Tapetal cells do not change in morphology and contain some large vacuoles. Starch grains diminish in abundance in the connective tissue, though sometimes seen in the three outermost wall layers of the anther, with the exception of the tapetum. Microspores released from the tetrads are densely cytoplasmic cells with a centrally located nucleus and densely labeled cell wall (Fig. 3a). Changes appearing in the anther wall include an enlargement and a filling of the cytoplasm of tapetal cells. Epidermis and endothecium are less highly cytoplasm than before, and starch grains are essentially depleted in the connective tissue. Microspores become progressively more highly vacuolated as small vacuoles coalesce to form a large central vacuole (Fig. 4a). The vacuole of the microspore occupies most of the cell and displaces the microspore nucleus to a peripheral position, which is a prelude to its asymmetric division. Microspores form a complete exine by this stage, with three apertures. The internal organization and external morphology of nearby tapetal cells become increasingly irregular. The other three anther wall layers remain highly vacuolated and continue their differentiation. Upon microspore mitosis, a large vegetative cell and smaller generative cell form, and both compose a bicellular pollen grain. The large vegetative cell vacuole progressively disappears and the bicellular pollen becomes densely cytoplasm as it matures (Fig. 5a). Tapetal cells become further irregular and ultimately these cells degenerate and are absorbed by pollen grains (Fig. 6a). Development of sterile anthers The early development of sterile anthers is essentially identical to that in fertile anthers during the MMC (Fig. 1b) and tetrad stages (Fig. 2b). After MMC meiosis, however, pollenkitt substances accumulate unevenly on the surface of early microspores and appear to have abnormal thick and thin regions on the cell (Fig. 3b). As the abnormal microspores continue to develop, their cytoplasm becomes less dense, and shrinks. Tapetum is initially abnormally thick (Fig. 4a) and then becomes vacuolated and degenerates as the microspores abort (Fig. 5b). Subsequently, cells collapse

3 77 Fig Microspore mother cells (MMCs). a MMCs in fertile anther with wall differentiated into four distinct layers; starch grains accumulate in connective tissue (arrowhead). b MMCs in sterile anther. Note starch grains in connective tissue (arrowhead). 2. Late meiosis. a In fertile anther, walls of tetrads have conspicuously labeled callose wall. b Meiosis in sterile anther displays fewer reproductive cells and some starch grains remain in connective tissues. 3. Early microspore stage. a In fertile anther, vacuoles in tapetal cells disappear as wall synthesis occurs in microspores. b In sterile anther, tapetal cells lose their vacuoles and the microspore wall formation proceeds abnormally and the anther shrivels to some degree as the locule collapses (Fig. 6a). Changes in polysaccharide distribution during the development of fertile and sterile anthers During fertile anther development, the distribution of polysaccharides in the anther correlates with the different stages of male gametophyte development. Prior to meiosis, starch grains accumulate in the connective tissue, with only a few accumulated starch grains present in the anther wall (data not shown). During meiosis, some starch grains appear in the cells of the epidermis, endothecium, and middle layer of the anther wall (Fig. 7a), but neither starch grains nor red coloring appears in tapetal cells. The callose wall, characteristic of the late MMC and tetrad is labeled red, indicating the presence of insoluble polysaccharides in the cell wall, but not within cells of the MMC or tetrad. During microspore development, the starch grains of connective tissue decrease in abundance, but some still occur in the anther wall (Fig. 8a). Tapetal cells do not display any red color, suggesting a low abundance of polysaccharidic materials in the cells. At this time, microspores in the locule actively synthesize pollen wall and some red labeling occurs in the pollen wall (Fig. 8a), presumably corresponding to the intine. After microspore mitosis, starch grains disappear from the anther. As bicellular pollen accumulates storage material, the cytoplasm and pollen wall display a diffuse red color, suggesting the occurrence of polysaccharidic material in the pollen. Then as tapetal cells mature, they disappear completely before anther dehiscence. No evident differences occur in starch distribution between fertile and sterile anthers before MMC meiosis and it appears as if there is less polysaccharide surrounding the tetrads of sterile anthers. The tapetal cells become bigger and no polysaccharide appears in the cells. There are numerous polysaccharide grains in the cells of epidermis, endothecium, middle layer of anther wall but not in the tapetum (Fig. 7b). After meiosis, microspores are released from tetrad. There are still numerous starch grains in the cells of epidermis, endothecium, middle layer of anther wall of sterile anthers (Fig. 8b). The secretions of tapetal cells in fertile plants are brighter pink (Fig 7a) than those present in sterile anthers (Fig. 7b). At the bicellular pollen stage sterile anthers contain only the remains of pollen in their locules (Fig. 8b), and at later stages, the tapetum displays intense pink coloration (Fig. 9b), suggesting an accumulation of a diffuse polysaccharidic material.

4 78 Fig Late microspore stage. a In fertile anthers, large vacuole in microspores, forcing nuclei to a peripheral position. Tapetal cell cannot be identified and some pollen invades into tapetal cells. b A sterile anther in relative late microspore stage comparing fertile anther, microspores appear abnormally. 5. Early bicellular pollen stage. a In fertile anthers, pollen grains accumulate dark material and tapetal cells become small. b In sterile anthers, pollen displays welldeveloped walls but lacks cellular contents. Note degeneration of tapetal cells. 6. Two days before anther dehiscence. a In fertile anthers, tapetal cells completely disappear, with some starch grains remaining in connective tissue. b In sterile anthers, some remnant of abortive microspores are evident, but no pollen grains Changes of lipid during the development of fertile and sterile anther Before and during meiotic division, neither fertile nor sterile anthers contain lipid droplets as a storage nutrient, rather the major storage materials are polysaccharide based (Fig. 7a, b). After meiosis, however, tapetal cells of fertile anthers begin to accumulate lipids, as indicated by the accumulation of blue tinted dark materials in the microspores (Fig. 8a). Bicellular pollen contains abundant lipid droplets in their cytoplasm (Fig. 9a) as well as the tapetum, indicating the importance of oil as an energy storage material in pollen as well as seeds. At the bicellular pollen stage, lipid droplet accumulation in the tapetal cells reaches a maximum. In sterile anthers at the MMC (Fig. 7b) and microspore stages (Fig. 8b), tapetal cells do not display lipid droplets, although abundant starch grains accumulate in cells of the epidermis, endothecium, and middle layer of the anther wall. At a stage that correlates to bicellular pollen stage in fertile anthers, the sterile anthers contain only cellular remnants (Fig. 9b) and no lipid droplets accumulate in these abortive anthers. Abortive pollen has an incomplete exine and contains no cytoplasm at late stages. Discussion Timing of developmental divergence in male-sterile anthers The onset of pollen abortion is critical to understanding mechanisms of control for male sterility in higher plants. In the Chinese cabbage used in the present study, morphological abnormalities in sterile anthers began in the developing microspores after tetrad separation. The first feature noted was that pollenkitt substances accumulated unevenly on the surface of the early microspores (Fig. 3b). Functional abnormalities during transfer and synthesis of lipid material are noted in the tapetum. Thus, in this male sterile plant observed using light microscopy, pollen abortion appears to occur in the early microspore stage. The function of tapetum and its relationship with pollen abortion Tapetal cells are physically the closest somatic cells to microspores, as they are positioned between male

5 79 Fig Meiosis. a Fertile anther with polysaccharide and starch stained red. Tetrads are enclosed in a thin callose wall (red). Some starch is evident in the connective tissue, but no label in tapetal cells (T). b In sterile anthers, abundant starch occurs in epidermis, endothecium, middle layer of anther walls, but not in tapetum. 8. Early microspore stage. a In fertile anthers, tapetal cells (T) synthesize lipid droplets (dark blue). b In sterile anthers, starch in epidermis, endothecium, and middle layers of the anther wall diminish and lipid droplets in tapetum are not evident. 9. Early bicellular pollen stage. a In fertile anthers, tapetal cells (T) and pollen grains contain abundant lipid material. b In sterile anthers, no lipid droplets accumulate in tapetal cells and polysaccharides are more diffusely labeled gametophytic tissues and surrounding sporophytic tissues. One function of tapetal cells of Brassica is to synthesize lipidic materials to support microspore development (Wu et al. 1999, 1997; Ting et al. 1998; Platt et al. 1998). In the present study, fertile and sterile anthers appear to have the same nutritional status before meiosis, as reflected in cytochemical localizations of lipids and polysaccharides in the anther. Abundant starch grains occur in the connective tissue, but no lipid droplets occur in meiotic anthers of either sterile or fertile plants. In later stages of microspore development, a large vacuole forms in the cell, starch grains in the connective tissue disappear, and lipid droplets begin to accumulate in the tapetum of fertile anthers (Fig. 8A), suggesting a shift from polysaccharidic storage products to lipidic storage products in anther somatic tissue. In sterile anthers, however, the tapetum displays diffuse red labeling, suggesting an abnormal distribution of polysaccharide in the cells, with few lipid droplets accumulating in the tapetal cells (Figs. 8b, 9b). The tapetum of fertile anthers appears to shift from polysaccharidic storage materials to lipid storage materials more effectively, as indicated by the accumulation of lipid droplets in the pollen (Figs. 8a, 9a). The disordered distribution of polysaccharides and few lipid droplets in the cytoplasm of sterile anthers may reflect a defect in metabolic shift that reflects that a normal shift from polysaccharide to lipid metabolism does not occur and ultimately results in a failure to synthesize lipid storage materials. Although many researchers have attributed male sterility in seed plants to deficiencies in the tapetum, which is intimately related with pollen nutrition (Laser and Lersten 1972; Raghavan 1997; Aarts et al. 1997; Jin et al. 1997), the abnormality of tapetal cells in this Chinese cabbage begins to occur earlier, during microspore stages. The question remains as to how microspore

6 80 abortion and tapetal cell abnormalities are related in the process of synthesizing lipid droplets. It is unclear for example whether the abortion of microspores may not provide a partial explanation for why tapetal cells fail to shift to a lipid-based mode of energy storage. Apparently, the abortion of microspores is not correlated with a depletion of energy stores in surrounding somatic tissues, as starch grains remain abundant in cells of the epidermis, endothecium, and middle layer of anther wall in sterile anthers (Fig 8b). In fertile anthers, however, the transition to lipidic storage materials may be stimulated by the further development and utilization of nutritional stores from the tapetum within the successfully maturing pollen grains in fertile anthers. Acknowledgements This work was supported by the National Natural Science Foundation of CHINA ( ). References Aarts MU, Hodge R, Kalantidis K, Florack D, Wilson ZA (1997) The Arabidopsis male sterility 2 protein shares similarity with reductases in elongation/condensation complexes. Plant J 12: Cao JS, Ye WZ, Zhang M, Zeng GW (2001) Differential display of flower bud mrna of genic male sterility (GMS) AB line in Chinese cabbage-pak-choi and analysis of differential cdna fragments. J Zhejiang Univ (Agri Life Sci) 27: Hu SY, Xu LY (1990) A cytochemical technique for demonstration of lipids, polysaccharides and protein bodies in thick resin sections. Acta Bot Sinica 32: Jin W, Horner HT, Palmer RG (1997) Genetics and cytology of a new genic male-sterile soybean (Glycine max L. Merr.). Sex Plant Reprod 10: Li RQ (1978) The cytological research of some crops male sterility. J Wuhan Univ (Nat Sci) 1: Laser KD, Lersten NR (1972) Anatomy and cytology of microsporogenesis in cytoplasmic male sterile angiosperms. Bot Rev 38: Meng XH, Wang JB, Li RQ (2000) Effect of photoperiod on calcium distribution in photoperiod-sensitive cytoplasmic malesterile wheat during anther development. Acta Bot Sin 42:15 22 Miao Y, Wu BH, Cao JS (2000) Partial cloning and sequence analysis of the genic male sterile gene in Brassica c ampestris. J Xiamen Uni (Nat Sci) 39: Platt KA, Huang AHC, Thomson WW (1998) Ultrastructural study of lipid accumulation in tapetal cells of Brassica napus L. cv. Wester during microsporogenesis. Int J Plant Sci 159: Raghavan V (1997) Molecular embryology of flowering plants. Cambridge University Press, Cambridge Tian HQ, Kuang A, Musgrave ME, Russell SD (1998) Calcium distribution in fertile and sterile anthers of a photoperiod-sensitive genic male-sterile rice. Planta 204: Ting JTL, Wu SSH, Ratnayake C, Huang AHC (1998) Constituents of the tapetosomes and elaioplasts in Brassica campestris tapetum and their degradation and retention during microsporogenesis. Plant J 16: Wu SSH, Moreau RA, Whitaker BD, Huang AHC (1999) Steryl esters in the elaioplasts of the tapetum in developing Brassica anthers and their recovery on the pollen surface. Lipids 34: Wu SSH, Platt KA, Ratnayake C, Wang TW, Ting JTL, Huang AHC (1997) Isolation and characterization of neutral-lipidcontaining organelles and globuli-filled plastids from Brassica napus tapetum. Proc Natl Acad Sci USA 94: