Histological dynamics of anther tapetum in Heuchera micrantha

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1 Proc. Indian Aead. Sei., Vol. 88 B, Part H, Humber 4, July 1979, pp , 9 printed in India. Histological dynamics of anther tapetum in Heuchera micrantha M R VIJAYARAGHAVAN and SHARADA RATNAPARKHI Department of Botany, University of Delhi, Delhi MS received 19 June 1978; revised 20 April 1979 Abstract. In Heuchera micrantha Dougl. the tapetum is secretory and its behaviour variable. The study of tapetum with reference to development and behaviour of meiocytes suggests a correlation between them. In a few sporangia meiocytes follow more than one way of development indicating that their behaviour is not totally controlled by the tapetum. Inner tangential walls of tapetal cells show thickening composed of sporopollenin-like substance. The thickening probably prevents transport of nutrients from tapetum to meiocytes resulting in hypertrophied tapetum and degeneration of meiocytes. Keywords. Heuchera micrantha; anther tapetum; hypertrophy; callose; sporopollenin. 1. Introduction The tapetal cells constitute a distinct component of the anther wall. Their close association with the developing meiocytes have made them an important object for investigation. The changes that occur in the tapetal cells during rrteiocyte development have received adequate attention (Maheshwari 1950; Wunderlich 1954; Vijnyaraghavan and Ratnaparkki 1973; Kapoor et al 1978), but little is known nbout the tapetal population that surrounds the meiocytes which deviate from the normnl path of development. In Heuchera micrantha Dougl. (Saxifragaceae), besides normal pollen grains, meiocytes arrested at various stages of development, aberrant tetrads, gigantic pollen grains and pollen-embryo sacs are encountered. The variation in the behaviour of meiocytes (Vijay~traghavan and Ratnaparkhi 1977) led us to investigate the histological dynamics of the anther tapetum which surrounds them. 2. Material and methods Buds and flowers of H. micrantha were fixed in formalin-acetic-alcohol. Dehydration, infiltration and embedding in paraffin wax were done in the conventional way. Sections were cut between 5 and 7 pm and stained with safranin-fast green 309

2 310 M R Vijayaraghavan and Sharada Ratnaparkhi combination and PAS-technique. Acetocarmine squashes and acetolysed pollen grains were examined. Sections were also observed under Reichert dark-field fluorescence microscope. 3. Observations 3.1. Ontogeny of anther tapetum A young anther consists of a mass of cells delimited by an epidermis. Later hypodermal archesporial cells differentiate in each anther lobe and cut off primary parietal and primary sporogenous cells. The cells of primary parietal layer divide periclinally to torm two secondary parietal layers of which the inner one matures directly into the tapetum. The cells of the connective lining the sporogenoas cells become distinct by their large-sized nuclei and nucleoli and mature into mpetum. These cells function like tapetal cells towards the inner side of the anther locale Behaviour of tapetal cells during the development of meiocytes During the formation of secondary sporogenous cells, the tapetal cells lying towards the wauward side of an anther locule remain small, quadrangular, densely cytoplasmic and uninucleate, whereas those situated on the connective side show poor cytoplasmic contents and prominent nucleoli. At microspore mother cell stage, the tapetal cells increase in size and later become binucleate. Sporangia with a two-layered tapetum towards the connective (figure 5, arrow) are also observed. During meiosis II and at the microspore tetrad stage, enlarged tapetal cells containing either two fusing nuclei or a single, large multinueleolate nucleus are observed. Both tangentially or radially elongated tapetal cells are recognised, In a sporangium the pattern of distribution of these two types of cells is not uniform. Tapetal cell degeneration is most pronounced during the formation of microspore tetrads. In a sporangium all tapetal cells either degenerate simultaneously or discordantly. No traces of tapetum are observed in dehisced anthers, 3.3. Structure of tapetum with reference to variable behaviour of meiocytes Meiocytes arrested at various stages of development, tetrads with enlarged or degenerated cells (figure 2) and pollen grains with variable number of nuclei have been observed. The structure of meiocytes may be uniform in all the sporangia of an anther (figures 1, 2), or uniform only in one pair of sporangia, or different in all the four sporangia, or variable even within the same sporangium Tapetal cell morphology in anthers showing uniform behaviour of meiocytes In an anther all the sporangia may contain normal pollen grains or degenerated microspore tetrads, or bi-, tetra- or multinucleate microspores or empty micro. spores. (i) In sporangia bearing normal pollen grains the tapetal ceils are either of normal size or slightly flattened, densely cytoplasmic and with two multinacleolate nuclei. (ii) Sporangia containing degenerated microspore tetrads

3 .4nther tapetum in H. micrantha 311 Figures Heuchera micrantha--structure of the anther tapetum. 1. Anther in transection; tapetal cells surrounding bi-, and tetranucleate microspores are small and densely cytoplasmic, x A part of transverse section of an anther showing large, vacuolated tapetal cells (t) around degenerated microspore tetrads. x Portion of an anther in ti'ansverse section. Note la~'ge tapetal ceils with distinct thickenings on their inner tangential walls (arrow) surrounding degenerating tetrads. Adjacent sporangium bearing normal uninucleate and a few large, binucleate microspores, however, shows one layer of small, densely cytopb, srnic tapetal cells, x Anther in transection to show striking resemblance between thickening on inner wails of tapetal cells and exine (arrows). x 779. (dmt--degenerated microspore tetrads; t--tapetum; thuthickening on tapetal wall.)

4 312 M R I/Tjayaraghavan and Sharada Ratnaparkhi Figures 5-7. Heuchera micrantha--xtructure of the anther tapetum. 5. An anther lobe; tapetal cells towards connective are biseriate, x A part of transverse section of an anther. In one sporangium enlarged structures (arrows) in association with compressed pollen grains and in the other bi- and tetranucleate microspores are seen. Note similarity in the structure of tapetal cells in the two sporangia, x Fluorescence micrograph of section of an anther mounted in plain distilled water and observed under ultraviolet light. Thickenings on inner tangential wails of tapetal cells display fluorescence, x 459. (bt--bilayered tapetum; t~tapetum; th--thickening on walls of tapetal cells.)

5 Anther tapetum in H. micrantha 313 show large tapetal cells. These cells are either poor in cell contents or empty. A prominent thickening is seen on their inner tangential walls which extends towards the radial walls forming a cap-like structure (figures 3, 4). (iii) Sporangia bearing 2- or 4-nucleate microspores (figure 1) consist of normal-sized, densely cytoplasmic and binucleate tapetal cells. (iv) Sporangia containing empty microspores reveal tapetal cells devoid of any cell contents Structure of tapetal cells in anthers showing nwre than one type of behaviour of meiocytes In an anther : (i) of the two juxtaposed sporangia one bears empty pollen grains, whereas the other bears degenerated microspore tetrads. Both the sporangia reveal large, empty tapetal cells; (ii) of the two juxtaposed sporangia one contains densely cytoplasmic, multinucleate microspores whereas the other degenerated microspore tetrads. Tapetal cells in the former are characterised by the presence of dense or vacuolated cytoplasm and barely visible nuclei; in the latter they are large with scanty contents and thickened inner walls. Comparative studies of several sporangia bearing either multinucleate structures or degenerated tetrads indicate that tapetal cells of such sporangia differ from each other mainly in the size and density of cytoplasm. Occasionally, two sporangia bear multinucleate structures while the other two normal pollen grains. Tapetal cells in such anthers appear to be normal Tapetal cell structure in sporangium revealing more than one kind of behariour of meiocytes Occasionall), within a single sporangium the following situations can be noted: (i) meiocytes following normal course of development, enlarged, bi-, or tetranucleate meiocytes and meiocytes arrested at various stages of development; (ii) normal or enlarging microspore tetrad and degenerated tetrads; (iii) a few small bi-, or tetranucleate pollen grains along with normal pollen grains; (iv) a few large uninacleate microspores with normal or compressed microspores (figure 6), and (v) multinucleate, sacciform structures with degenerated microspores. Tapetal cells surrounding such heterogenous populations of meiocytes do not differ from those investing normal meiocytes Nature of thickening on the inner tangential walls of tapetal cells Tapetal cells surrounding degenerated microspore tetrads show a very distinct thickening on their inner tangential and a portion of radial walls (figures 3, 4). The thickenings resemble exine of normal pollen grains (figure 4) and are PAS negative. These autofluorescence are then irradiated with ultraviolet rays (figure 7). Thus the nature of the thickenings is different from that of outer tangential and radial walls of tapetal cells which are PAS positive and non-autofluorescent. The intensity of fluorescence of the pollen wall and the coating on the tapetal cell walls is curiously the same.

6 314 M R Vijayaraghavan and Sharada Ratnaparkhi 4. l)iseussioa 4.1. Correlation between anther tapetum and meiocytes There are not many studies to ascertain the relation between anther tapetum and the variable behaviour of meiocytes. In Heuchera micrantha the meiocytes in all the sporangia of an anther generally follow uniform course of development. Occasionally, however, in adjacent sporangia or even within the same sporangium of an anther, meiocytes following different paths of development are observed. Such variations provide an ideal situation for comparative studies of the behaviour of meiocytes and the tapetum. Present observations indicate that if the meiocytes in all the fottr sporangia of an anther behave uniformly then the tapetal cells surrounding them also exhibit uniformity in their tectonics. Morphological features of tapetal cells can be regarded as an expression of their functional state. Association of the meiocytes behaving in a particular way with particular type of tapetum indicates a positive correlation between them. Morphological variations in the tapetal cells of adjacent sporangia containing meiocytes following different trends of development (present work) as structural and behavioural differences in the tapetum of male-fertile and male-sterile plants (Zenkteler 1962; Hayward and Manthriratna 1972; Whyte 1975; Kalyani 1978) further indicate correlation between the tapetum and the meiocytes (see Steil 1949). In H. micrantha, occasionally, meiocytes following different courses of development are encountered within the same sporangium. This indicates that meiocyte morphogenesis is not always controlled by the tapetum. This is revealed further by anthers in which sporangia bearing normal pollen grains and multint~cleate microspores show morphologically similar tapetal cells Tapetal cell hypertrophy Hypertroptty of tapetal cells leading to the occlusion of the sporangial cavity has been regarded as ~ cause of pollen sterility. How hypertrophy brings about degeneration of meiooyte~ is variously explained (Rustagi and Mohan Ram 1971). Artschwager (1947) believes that meiocytes degenerate owing to lack of nutrition and excessive pressure exerted by hypertrophied tapetal cells. Higher callase activity of the hypertrophied tapetum may also result in precocious degradation of callose and production of sterile pollen grains (Nanda and Gupta 1974). In H. micrantha (present work) it is envisaged that diffusion of nutrients to the meiocytes is prevented by the thick coating on the inner wails of tapetal cells. The nutrients are most probably used op in the enlargement of the tapetal cells themselves and consequently these cells extend up to the centre of the locule. By the pressttre imposed by these cells, the degenerating tetrads coalesce and form an amorphotts mass. It is unlikely that ~petal cells absorb nutrients from degenerating tetrads as the density of their cell contents does not increase during tetrad degeneration. The coating on the inner wails of tapetal cells resembles exine and fluoresces with the same intensity suggesting that it is composed of sporopollenin-like substances. Dickinson and Bell (1976) observed accumulation of sporopollenin

7 Anther tapetum in H. micrantha 315 in tapetal walls and believed it to be due to local atttopolymerisation of sporopollenin precursors (see Echlin 1968). That sporopollenin precursors are synthesised in the tapetum (Rowley 1963; Risueno etal 1969; Heslop-Harrison 1971) and that tapetal products like orbicales (Vijayaraghavan and Marwah 1969) resemble pollen wall (Ubisch 1927 ; Christensen et al 1972) are well known. In H. micrantha precocious synthesis of sporopollenin precursors followed by their polymerisation on the inner walls of tapetal cells may impede transport of nutrients from tapetum to meiocytes resulting in hypertrophy of tapetum and degeneration of meiocytes. We believe that not only the imbalance in the nutritional mechanism in the tapetum but any irregularity in the period of synthesis and transport of wall materials may also bring about degeneration of meiocytes. Acknowledgement One at" us (SR) thanks the University Grants Commission for award of a Postdoctoral Fellowship. References Artschwager E 1947 Pollen degeneration in male sterile sugar beets, with special reference to the tapetal plasmodium; Y. Agric. Res Christensen J E, Homer Jr H T and Lersten N R 1972 Pollen wall and tapetal orbicular wall development in Sorghum bicolor (Gramineae); Am. J. Bet Dickinson H G and Bell P R 1976 The changes in the tapetum of Pinus banksiana accompanying formation and maturation of the pollen; Ann. Bet Echlin P 1968 Development of the pollen grain wall ; Bet. Dtsch. Bet. Ges Hayward M P and Manthriratna M A P P 1972 Pollen development and variation in the genus Lolium. 1. Pollen size and tapetal relationships in male fertiles and male steriles; Z. Pflanzenzuchtg Heslop--Harrison J 1971 Sporopollenin in the biological context; in Sporopollenin eds. J Brooks, P R Grant, M D Muir, P Van Gijzel and G Shaw (New York: Academic Press), pp 1-30 Kalyani Y 1978 Ontogenetical and histochemical studies on anthers of male-fertile and malesterile Pennisetum typhoides (Burro. F.) Stapf and Hubb; M.Sc. Thesis, Delhi University, Delhi Kapoor Tripat, Vijayaraghavan M R and Parulekar N K 1978 Ontogeny, structure and differentiation of anther tapetum in Celsia coromandeliana; Phyton (Austria) Maheshwari P 1950 An introduction to the embryology of angiosperms (New York: McGraw-Hill Book Co. Inc.) Nanda K and Gupta S C 1974 Malfunctioning tapetum and callose wall behaviour in Allium cepa microsporangia; Beitr. Biol. Pflanz Risueno M C, Giminez-Martin G, Lope-Suez J F and Garcia M I R 1969 Origin and developmeat of sporopollenin bodies; Protoplasma Rowley J R 1963 Ubisch body development in Pea annua; Grana Palynol Rustagi P N and Mohan Ram H Y 1971 Evaluation of mendok and dalapon as male gametocides and their effects on growth and yield of linseed; New Phytol Steil W N 1949 Some evidence for the interaction of tapetal and sporogenous ceils in certain vascular plants; Bet. Gaz Ubisch G V 1927 Zur Entwicklungsgeschichte der Antheren; Planta Vijayaraghavan M R and Marwah K N 1969 Studies in the family Ranunculaceae--microsporangium, microsporogenezis and ubisch granules in Nigella damascena; Phyton p. (B)--6

8 316 M R Vijayaraghavan and Sharada Ratnaparkhi Vijayaraghavan M R and Ratnaparkhi S 1973 Dual origin and dimorphism of the anther tapetum in Alectra thomsoni Hook.; Ann. Bot Vijayaraghavan M R and Ratnaparkhi S 1977 Pollen-embryo sacs in Heuchera micrantha Dougl. ; Caryologia Whyte R O 1975 The geography of abnormal meiosis in plants; Nucleus Wunderlich R bet das Antbertapctum mit besonderer Bcrucksichtigung kernzahl; Ost. Bot. Z Zenkteler M 1962 Microsporogenesis and tapetal development in normal and male-sterile carrots (Daucus carota); Am. J. Bot