Protoplasma DOI 10.1007/s00709-013-0567-y
SHORT COMMUNICATION
Classical macroautophagy in Lobivia rauschii (Cactaceae) and possible plastidial autophagy in Tillandsia albida (Bromeliaceae) tapetum cells Alessio Papini & Stefano Mosti & Wouter G. van Doorn
Received: 13 July 2013 / Accepted: 9 October 2013 # Springer-Verlag Wien 2013
Abstract The tapetum in anthers is a tissue that undergoes programmed cell death (PCD) during the production of pollen. We observed two types of autophagy prior to cell death. In Lobivia rauschii (Cactaceae), tapetum cells showed plant-type autophagosomes–autolysosomes, which have been found previously exclusively in root meristem cells. The autophagic structures were formed by a network of tubules which apparently merged laterally, thereby sequestering a portion of the cytoplasm. The organelles observed in the sequestered material included multilamellar bodies, which have not been reported earlier in these organelles. By contrast, Tillandsia albida (Bromeliaceae) tapetum cells contained no such organelles but showed plastids that might possibly carry out autophagy, as they contained portions of the cytoplasm similar to the phenomenon reported earlier in Phaseolus and Dendrobium. However, the ultrastructure of the T. albida plastids was different from that in the previous reports. It is concluded that in L. rauschii classical plant macroautophagy was involved in degradation of the cytoplasm, while in T. albida such classical macroautophagy was not observed. Instead, the data in T. albida suggested the hypothesis that plastids are able to carry out degradation of the cytoplasm. Keywords Autolysosomes . Autophagosomes . Cell death . Macroautophagy . Degeneration . Plastid . Tapetum
Handling Editor: Peter Nick A. Papini (*) : S. Mosti Dipartimento di Biologia Vegetale, Università di Firenze, Via La Pira, 4, 50132 Florence, Italy e-mail: [email protected] W. G. van Doorn Mann Laboratory, Department of Plant Sciences, University of California, Davis, CA 95616, USA
Abbreviations PCD TEM
Programmed cell death Transmission electron microscopy
Introduction Programmed cell death (PCD) takes place in many types of plant cells, including the tapetum, a tissue that surrounds the male gametophytes in plants (Parish and Li 2010). The tapetum in anthers serves a number of functions, the most important of which are pollen nourishment and the production of the main component of the exine layer. Therefore, pollen development is strictly related to the development of the tapetum. The next description follows Brighigna and Papini (1997), Milocani et al. (2006), Papini and Brighigna (1993), Papini et al. (1999) for Lobivia and Tillandsia anthers. At the stage of very young anther, the tapetum cells surround the microspores mother cells (mmcs) and do not differ much from normal parenchymatic cells. This stage lasts until the meiosis of the mmcs. In this stage, the meiosis of the mmcs leads to the formation of dyads and later tetrads of microspores. The tetrads remain for a given time connected among themselves through cytomictic channels and enveloped by a callose layer that filters the exchange with the tapetum cells. At this stage, the tapetum has detached from the mmcs forming a cavity, the loculus. The tapetal cells have now a more radially elongated shape and pave the loculus cavity. Normally, the tapetal cells at this stage show two nuclei in relation to their high metabolic activity. This is the case, at least, in secretory tapeta (other plants show tapeta that do not form a loculus; see Pacini et al. 1985), as those of Lobivia rauschii and Tillandsia albida.
A. Papini et al.
At a later development stage, the callose layer disappears and the microspores remain free in the loculus. They will undergo a mitosis to form the generative cell and the pollen tube cell in the two species here considered (in some plants the generative cell undergoes mitosis in the anther to form the sperm cells and the pollen is hence released as three-cell pollen. This stage is that in which the nutrients and signals exchange between pollen and tapetum is the highest during the development. During the last stage, the loculus contains mature pollen grains with a fully formed exine layer. In this stage the tapetum undergoes a PCD genetic program. Tapetum degeneration results in deposition of material on the outside of the pollen, called pollen coating, and helps to loosen the pollen from the surrounding tissue (Pacini 1997; Papini et al. 1999; Vizcay-Barrena and Wilson 2006). This developmental program requires that pollen and tapetal cells undergo a continuous cell reorganization following the changes in developmental stages. Such cell reorganization is largely due to autophagic processes that allow to recycle organelles and the cytoskeleton components. PCD in plants is often accompanied by a decrease of cytoplasm volume. It has been suggested that this decrease involves autophagy (Liu and Bassham 2012), but the evidence is still quite scarce. Microautophagy is the uptake of cellular constituents at the lysosomal (or vacuolar) membrane (Bassham et al. 2006). Macroautophagy is carried out further away from the vacuole (van Doorn and Papini 2013). It usually involves larger parts of the cytoplasm than microautophagy. In animals, macroautophagy is carried out by a double membrane (ER-like) structure that extends and eventually surrounds a portion of the cytoplasm. Once a portion of the cytoplasm has been completely surrounded, the structure is called autophagosome. In animal cells, the autophagosome joins a vesicle that contains hydrolases, called lysosome. The merged structure is called autolysosome. The hydrolases from the lysosome destroy the inner membrane of the autophagosome and the sequestered portion of the cytoplasm (Yang and Klionsky 2010). In plants a similar — but clearly different — macroautophagic process seems to occur. The following sequence of events has been suggested. Small tubes, containing hydrolases, are formed. The tubes form a cage-like network, and merge laterally. The final result is a continuous doublemembrane-bound spherical space in the cytoplasm, or cupshaped, after Avin-Wittenberg et al. (2012) terminology. A portion of the cytoplasm becomes sequestered inside this space. The inner membrane becomes degraded as well as the portion of sequestered cytoplasm. The result is a small vacuole. This process has been described mainly in root meristem cells, as summarized by Marty (1999). Plants seem to have at least one additional way of destroying the cytoplasm. Plastids seemed to contain portions of the cytoplasm. These portions inside plastids showed hydrolytic
activity, indicating that the material became degraded. The plastids apparently take up parts of cytoplasm, containing at least endoplasmatic reticulum and membranes. Evidence for this type of macroautophagy has been found only in two genera, suspensor cells in Phaseolus (dicotyledons; Nagl 1977; Gärtner and Nagl 1980) and tepal mesophyll cells in Dendrobium (monocotyledons; van Doorn et al. 2011). This report examines macroautophagy prior to cell death in the tapetum in T. albida (Bromeliaceae, monocotyledons) and L . rauschii (Cactaceae, dicotyledons), using transmission electron microscopy (TEM). We found plant-type autophagosome–autolysosomes only in L . rauschii. The data led to the hypothesis that plastids carry out autophagy in T. albida. The data in this study are from what was called the first phase of pollen development, until the meiotic prophase in Lobivia and at the stage of free microspores in Tillandsia, both in the tapetum and the pollen grain (Papini et al. 1999).
Materials and methods Parts of the anthers of T. albida Mez et Purpus and L . rauschii Zecher, about 2 mm in length, were prefixed in 2.5 % glutaraldehyde and 4 % paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). The samples were fixed in OsO4 1 % in the same buffer for 1 h. After dehydration in an ethanol series, the samples were embedded in Spurr's epoxy resin. Sections, approximately 800 nm thick, were cut with a diamond knife, stained with uranyl acetate and lead citrate, and were examined using a Philips EM300 transmission electron microscope at 80 kV.
Results Plant-type autophagosomes–autolysosomes in Lobivia tapetum cells Two stages of plant-type macroautophagy of the type described by the school of Buvat (see Introduction) are shown in Fig. 1a. The arrowhead points to a portion of the cytoplasm that has become surrounded by an electron-translucent structure. The portion of the cytoplasm inside this structure is bound by a membrane. The outer part of the translucent structure is also bound by a membrane. A similar structure, although at an earlier stage of development, is indicated by a dashed arrow. The white arrow points at tubules, which form a circle contiguous with the larger half-moon shaped structure indicated by the dashed arrow. The arrowhead in Fig. 1b also indicates a late developmental stage of a macroautophagous structure. However, the translucent area around a portion of the cytoplasm is here considerably thinner than the ones shown in Fig. 1a. The arrows
Autophagy in tapetum cells Fig. 1 Macroautophagy in Lobivia rauschii tapetum cells. The tapetum was at a late stage during the first phase of tapetum development (see text), whereby the microsporemother cell was surrounded by a thin layer of callose. a Two stages of plant-type macroautophagy. A portion of the cytoplasm has almost become surrounded by an electrontranslucent structure, bound by two membranes (white arrowhead). Tubules (provacuoles; white arrow) are present around another portion of the cytoplasm. Some of these tubules have apparently already merged (dashed white arrow). b A portion of the cytoplasm has almost become surrounded by a thin electron-translucent structure, bound by two membranes (white arrowhead). Loose tubules (white arrows) are also found. c The upper structure indicated by a white arrowhead consists of several tubules, most of which have merged to form an electrontranslucent structure around a portion of the cytoplasm. In the lower structure indicated by a white arrowhead, the tubules have apparently completely merged. The white arrows indicate places in the cytoplasm where tubules seem to start surrounding portions of the cytoplasm. The thick white arrow indicates a possible multilamellar body. d An autophagous structure (white arrow) in which the merging of the tubules is complete. This structure includes a multilamellar body (black arrow). e Another example of a thin electrontranslucent structure around a portion of the cytoplasm, which is almost closed (white arrowhead). The arrows indicate places in the cytoplasm where tubules seem to start surrounding portions of the cytoplasm
in Fig. 1b indicate tubules. The upper arrowhead in Fig. 1c shows an autophagous structure which is almost complete, but in which the tubules are in the process of merging laterally. Some tubules — especially at the lower end of the structure — have already merged, while others (towards the top of the structure) have not. The other arrowhead in Fig. 1c indicates a
a
b
c
d
e
more advanced stage of tubule merging, whereby an almost contiguous electron-translucent area has been formed. The small arrows in Fig. 1c indicate several other tubules. The cytoplasm inside the observed macroautophagic structures often contain only ribosomes (Fig. 1a,b,e). In the lower autophagous structure in Fig. 1c (large arrow) an
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electron-dense body is present in addition to a portion of cytoplasm mainly containing ribosomes. This might be a multilammellar body (i.e., a body consisting of concentric membranes). Figure 1d shows a clear example of a multilamellar body (black arrow) in the autophagous structure (arrowhead) in addition to a portion of cytoplasm mainly containing ribosomes. The white arrowhead Fig. 1e indicates another example of a developing autophagosome in which the translucent area around a portion of the cytoplasm is thin, similar to the structure observed in Fig. 1b. Arrows in Fig. 1e indicate tubules. No classical macroautophagy in Tillandsia tapetum cells, possible role of plastids in cytoplasm degradation The autophagous structures shown in Fig. 1a–e were absent in the tapetum cells of Tillandsia. Tillandsia, by contrast, contained what we hypothesize to be autophagous plastids, which were absent in Lobivia tapetum cells. In Fig. 2a–d, the plastids are indicated by big black arrows. The plastids contain no grana, only a few membranes, and some plastoglobuli. Some plastids show what might be starch granules (Fig. 2c, arrowhead; Fig. 2e, arrowheads). Double-membrane-bound structures were found inside these plastids (white arrows in Fig. 2a,b). These structures, called intraplastidial spaces, seemed to hold a portion of the cytoplasm as they contained ribosomes and membranes. In the intraplastidial spaces, some areas are less electrondense than others. Some of these less electron-dense areas are bound by a membrane (dashed arrows in Fig. 2b and d, arrows in Fig. 2e and f). Possible degeneration of the cytoplasm inside the intraplastidial spaces might be suggested from the data of Fig. 2d (white arrow), as some parts are less electron-dense than others. A similar structure is observed in Fig. 2e (white arrow) and in Fig. 2f (thick black and thick white arrow). Two membranes located closely together in the intraplastidial space of Fig. 2f are indicated with an arrowhead. What might be hypothesized to be a stage of disintegration of the sequestered cytoplasm is shown in Fig. 2f (dashed arrow). Figure 2g exhibits two plastids. The one at the left of the micrograph contains a large crystalloid (thick white arrow). This plastid also contains several spaces with what seems to be debris, which might therefore be intraplastidial spaces, but it cannot be excluded that they are starch granules (black arrows). Other such areas have no debris and might therefore be intraplastidial spaces after degradation of its cytoplasm contents. They might also possibly be starch grains (thin white arrows). The plastid at the right side of the micrograph shows what is apparently an early stage of autophagy (arrowhead).
Discussion We distinguished the same phases of tapetum and pollen development in T. albida and L . rauschii as described previously (Papini and Brighigna 1993; Papini et al. 1999). In the first phase of development the microspore mother cell is surrounded by a thin layer of callose. The tapetum cells assume a prismatic shape with the main axis in radial position with respect to the center of the anther. Plasmodesmata connect the tapetum cells. All data in the present paper refer to this first phase. Early during tapetum development, proplastids are present. These generally develop into elaioplasts, i.e., plastids that store lipidic material (Pacini 1997). In T. albida, during the early stage of tapetum development (studied here), we did not observe plastids with lipid storage compartments. The plastids thus were not elaioplasts (unpublished data). We rather found plastids that possibly contained some starch granules, but mostly intraplastidial spaces. In the species studied here, the pollen are covered by pollenkitt. This derives from collapsed tapetal cells. These cells die, whereby their contents fill the loculus and coat the pollen. Pollenkitt is usually deposited on the pollen just before the anther opens (Pacini 1997). The type of tapetum in both species studied can be classified as parietal, i.e., secretory. This confirms the data of Pacini et al. (1985), who called the type of tapetum found in Bromeliaceae parietal. Autophagy-like structures like those shown in Fig. 1 have been described previously only in root meristem cells. The cells were from Hordeum sativum (Poaceae; Buvat 1977; Buvat and Robert 1979), Cucurbita pepo (Cucurbitaceae; e.g., Coulomb et al. 1982), Euphorbia characias (Euphorbiaceae; Marty 1978) and Scorzonera hispanica (Asteraceae; Coulomb 1973). These papers are from the school of Roger Buvat in Marseille, France. In this work, we report, independently, similar structures in a very different tissue: the tapetum of L. rauschii (Cactaceae). We confirm the work by the school of Buvat in root meristems: their interpretation, as far as we can see, was correct. It should be noted that TEM micrographs in a few other papers also showed autophagic structures similar to the ones provided by the school of Buvat and the ones found here (Toyooka et al. 2006; Gaffal et al. 2007; Avci et al. 2008; Hiratsuka and Terasaka 2010). However, these papers did not point out what these structures might be and thus did not give a proper interpretation. Previously, multilamellar bodies have apparently not been reported inside the portion of cytoplasm that becomes sequestered by the plant type autophagosome–autolysosome. The present data show such a body unequivocally in Fig. 1d, and less clearly in Fig. 1c. For a three-dimensional interpretation of our data on this type of autophagy, we refer to the study of Marty (1978),
Autophagy in tapetum cells
Fig. 2 Macroautophagy in Tillandsia albida tapetum cells. The tapetum was at a late stage during the first phase of tapetum development (see text), whereby the microspore-mother cell was surrounded by a thin layer of callose. a–g Plastids in Tillandsia albida tapetum showing apparent plastidial autophagy. Plastids in a–d are indicated with black arrows. Intraplastidial spaces in a are shown using white arrows. In b and d–f, electron-translucent areas in the intraplastidial space are indicated with dashed white or black arrows. The arrowheads in c and e point at possible starch grains or intraplastidial spaces at are electron-translucent, and therefore at a late stage of cytoplasm degeneration. The arrows in e and f indicate areas with less electron-dense material in the intraplastidial
spaces, compared with more grainy material in the other parts of these spaces. In f, the dashed arrow indicates an area in the intraplastidial space in which the electron density is low but not yet electron-translucent. The arrowhead in f points at two membranes close together. The plastid at the left of micrograph g contains a large crystalloid in its centre (thick arrow). It also has some spaces showing apparent debris, which might therefore be remnants of degradation of a portion of the cytoplasm (black arrows), but might also be starch grains. In other such spaces, no debris is found (white arrows). These might be remnants of degradation of a portion of the cytoplasm, or are starch grains. The plastid at the right shows a structure that seems to be an early stage of autophagy (arrowhead)
although we do not know if in our system the autophagous tubules derive from the interface of the ER and the Golgi apparatus or from another location. We observed a large crystalloid in an organelle of Tillandsia. Such crystalloids are only found in plastids (Nguyen et al.
2001), peroxisomes (Evert 2006), and sometimes in protein storage vacuoles (Jiang et al. 2000). As the structure of vacuoles and peroxisomes is quite different from the one shown in Fig. 2g, it is concluded that the organelle at the left side of the figure is a plastid. This plastid also contained several
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intraplastidial spaces, as some debris was found inside. Other such areas had no debris but seemed also intraplastidial spaces, although it cannot be ruled out that they were starch granules. The plastids depicted contained double-membrane bound intraplastidial spaces. These spaces were interpreted to be due to autophagy, in line with previous data (Nagl 1977; Gärtner and Nagl 1980; van Doorn et al. 2011). Both in Phaseolus (Nagl 1977; Gärtner and Nagl 1980) and Dendrobium (van Doorn et al. 2011) acid phosphatase was demonstrated inside such intraplastidial spaces. Similar plastids were observed also during the functional megaspore growth in the ovule of other species of Tillandsia (Papini et al. 2011). Based on the present data, we hypothesize that plastids in T. albida tapetum cells carry out autophagy. This hypothesis is based on extrapolation from similar phenomena in Phaseolus and Dendrobium (see Introduction). We observed a double membrane around a space inside several T. albida plastids. Both in Phaseolus (Gärtner and Nagl 1980) and Dendrobium (van Doorn et al. 2011), acid phosphatase was demonstrated inside the space that was bound by a double membrane. The partial disappearance of electron-opaque material in these spaces in T. albida, it is here hypothesized, might also be due to hydrolase activity. Nonetheless, several features of the plastids in T. albida were different from the autophagous plastids previously reported in Phaseolus and Dendrobium . The doublemembrane-bound spaces in T. albida had a more irregular shape, as shown in Fig. 2f. Second, electron-translucent areas, bound by a membrane, were observed inside the spaces of T. albida (Fig. 2, dashed arrows). For a three-dimensional interpretation of our data on this possible type of autophagy, we refer to a previous paper (van Doorn et al. 2011). The present data do not show a causal relationship between autophagy and PCD. It is possible that the macroautophagy we observed in L . rauschii tapetum cells has (a) little to do with the cause of death, (b) is a cause of death, or even (c) delays death. It is concluded that in L . rauschii classical plant macroautophagy was involved in degradation of the cytoplasm, while in T. albida such classical macroautophagy was not observed. Instead, the data in T. albida suggested the hypothesis that plastids are able to carry out some degradation of the cytoplasm. Acknowledgements The work was supported by grants from the Fondi di Ateneo of the University of Florence and from the Fondazione Cassa di Risparmio di Pistoia e Pescia. Conflict of interest The authors declare that they have no conflict of interest.
References Avci U, Petzold HE, Ismail IO, Beers EP, Haigler CH (2008) Cysteine proteases XCP1 and XCP2 aid micro-autolysis within the intact central vacuole during xylogenesis in Arabidopsis roots. Plant J 56:303–315 Avin-Wittenberg T, Honig A, Galili G (2012) Variations on a theme: plant autophagy in comparison to yeast and mammals. Protoplasma 249: 285–299 Bassham DC, Laporte M, Marty F, Moriyasu Y, Ohsumi Y, Olsen LJ, Yoshimoto K (2006) Autophagy in development and stress responses of plants. Autophagy 2:2–11 Brighigna L, Papini A (1997) Plastidial dynamics during pollen development in Tillandsia albida Mez et Purpus (Bromeliaceae) before anthesis. Phytomorphology 47(1):59–65 Buvat R (1977) Origine golgienne et lytique des vacuoles dans les cellules méristématiques des racines d’orge (Hordeum sativum). Comptes Rendues de l’Académie des Sciences, Paris, 284:167–70 Buvat R, Robert G (1979) Vacuole formation in the actively growing root meristem of barley (Hordeum sativum). Am J Bot 66:1219–1237 Coulomb C (1973) Diversité des corps multivesiculaires et notion d'hétérophagie dans le méristème radiculaire de scorsonère (Scorzonera hispanica). J Microsc 16:345–360 Coulomb PJ, Coulomb C, Coulomb PJ, Michel G (1982) Use of Triton WR-1339 in cytochemical and biochemical characterization of phytolysomes. Histochem J 14:175–196 Evert RF (2006) Esau's plant anatomy, 3rd edn. Wiley, Hoboken Gaffal KP, Friedrichs GJ, El-Gammal S (2007) Ultrastructural evidence for a dual function of the phloem and programmed cell death in the floral nectary of Digitalis purpurea. Ann Bot 99:593–607 Gärtner PJ, Nagl W (1980) Acid phosphatase activity in plastids (plastolysomes) of senescing embryo-suspensor cells. Planta 149: 341–349 Hiratsuka R, Terasaka O (2010) Pollen tube reuses intracellular components of nucellar cells undergoing programmed cell death in Pinus densiflora. Protoplasma 248:339–351 Jiang L, Phillips TE, Rogers SW, Rogers JC (2000) Biogenesis of the protein storage vacuole crystalloid. J Cell Biol 150:755– 770 Liu Y, Bassham DC (2012) Autophagy: pathways for self-eating in plant cells. Annu Rev Plant Biol 63:215–237 Marty F (1978) Cytochemical studies on GERL, provacuoles, and vacuoles in root meristematic cells of Euphorbia. Proc Natl Acad Sci U S A 75:852–856 Marty F (1999) Plant vacuoles. Plant Cell 11:587–599 Milocani E, Papini A, Brighigna L (2006) Ultrastructural studies on bicellular pollen grains of Tillandsia seleriana Mez (Bromeliaceae), a neotropical epiphyte. Caryologia 59(1):88–97 Nagl W (1977) 'Plastolysomes' — plastids involved in the autolysis of the embryo-suspensors in Phaseolus. Z Pflanzenphysiol 85:45–51 Nguyen M, Francis D, Schwartz S (2001) Thermal isomerisation susceptibility of carotenoids in different tomato varieties. J Sci Food Agric 81:910–917 Pacini E (1997) Tapetum character states: analytical keys for tapetum types and activities. Can J Bot 75:1448–1459 Pacini E, Franchi GG, Hesse M (1985) The tapetum: its form, function and possible phylogeny in Embryophyta. Plant Syst Evol 149:155–185 Papini A, Brighigna L (1993) The ultrastructure of the tapetum of Tillandsia albida Mez et Purpus. Phytomorphol 43:261–275 Papini A, Mosti S, Brighigna L (1999) Programmed-cell-death events during tapetum development of angiosperms. Protoplasma 207: 213–221
Autophagy in tapetum cells Papini A, Mosti S, Milocani E, Tani G, Di Falco P, Brighigna L (2011) Megasporogenesis and programmed cell death in Tillandsia (Bromeliaceae). Protoplasma 248:651–662 Parish RW, Li SF (2010) Death of a tapetum: a programme of developmental altruism. Plant Sci 178:73–89 Toyooka K, Moriyasu Y, Goto Y, Takeuchi M, Fukuda H, Matsuoka K (2006) Protein aggregates are transported to vacuoles by a macroautophagic mechanism in nutrient-starved plant cells. Autophagy 2:96–106
van Doorn WG, Kirasak K, Sonong A, Srihiran Y, van Lent J, Ketsa S (2011) Do plastids in Dendrobium cv. Lucky Duan petals function similar to autophagosomes and autolysosomes? Autophagy 7:584–597 van Doorn WG, Papini A (2013) Ultrastructure of autophagy in plant cells: a review. Autophagy: 9 (online First) Vizcay-Barrena G, Wilson ZA (2006) Altered tapetal PCD and pollen wall development in the Arabidopsis ms1 mutant. J Exp Bot 57:2709–2717 Yang Z, Klionsky DJ (2010) Eaten alive: a history of macroautophagy. Nat Cell Biol 12:814–822
SHORT COMMUNICATION
Classical macroautophagy in Lobivia rauschii (Cactaceae) and possible plastidial autophagy in Tillandsia albida (Bromeliaceae) tapetum cells Alessio Papini & Stefano Mosti & Wouter G. van Doorn
Received: 13 July 2013 / Accepted: 9 October 2013 # Springer-Verlag Wien 2013
Abstract The tapetum in anthers is a tissue that undergoes programmed cell death (PCD) during the production of pollen. We observed two types of autophagy prior to cell death. In Lobivia rauschii (Cactaceae), tapetum cells showed plant-type autophagosomes–autolysosomes, which have been found previously exclusively in root meristem cells. The autophagic structures were formed by a network of tubules which apparently merged laterally, thereby sequestering a portion of the cytoplasm. The organelles observed in the sequestered material included multilamellar bodies, which have not been reported earlier in these organelles. By contrast, Tillandsia albida (Bromeliaceae) tapetum cells contained no such organelles but showed plastids that might possibly carry out autophagy, as they contained portions of the cytoplasm similar to the phenomenon reported earlier in Phaseolus and Dendrobium. However, the ultrastructure of the T. albida plastids was different from that in the previous reports. It is concluded that in L. rauschii classical plant macroautophagy was involved in degradation of the cytoplasm, while in T. albida such classical macroautophagy was not observed. Instead, the data in T. albida suggested the hypothesis that plastids are able to carry out degradation of the cytoplasm. Keywords Autolysosomes . Autophagosomes . Cell death . Macroautophagy . Degeneration . Plastid . Tapetum
Handling Editor: Peter Nick A. Papini (*) : S. Mosti Dipartimento di Biologia Vegetale, Università di Firenze, Via La Pira, 4, 50132 Florence, Italy e-mail: [email protected] W. G. van Doorn Mann Laboratory, Department of Plant Sciences, University of California, Davis, CA 95616, USA
Abbreviations PCD TEM
Programmed cell death Transmission electron microscopy
Introduction Programmed cell death (PCD) takes place in many types of plant cells, including the tapetum, a tissue that surrounds the male gametophytes in plants (Parish and Li 2010). The tapetum in anthers serves a number of functions, the most important of which are pollen nourishment and the production of the main component of the exine layer. Therefore, pollen development is strictly related to the development of the tapetum. The next description follows Brighigna and Papini (1997), Milocani et al. (2006), Papini and Brighigna (1993), Papini et al. (1999) for Lobivia and Tillandsia anthers. At the stage of very young anther, the tapetum cells surround the microspores mother cells (mmcs) and do not differ much from normal parenchymatic cells. This stage lasts until the meiosis of the mmcs. In this stage, the meiosis of the mmcs leads to the formation of dyads and later tetrads of microspores. The tetrads remain for a given time connected among themselves through cytomictic channels and enveloped by a callose layer that filters the exchange with the tapetum cells. At this stage, the tapetum has detached from the mmcs forming a cavity, the loculus. The tapetal cells have now a more radially elongated shape and pave the loculus cavity. Normally, the tapetal cells at this stage show two nuclei in relation to their high metabolic activity. This is the case, at least, in secretory tapeta (other plants show tapeta that do not form a loculus; see Pacini et al. 1985), as those of Lobivia rauschii and Tillandsia albida.
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At a later development stage, the callose layer disappears and the microspores remain free in the loculus. They will undergo a mitosis to form the generative cell and the pollen tube cell in the two species here considered (in some plants the generative cell undergoes mitosis in the anther to form the sperm cells and the pollen is hence released as three-cell pollen. This stage is that in which the nutrients and signals exchange between pollen and tapetum is the highest during the development. During the last stage, the loculus contains mature pollen grains with a fully formed exine layer. In this stage the tapetum undergoes a PCD genetic program. Tapetum degeneration results in deposition of material on the outside of the pollen, called pollen coating, and helps to loosen the pollen from the surrounding tissue (Pacini 1997; Papini et al. 1999; Vizcay-Barrena and Wilson 2006). This developmental program requires that pollen and tapetal cells undergo a continuous cell reorganization following the changes in developmental stages. Such cell reorganization is largely due to autophagic processes that allow to recycle organelles and the cytoskeleton components. PCD in plants is often accompanied by a decrease of cytoplasm volume. It has been suggested that this decrease involves autophagy (Liu and Bassham 2012), but the evidence is still quite scarce. Microautophagy is the uptake of cellular constituents at the lysosomal (or vacuolar) membrane (Bassham et al. 2006). Macroautophagy is carried out further away from the vacuole (van Doorn and Papini 2013). It usually involves larger parts of the cytoplasm than microautophagy. In animals, macroautophagy is carried out by a double membrane (ER-like) structure that extends and eventually surrounds a portion of the cytoplasm. Once a portion of the cytoplasm has been completely surrounded, the structure is called autophagosome. In animal cells, the autophagosome joins a vesicle that contains hydrolases, called lysosome. The merged structure is called autolysosome. The hydrolases from the lysosome destroy the inner membrane of the autophagosome and the sequestered portion of the cytoplasm (Yang and Klionsky 2010). In plants a similar — but clearly different — macroautophagic process seems to occur. The following sequence of events has been suggested. Small tubes, containing hydrolases, are formed. The tubes form a cage-like network, and merge laterally. The final result is a continuous doublemembrane-bound spherical space in the cytoplasm, or cupshaped, after Avin-Wittenberg et al. (2012) terminology. A portion of the cytoplasm becomes sequestered inside this space. The inner membrane becomes degraded as well as the portion of sequestered cytoplasm. The result is a small vacuole. This process has been described mainly in root meristem cells, as summarized by Marty (1999). Plants seem to have at least one additional way of destroying the cytoplasm. Plastids seemed to contain portions of the cytoplasm. These portions inside plastids showed hydrolytic
activity, indicating that the material became degraded. The plastids apparently take up parts of cytoplasm, containing at least endoplasmatic reticulum and membranes. Evidence for this type of macroautophagy has been found only in two genera, suspensor cells in Phaseolus (dicotyledons; Nagl 1977; Gärtner and Nagl 1980) and tepal mesophyll cells in Dendrobium (monocotyledons; van Doorn et al. 2011). This report examines macroautophagy prior to cell death in the tapetum in T. albida (Bromeliaceae, monocotyledons) and L . rauschii (Cactaceae, dicotyledons), using transmission electron microscopy (TEM). We found plant-type autophagosome–autolysosomes only in L . rauschii. The data led to the hypothesis that plastids carry out autophagy in T. albida. The data in this study are from what was called the first phase of pollen development, until the meiotic prophase in Lobivia and at the stage of free microspores in Tillandsia, both in the tapetum and the pollen grain (Papini et al. 1999).
Materials and methods Parts of the anthers of T. albida Mez et Purpus and L . rauschii Zecher, about 2 mm in length, were prefixed in 2.5 % glutaraldehyde and 4 % paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). The samples were fixed in OsO4 1 % in the same buffer for 1 h. After dehydration in an ethanol series, the samples were embedded in Spurr's epoxy resin. Sections, approximately 800 nm thick, were cut with a diamond knife, stained with uranyl acetate and lead citrate, and were examined using a Philips EM300 transmission electron microscope at 80 kV.
Results Plant-type autophagosomes–autolysosomes in Lobivia tapetum cells Two stages of plant-type macroautophagy of the type described by the school of Buvat (see Introduction) are shown in Fig. 1a. The arrowhead points to a portion of the cytoplasm that has become surrounded by an electron-translucent structure. The portion of the cytoplasm inside this structure is bound by a membrane. The outer part of the translucent structure is also bound by a membrane. A similar structure, although at an earlier stage of development, is indicated by a dashed arrow. The white arrow points at tubules, which form a circle contiguous with the larger half-moon shaped structure indicated by the dashed arrow. The arrowhead in Fig. 1b also indicates a late developmental stage of a macroautophagous structure. However, the translucent area around a portion of the cytoplasm is here considerably thinner than the ones shown in Fig. 1a. The arrows
Autophagy in tapetum cells Fig. 1 Macroautophagy in Lobivia rauschii tapetum cells. The tapetum was at a late stage during the first phase of tapetum development (see text), whereby the microsporemother cell was surrounded by a thin layer of callose. a Two stages of plant-type macroautophagy. A portion of the cytoplasm has almost become surrounded by an electrontranslucent structure, bound by two membranes (white arrowhead). Tubules (provacuoles; white arrow) are present around another portion of the cytoplasm. Some of these tubules have apparently already merged (dashed white arrow). b A portion of the cytoplasm has almost become surrounded by a thin electron-translucent structure, bound by two membranes (white arrowhead). Loose tubules (white arrows) are also found. c The upper structure indicated by a white arrowhead consists of several tubules, most of which have merged to form an electrontranslucent structure around a portion of the cytoplasm. In the lower structure indicated by a white arrowhead, the tubules have apparently completely merged. The white arrows indicate places in the cytoplasm where tubules seem to start surrounding portions of the cytoplasm. The thick white arrow indicates a possible multilamellar body. d An autophagous structure (white arrow) in which the merging of the tubules is complete. This structure includes a multilamellar body (black arrow). e Another example of a thin electrontranslucent structure around a portion of the cytoplasm, which is almost closed (white arrowhead). The arrows indicate places in the cytoplasm where tubules seem to start surrounding portions of the cytoplasm
in Fig. 1b indicate tubules. The upper arrowhead in Fig. 1c shows an autophagous structure which is almost complete, but in which the tubules are in the process of merging laterally. Some tubules — especially at the lower end of the structure — have already merged, while others (towards the top of the structure) have not. The other arrowhead in Fig. 1c indicates a
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more advanced stage of tubule merging, whereby an almost contiguous electron-translucent area has been formed. The small arrows in Fig. 1c indicate several other tubules. The cytoplasm inside the observed macroautophagic structures often contain only ribosomes (Fig. 1a,b,e). In the lower autophagous structure in Fig. 1c (large arrow) an
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electron-dense body is present in addition to a portion of cytoplasm mainly containing ribosomes. This might be a multilammellar body (i.e., a body consisting of concentric membranes). Figure 1d shows a clear example of a multilamellar body (black arrow) in the autophagous structure (arrowhead) in addition to a portion of cytoplasm mainly containing ribosomes. The white arrowhead Fig. 1e indicates another example of a developing autophagosome in which the translucent area around a portion of the cytoplasm is thin, similar to the structure observed in Fig. 1b. Arrows in Fig. 1e indicate tubules. No classical macroautophagy in Tillandsia tapetum cells, possible role of plastids in cytoplasm degradation The autophagous structures shown in Fig. 1a–e were absent in the tapetum cells of Tillandsia. Tillandsia, by contrast, contained what we hypothesize to be autophagous plastids, which were absent in Lobivia tapetum cells. In Fig. 2a–d, the plastids are indicated by big black arrows. The plastids contain no grana, only a few membranes, and some plastoglobuli. Some plastids show what might be starch granules (Fig. 2c, arrowhead; Fig. 2e, arrowheads). Double-membrane-bound structures were found inside these plastids (white arrows in Fig. 2a,b). These structures, called intraplastidial spaces, seemed to hold a portion of the cytoplasm as they contained ribosomes and membranes. In the intraplastidial spaces, some areas are less electrondense than others. Some of these less electron-dense areas are bound by a membrane (dashed arrows in Fig. 2b and d, arrows in Fig. 2e and f). Possible degeneration of the cytoplasm inside the intraplastidial spaces might be suggested from the data of Fig. 2d (white arrow), as some parts are less electron-dense than others. A similar structure is observed in Fig. 2e (white arrow) and in Fig. 2f (thick black and thick white arrow). Two membranes located closely together in the intraplastidial space of Fig. 2f are indicated with an arrowhead. What might be hypothesized to be a stage of disintegration of the sequestered cytoplasm is shown in Fig. 2f (dashed arrow). Figure 2g exhibits two plastids. The one at the left of the micrograph contains a large crystalloid (thick white arrow). This plastid also contains several spaces with what seems to be debris, which might therefore be intraplastidial spaces, but it cannot be excluded that they are starch granules (black arrows). Other such areas have no debris and might therefore be intraplastidial spaces after degradation of its cytoplasm contents. They might also possibly be starch grains (thin white arrows). The plastid at the right side of the micrograph shows what is apparently an early stage of autophagy (arrowhead).
Discussion We distinguished the same phases of tapetum and pollen development in T. albida and L . rauschii as described previously (Papini and Brighigna 1993; Papini et al. 1999). In the first phase of development the microspore mother cell is surrounded by a thin layer of callose. The tapetum cells assume a prismatic shape with the main axis in radial position with respect to the center of the anther. Plasmodesmata connect the tapetum cells. All data in the present paper refer to this first phase. Early during tapetum development, proplastids are present. These generally develop into elaioplasts, i.e., plastids that store lipidic material (Pacini 1997). In T. albida, during the early stage of tapetum development (studied here), we did not observe plastids with lipid storage compartments. The plastids thus were not elaioplasts (unpublished data). We rather found plastids that possibly contained some starch granules, but mostly intraplastidial spaces. In the species studied here, the pollen are covered by pollenkitt. This derives from collapsed tapetal cells. These cells die, whereby their contents fill the loculus and coat the pollen. Pollenkitt is usually deposited on the pollen just before the anther opens (Pacini 1997). The type of tapetum in both species studied can be classified as parietal, i.e., secretory. This confirms the data of Pacini et al. (1985), who called the type of tapetum found in Bromeliaceae parietal. Autophagy-like structures like those shown in Fig. 1 have been described previously only in root meristem cells. The cells were from Hordeum sativum (Poaceae; Buvat 1977; Buvat and Robert 1979), Cucurbita pepo (Cucurbitaceae; e.g., Coulomb et al. 1982), Euphorbia characias (Euphorbiaceae; Marty 1978) and Scorzonera hispanica (Asteraceae; Coulomb 1973). These papers are from the school of Roger Buvat in Marseille, France. In this work, we report, independently, similar structures in a very different tissue: the tapetum of L. rauschii (Cactaceae). We confirm the work by the school of Buvat in root meristems: their interpretation, as far as we can see, was correct. It should be noted that TEM micrographs in a few other papers also showed autophagic structures similar to the ones provided by the school of Buvat and the ones found here (Toyooka et al. 2006; Gaffal et al. 2007; Avci et al. 2008; Hiratsuka and Terasaka 2010). However, these papers did not point out what these structures might be and thus did not give a proper interpretation. Previously, multilamellar bodies have apparently not been reported inside the portion of cytoplasm that becomes sequestered by the plant type autophagosome–autolysosome. The present data show such a body unequivocally in Fig. 1d, and less clearly in Fig. 1c. For a three-dimensional interpretation of our data on this type of autophagy, we refer to the study of Marty (1978),
Autophagy in tapetum cells
Fig. 2 Macroautophagy in Tillandsia albida tapetum cells. The tapetum was at a late stage during the first phase of tapetum development (see text), whereby the microspore-mother cell was surrounded by a thin layer of callose. a–g Plastids in Tillandsia albida tapetum showing apparent plastidial autophagy. Plastids in a–d are indicated with black arrows. Intraplastidial spaces in a are shown using white arrows. In b and d–f, electron-translucent areas in the intraplastidial space are indicated with dashed white or black arrows. The arrowheads in c and e point at possible starch grains or intraplastidial spaces at are electron-translucent, and therefore at a late stage of cytoplasm degeneration. The arrows in e and f indicate areas with less electron-dense material in the intraplastidial
spaces, compared with more grainy material in the other parts of these spaces. In f, the dashed arrow indicates an area in the intraplastidial space in which the electron density is low but not yet electron-translucent. The arrowhead in f points at two membranes close together. The plastid at the left of micrograph g contains a large crystalloid in its centre (thick arrow). It also has some spaces showing apparent debris, which might therefore be remnants of degradation of a portion of the cytoplasm (black arrows), but might also be starch grains. In other such spaces, no debris is found (white arrows). These might be remnants of degradation of a portion of the cytoplasm, or are starch grains. The plastid at the right shows a structure that seems to be an early stage of autophagy (arrowhead)
although we do not know if in our system the autophagous tubules derive from the interface of the ER and the Golgi apparatus or from another location. We observed a large crystalloid in an organelle of Tillandsia. Such crystalloids are only found in plastids (Nguyen et al.
2001), peroxisomes (Evert 2006), and sometimes in protein storage vacuoles (Jiang et al. 2000). As the structure of vacuoles and peroxisomes is quite different from the one shown in Fig. 2g, it is concluded that the organelle at the left side of the figure is a plastid. This plastid also contained several
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intraplastidial spaces, as some debris was found inside. Other such areas had no debris but seemed also intraplastidial spaces, although it cannot be ruled out that they were starch granules. The plastids depicted contained double-membrane bound intraplastidial spaces. These spaces were interpreted to be due to autophagy, in line with previous data (Nagl 1977; Gärtner and Nagl 1980; van Doorn et al. 2011). Both in Phaseolus (Nagl 1977; Gärtner and Nagl 1980) and Dendrobium (van Doorn et al. 2011) acid phosphatase was demonstrated inside such intraplastidial spaces. Similar plastids were observed also during the functional megaspore growth in the ovule of other species of Tillandsia (Papini et al. 2011). Based on the present data, we hypothesize that plastids in T. albida tapetum cells carry out autophagy. This hypothesis is based on extrapolation from similar phenomena in Phaseolus and Dendrobium (see Introduction). We observed a double membrane around a space inside several T. albida plastids. Both in Phaseolus (Gärtner and Nagl 1980) and Dendrobium (van Doorn et al. 2011), acid phosphatase was demonstrated inside the space that was bound by a double membrane. The partial disappearance of electron-opaque material in these spaces in T. albida, it is here hypothesized, might also be due to hydrolase activity. Nonetheless, several features of the plastids in T. albida were different from the autophagous plastids previously reported in Phaseolus and Dendrobium . The doublemembrane-bound spaces in T. albida had a more irregular shape, as shown in Fig. 2f. Second, electron-translucent areas, bound by a membrane, were observed inside the spaces of T. albida (Fig. 2, dashed arrows). For a three-dimensional interpretation of our data on this possible type of autophagy, we refer to a previous paper (van Doorn et al. 2011). The present data do not show a causal relationship between autophagy and PCD. It is possible that the macroautophagy we observed in L . rauschii tapetum cells has (a) little to do with the cause of death, (b) is a cause of death, or even (c) delays death. It is concluded that in L . rauschii classical plant macroautophagy was involved in degradation of the cytoplasm, while in T. albida such classical macroautophagy was not observed. Instead, the data in T. albida suggested the hypothesis that plastids are able to carry out some degradation of the cytoplasm. Acknowledgements The work was supported by grants from the Fondi di Ateneo of the University of Florence and from the Fondazione Cassa di Risparmio di Pistoia e Pescia. Conflict of interest The authors declare that they have no conflict of interest.
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