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Morphological and ultrastructural examination of senescence in Morchella elata
Micron 78 (2015) 79–84
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Morphological and ultrastructural examination of senescence in Morchella elata Peixin He a , Yingli Cai b , Sumeng Liu a , Li Han a , Lina Huang a , Wei Liu a,c,∗ a b c
School of Food and Biological Engineering, Zhengzhou University of Light Industry, Zhengzhou 450002, China Institute of Applied Mycology, Huazhong Agricultural University, Wuhan 430070, China Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430070, China
a r t i c l e
i n f o
Article history: Received 16 July 2015 Accepted 23 July 2015 Available online 26 July 2015 Keywords: Aging Apoptosis Autophagy Morel Necrosis
a b s t r a c t In recent years, the artificial cultivation of Morchella mushrooms that belong to Elata Clade, including Morchella elata, has been developed rapidly in China. However, the prominent problem of spawn aging has been frustrating the morel farming. In this paper, aging in M. elata was achieved from 12 to 17 subcultures and lifespan of 1536–2256 h by successive subculturing. The lifespan can be roughly divided into juvenile phase and senescent phase with respect to the mycelia linear growth rate. After a certain period of rapid growth with almost constant rate at the juvenile phase, the isolate entered the senescent phase characterized by slow down of mycelia growth, producing pigments ahead of time and final death of the apical hyphae. The period of the senescent phase was definitely 240–288 h; while that of the juvenile phase was diverse relying on different isolates. Moreover, microscopic study showed that angles between the leading and primary hyphae increased constantly with aging. In senesced hyphal cells of M. elata, the typical characteristics of autophagy (enlargement of vacuoles and existence of organelles sequestrated lysosomes) and apoptosis (condensation of the cytoplasm and nuclear and plasmolysis) were observed. In addition, in the final stage of senescence, the apical hyphae collapsed with the plasma membrane and all the cellular organelles disrupted, indicated a necrotic mode of cell death. Taken together, these data revealed the involvement of autophagy, apoptosis and necrosis in senescence of M. elata. The characterization and molecular mechanism of autophagy, apoptosis and necrosis need further study and the systematic study of morel aging will be beneficial for the healthy development of morel farming. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction Aging is a process of progressive decline in the ability to withstand stress, damage and disease (Sharon et al., 2009). Senescence (aging process), especially replicative senescence achieved by successive subculturing, has been extensively studied in human, higher animals and plants (Cristofalo et al., 2004; Humbeck, 2014; van Deursen, 2014). In filamentous fungi, aging of Podospora anserina was firstly described in the early 1950s. Cultures of P. anserina do not grow indefinitely but senesce after a strain-specific period of growth (Rizet, 1953). Similar phenomena were also found in Neurospora spp. (Maheshwari and Navaraj, 2008). Moreover, in entomopathogenic fungus Beauveria bassiana, positively correlated with the decline of spore germination rate and spore-bound sub-
∗ Corresponding author at: School of Food and Biological Engineering, Zhengzhou University of Light Industry, Zhengzhou 450002, China. Fax: +86 37186609631. E-mail address: [email protected] (P. He). http://dx.doi.org/10.1016/j.micron.2015.07.010 0968-4328/© 2015 Elsevier Ltd. All rights reserved.
tilisin (Pr1) activity, the virulence of subcultures decreased (Safavi, 2011). True morels (Morchella, phylum Ascomycota) are highly prized for their medicinal and nutritional values and intensively collected around the world by mycophiles (Kuo, 2005). In recent years, the artificial cultivation of Morchella mushrooms, especially those belong to Elata Clade, including M. elata, M. importuna, M. septimelata and M. sextelata, has been developed rapidly in China (Du et al., 2014). However, along with the rapid expansion of artificial cultivation, the prominent problem of spawn aging has been frustrating the morel farming. Application of the senesced spawns characterized by slim mycelia, reduced growth rate and untidy growth in cultivation may result in remarkable decrease of production. Unfortunately, aging in Morchella mushrooms has not been systematically studied. Programmed cell death (PCD) represents a highly conserved cellular suicide programme in multicellular organisms (Hamann et al., 2008). The main types of PCD of apoptosis, autophagy and necrosis (necroptosis) may be involved in fungal senescence (Bursch, 2001; Hamann et al., 2008; Pinan-Lucarré et al., 2007; Pollack
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et al., 2009; Sharon et al., 2009; Shlezinger et al., 2012; Sridharan and Upton, 2014 Sridharan and Upton, 2014). Apoptosis has been characterized ultrastructurally by cell shrinkage and chromatin condensation, and plasma membrane blebbing (Sharon et al., 2009). The major characteristics of apoptosis except membrane blebbing were observed by electron microscopy in vegetative incompatibility of N. crassa, which implied the possible involvement of apoptosis in heterokaryon incompatibility of the filamentous fungus (Jacobson et al., 1998). Moreover, the typical characteristics of autophagy of vacuolar enlargement and cytoplasmic vacuolization were induced to appear by starvation in Aspergillus fumigates (Richie et al., 2007) and A. oryzae (Pollack et al., 2008) and were also observed in vegetative incompatibility of P. anserina (PinanLucarré et al., 2003). In addition, the ultrastructural characteristics of swollen mitochondria lacking cristae and disruption of nuclear and mitochondrial membranes indicated a necrotic mode of cell death in senescence induced by kalilo plasmids of Neurospora cells (Bok et al., 2003). In this study, we achieved aging in M. elata through successive subculturing. The morphological and ultrastructural characteristics of cultures at different phase of senescence were studied. The results revealed the involvement of autophagy, apoptosis and necrosis in senescence of M. elata. The molecular mechanism of aging in Morchella spp. need further study. The systematic study of morel aging will promote the breeding and spawn production of Morchella mushrooms and thus beneficial for the development of morel farming. 2. Materials and methods 2.1. Fungal isolation The ascocarps of M. elata occurring on forest land were collected from Chengdu, Sichuan province of China. Strains were isolated from the stipe and pileus of a dry ascoma by tissue isolation (Pacumbaba and Pacumbaba, 1999) onto Complete Yeast extracts Medium (CYM) plates (glucose 20 g l−1 , yeast extracts 2 g l−1 , peptone 2 g l−1 , K2 HPO4 1 g l−1 , MgSO4 0.5 g l−1 , KH2 PO4 0.46 g l−1 , and agar 20 g l−1 ). Briefly, the stipe and pileus were cut with a flame-sterilized scalpel into segments of about 1 cm2 . Each segment was dipped into a 10 ml centrifuge tube with 5 ml sterile water and shaken for 60 s by hand. The shaking for wash was repeated four times. Then, the tissue segment was blotted with aseptic filter papers, cut into smaller pieces of about 1 mm2 with a sterilized scalpel. The small pieces of tissue were transferred with a flame-sterilized inoculating hoop onto CYM plates and incubated at 20 ◦ C for 3–5 days away from light. Newly-grown hyphae were transferred to CYM slants as pure cultures to be used in successive subculturing. The isolates designated as Ms (Ms04-1 and 04-2) were isolated from stipe and a serial of Mp isolates (Mp04-1, 04-2 and 04-3) were from pileus of the same sporocarp. The taxonomic status of all tested isolates was confirmed by internal transcribed spacers (ITS) sequence analysis (He and Liu, 2010) before further study. 2.2. Successive subculturing To obtain cultures of different ages, the mycelia were inoculated on one side of CYM plates directly and on autoclaved cellophane membranes placed on the surface of CYM plates. It should be noted that the volume of medium in a Petri dish must be fixed, i.e., 18 ml medium in a Petri dish of 9 cm in diameter. The mycelia grown on cellophane membranes were used for light microscopy and those from CYM plates without cellophane membranes were also used for colony observation and transmission electron microscopy (see
below). All cultures were incubated at 24 ◦ C in dark place. The growth front was marked every 12 h. Before the mycelia reaching the other side of a Petri dish, the hyphal tips excised from colony edges were transferred to fresh plates with growth tips toward the margin of medium and continued to cultivate until complete stop of mycelia growth. Each subculture was repeated three times with inocula of the same age and from the same plate. 2.3. Linear growth rate and total growth length The period of mycelia linear growth before cessation was recorded as its lifespan in hours. During this period, the growth rate was measured in millimeters per hour and presented as the mean value +SD. The total growth length was recorded as the sum of mean value of each batch of subculture before growth cessation. 2.4. Light microscopy The colony on the plate was observed and recorded directly. For microscopic observation, cellophane membranes with hyphal tips adhering were cut off and placed on glass slides. At least three slides for each sample were examined under bright field illumination on an optical microscope with DIC module (Zeiss Axio Observer Z1, Oberkochen, Germany). 2.5. Transmission electron microscopy The agar in the region of the hyphal tips was chopped into 1 mm blocks. After cleaning with distilled water, the blocks were fixed in 2.5% glutaraldehyde buffered with 0.1 M phosphate buffered saline (PBS, pH 7.0) for more than 4 h at 4 ◦ C, rinsed for 30 min with three changes of distilled water, and then post-fixed in 1% osmium tetroxide for 2 h at 4 ◦ C, followed by rinsing with PBS three times, dehydration in a graded ethanol series, and permutation with absolute acetone 2 times with 15 min each time. Fixed samples were embedded overnight at 37 ◦ C in a mixture of acetone and embedding liquid (SPI-PON 812 Kit, USA) (2:1, v/v), and in pure embedding liquid for 2 h. Subsequently, they were transferred into embedding mold and polymerized for 48 h at 65 ◦ C. Ultra-thin sections were prepared by Leica UC7 Ultramicrotome with a diamond knife. Prior to viewing with a transmission electron microscope (Hitachi HT-7700, Japan), ultra-thin sections were stained with 2% uranyl acetate for 25 min, followed by rinsing with 75% ethanol and distilled water, and stained again with 2% lead citrate for 15 min. 3. Results and discussion 3.1. Systemic senescence within a distinct time frame Both isolates from stipe and pileus of M. elata showed systemic senescence within a distinct time frame. The lifespan was different from 1536 h (Mp04-3, Fig. 1) to 2256 h (Mp04-2 and Ms04-2, Fig. 2). The subculture times were 12, 13, and 17 for Mp04-3, Mp04-1, and Ms04-1 (Fig. 1) and Mp04-2 and Ms04-2 (Fig. 2), respectively. The total growth length was from 71.6 cm (Mp04-1) to 110.7 cm (Mp042) (Table 1). In addition, there was no obvious correlation between sites (stipe or pileus) for isolation of fungal strains and physiological state of the isolates reflected by linear growth rate and total growth length (Figs. 1 and 2, Table 1). The lifespan can be roughly divided into two phases with respect to mycelia linear growth rate: juvenile phase and senescent phase. At the juvenile phase, the isolates grew fast at a almost identical linear growth rate. However, once entering the senescent phase, they grew at constant reduced rate until cessation within 12 days. The period of the senescent phase was definitely 240–288 h; while that of the juvenile phase was diverse relying on different isolates (Figs. 1 and 2). Therefore, the lifespan
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Fig. 1. Growth curve of isolate Mp04-1, Mp04-3, and Ms04-1 of Morchella elata successively subcultured on CYM plates.
Fig. 2. Growth curve of isolate Mp04-2 and Ms04-2 of Morchella elata successively subcultured on CYM plates.
Table 1 Total growth length of Morchella elata through successive subculturing. Isolate
Mp04-1
Mp04-2
Mp04-3
Ms04-1
Ms04-2
Total length (cm)
71.6
110.7
76.3
73.4
110.1
and total growth length mainly depended on the period of juvenile phase. In this study, several isolates of M. elata exihibited systemic senescence through successive subculturing, manifested in 12–17 subcultures and lifespan of 1536–2256 h. Successive subculturing has been used in animal and human cells for study of replicative senescence in vitro (Cristofalo et al., 2004). In filamentous
ascomycetes, senescence usually manifests in 5 30 subcultures made at weekly intervals. A strain that has not senesced in some 50 subcultures is commonly regarded as immortal (Maheshwari and Navaraj, 2008). In Neurospora, senescing strains have been found in populations of N. crassa, N. intermedia and N. tetrasperma (Griffiths and Bertrand, 1984; Maheshwari et al., 1994; Maas et al., 2005). The mean lifespan of wild-type strains of P. anserina is 25 days when
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grown on rich-medium at 27 ◦ C (Osiewacz, 2002; Rizet, 1953). While the replicative lifespan of Ascobolus immerses is 35–40 days (Barra et al., 2000). 3.2. Morphological characteristics During the juvenile phase, the isolates grew fast and evenly at the rate of 0.35–0.5 mm/h and the mycelium covered the whole plate only in 6 days. The culture produced white to light grayish colony with abundant fluffy aerial mycelium. Extended culture period to 7 days, the isolates began to secrete light brown pigments, giving the colony a characteristic brownish appearance (Fig. 3A). However, during the senescent phase, the culture produced pigments earlier (3–5 days after inoculation), first from the older mycelium near inocula and progressed to the periphery of the plate with aging (Fig. 3B). In the late phase of senescence, the formation of aerial hyphae decreased and the colony margin was plumose rather than even. The aerial hyphae were more slender than those from younger ones. Finally, the growth of a senescent culture stopped completely and the hyphae died at the front growth (Fig. 3C). During the juvenile phase, the leading hyphae grew vigorously. These hyphae were 12–17 m wide and were divided by septa into internodes. Primary branches grew out of the leading hyphae at angles less than 30◦ (with wider angles in rare cases) to form the primary hyphae. Short hyphae branched off at right angles from these primary branches, and in rare cases from secondary branches, to form the thinner tertiary branches (5–10 m) (Fig. 3D). In the early senescent phase, the leading hyphae grew sparsely. The angles between leading and primary hyphae were bigger (30–45◦ ) than those in the juvenile phase. The tertiary hyphae that branched off at right angles from primary and secondary branches were more abundant (Fig. 3E). In the late senescent phase, the primary branches grew out of the leading hyphae at bigger angles of 45–60◦ (at right angles in rare cases). The abundant tertiary hyphae were thinner (4–8 m). Most significantly, some apical hyphae collapsed in the final stage of senescence (Fig. 3F). After certain periods of rapid growth with almost constant rate, the isolates of M. elata entered the senescent stage characterized by slow down of mycelia growth, final death at the front growth and some phenotype changes. M. elata shared some morphological features with P. anserina during senescence: irregular growth prior to a complete growth arrest and final burst of apical hyphal cells from senescent cultures (Osiewacz, 2002; Rizet, 1953). However, increased pigmentation in senescent culture of P. anserina (Rizet, 1953) was not observed in M. elata. Alternatively, the time of pigmentation in senescent cultures of M. elata was advanced. Furthermore, the fact that the angles between the leading and primary hyphae increased constantly with aging, has not been found in other filamentous ascomycetes.
shrink away from the cell wall resulted in plasmolyzed cells. The nucleus and mitochondrium remained visible in some cells, but became irregularly shaped. Other cellular organelles were difficult to distinguish. The membrane did not always pull away uniformly and was often ruptured, causing leakage of the degraded cellular contents into the gap between the plasma membrane and cell wall. The membranes around some vacuoles were still intact, but more were obscure. In addition, many vacuoles appeared to be coalescing resulted in bigger ones. The lysosome containing recognizable remnants of autophagocytosed mitochondria and other cytoplasmic elements could be observed (Fig. 4C). Along with aging proceeded, the cells were extensively vacuolated and the organelles began to be degraded, causing most of the organelles in these cells unidentifiable. However, the cell wall was still intact and the lipid storage vesicles were easily distinguishable (Fig. 4D). In the final stage of senescence, the plasma membrane and all the cellular organelles were broken up and disappeared. The hyphal cell became an empty shell within some membrane fragments, although still remained its original shape for sustaining of the intact rigid cell wall. Rupture of the plasma membrane may lead to the release of cytoplasmic contents out of the cell, because the cell wall is not the main barrier for movement of many substances (Fig. 4E and F). The ultrastructural characteristics of M. elata in the final stage of senescence were similar to those of M. esculenta (Hervey et al., 1978). In the previous study, Hervey et al. found that the growth of subculture of GB groups of single ascospore isolates was determinate. Microscopic examination revealed that the cell wall of the apical cells of determinant cultures was still intact but devoid of both nuclei and cytoplasm. In senesced hyphal cells of M. elata, the characterized enlargement of vacuoles and the existence of organelle-sequestrated lysosomes imply the involvement of autophagy in senescence. Autophagy is a broad term for catabolic processes involving the lysosomal/vacuolar pathway (Pollack et al., 2009) and was identified as a longevity assurance mechanism in the aging model P. anserina (Knuppertz et al., 2014; Philipp et al., 2013). Moreover, some ultrastructural details of aging in M. elata corresponded with apoptotic events, for example, condensation of the nucleus and cytoplasm and cytoplasmolysis. However, the fragmentation of the cell contents into apoptotic bodies by membrane blebbing was not seen at a high frequency in this study, which is similar to the ultrastructural changes in dying cells of vegetative incompatibility in N. crassa (Jacobson et al., 1998). In addition, in the final stage of senescence, some apical hyphal cells from senescent cultures of M. elata burst with plasma membrane and all cellular organelles disrupted, which indicated a necrotic mode of cell death. This mode of cell death was also found in senescence of N. crassa and N. intermedia induced by kalilo plasmids (Bok et al., 2003). 3.4. The mechanism of aging in M. elata needs further study
3.3. Ultrastructural features The cytoplasm of juvenile cultures stained lightly and evenly. The nucleus and mitochondrium were easily distinguishable and the membranes were intact. Other organelles and structures that were visible in various cells included plasma membrane, endoplasmic reticulum and large amounts of small vacuoles with intact membranes (Fig. 4A and B). The septum that separate neighbor cells was obvious. Surprisingly, most septal pores were sealed by the woronin bodies. Two types of inclusion bodies lack of bounded membrane were distinguished and shared in juvenile hyphal cells. The near-spherical structures were lipid storage vesicles. However, the chemical composition and function of some rod-like crystal bodies that often appeared near the woronin body need further studies (Fig. 4B). During the senescent phase, the cytoplasm and nucleus were condensed and the plasma membrane appeared to
In this study, ultrastructural examination revealed the involvement of autophagy, apoptosis and necrosis in senescence of M. elata achieved by successive subculturing. In P. anserina, it is clear that oxidative stress, in particular as the result of reactive oxygen species (ROS) generation at the mitochondrial respiratory chain, plays a crucial role in healthy aging (Lorin et al., 2006). ROS is locally accumulated in the apical region of filamentous ascomycetes accompanied by the generation of adenosine triphosphate (ATP) (Semighini and Harris, 2008). Increased ROS levels during aging trigger mitochondrion-dependent PCD in senescent cultures and subsequently result in ATP depletion (Osiewacz, 2002). Intracellular ATP levels have a determining role in the interplay between apoptosis and necrosis. Depletion of intracellular ATP levels switches the energy requiring apoptotic cell death to necrosis (Skulachev, 2006). Furthermore, oxidative stress is a common stim-
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Fig. 3. Colony morphology (A–C) and light microscopy images (D–F) of isolate Ms04-1 (Morchella elata) cultures growing in 9 cm diameter Petri dish. (A) and (D), (B) and (E), (C) and (F) were cultures cultivated for 144 h, 1392 h and 1536 h, respectively, and were representative of those at the juvenile phase, in the early and final stage of senescence, respectively. The collapsed apical hyphae in the final stage of senescence (F) were marked with arrows.
Fig. 4. Transmission electron microscopy images of ultrathin sections of isolate Ms04-1 (Morchella elata) cultures. (A) and (B), (C) and (D), (E) and (F) were cultures cultivated for 144 h, 1392 h, and 1536 h, respectively, representing those at the juvenile phase, in the early and final stage of senescence, respectively. At the juvenile phase, most septal pores were sealed by the woronin bodies (WB) (Fig. 4B, arrow). In the early stage of senescence, the plasma membrane appeared to shrink away from the cell wall (Fig. 4C and D, arrows). In some apical hyphal cells, the lysosome containing recognizable remnants of autophagocytosed mitochondria and other cytoplasmic elements could be observed (Fig. 4C, lightning). In addition, the membrane apart from the cell wall often ruptured, causing leakage of the degraded cellular contents into the gap between the plasma membrane and cell wall (Fig. 4C, arrow head). C–crystal body, E–endoplasmic reticulum, L–lipid storage vesicle, M–mitochondrium, MF–membrane fragments, N–nuclear, S–septum, V–vacuole, W–cell wall. Scale bars = 0.5 m.
ulus for apoptosis, necrosis and autophagy (Nikoletopoulou et al., 2013). Under laboratory conditions, the vegetative growth and pseudosclerotial development of Morchella spp. are rapidly accomplished in several days (Figs. 1 and 2). Rapid growth and development are accompanied with increased ROS levels or oxidative stress (Georgiou et al., 2006; He et al., 2014; Király and Czövek, 2007), which may be the direct or indirect stimulus for apoptosis, necrosis and autophagy. Furthermore, the induction of autophagy in apical hyphal cells could contribute together with plugging of septal pores (Fig. 4) to the protection of neighboring cells from cell death (Pinan-Lucarré et al., 2005). Finally, it should be noted
that conventional microscopy is not always possible to distinguish autophagy, apoptosis and necrosis. The characterizations and molecular mechanisms of autophagy, apoptosis and necrosis in senescence of M. elata need further extensive study.
Acknowledgements This work was financially supported by the Science and Technology Innovatory Fund for Young Distinguished Scholars in Henan Province, China (No. 134100510017). We sincerely thank Mr. Ya Zhang for the provision of some information on morel cultivation
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Morphological and ultrastructural examination of senescence in Morchella elata Peixin He a , Yingli Cai b , Sumeng Liu a , Li Han a , Lina Huang a , Wei Liu a,c,∗ a b c
School of Food and Biological Engineering, Zhengzhou University of Light Industry, Zhengzhou 450002, China Institute of Applied Mycology, Huazhong Agricultural University, Wuhan 430070, China Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430070, China
a r t i c l e
i n f o
Article history: Received 16 July 2015 Accepted 23 July 2015 Available online 26 July 2015 Keywords: Aging Apoptosis Autophagy Morel Necrosis
a b s t r a c t In recent years, the artificial cultivation of Morchella mushrooms that belong to Elata Clade, including Morchella elata, has been developed rapidly in China. However, the prominent problem of spawn aging has been frustrating the morel farming. In this paper, aging in M. elata was achieved from 12 to 17 subcultures and lifespan of 1536–2256 h by successive subculturing. The lifespan can be roughly divided into juvenile phase and senescent phase with respect to the mycelia linear growth rate. After a certain period of rapid growth with almost constant rate at the juvenile phase, the isolate entered the senescent phase characterized by slow down of mycelia growth, producing pigments ahead of time and final death of the apical hyphae. The period of the senescent phase was definitely 240–288 h; while that of the juvenile phase was diverse relying on different isolates. Moreover, microscopic study showed that angles between the leading and primary hyphae increased constantly with aging. In senesced hyphal cells of M. elata, the typical characteristics of autophagy (enlargement of vacuoles and existence of organelles sequestrated lysosomes) and apoptosis (condensation of the cytoplasm and nuclear and plasmolysis) were observed. In addition, in the final stage of senescence, the apical hyphae collapsed with the plasma membrane and all the cellular organelles disrupted, indicated a necrotic mode of cell death. Taken together, these data revealed the involvement of autophagy, apoptosis and necrosis in senescence of M. elata. The characterization and molecular mechanism of autophagy, apoptosis and necrosis need further study and the systematic study of morel aging will be beneficial for the healthy development of morel farming. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction Aging is a process of progressive decline in the ability to withstand stress, damage and disease (Sharon et al., 2009). Senescence (aging process), especially replicative senescence achieved by successive subculturing, has been extensively studied in human, higher animals and plants (Cristofalo et al., 2004; Humbeck, 2014; van Deursen, 2014). In filamentous fungi, aging of Podospora anserina was firstly described in the early 1950s. Cultures of P. anserina do not grow indefinitely but senesce after a strain-specific period of growth (Rizet, 1953). Similar phenomena were also found in Neurospora spp. (Maheshwari and Navaraj, 2008). Moreover, in entomopathogenic fungus Beauveria bassiana, positively correlated with the decline of spore germination rate and spore-bound sub-
∗ Corresponding author at: School of Food and Biological Engineering, Zhengzhou University of Light Industry, Zhengzhou 450002, China. Fax: +86 37186609631. E-mail address: [email protected] (P. He). http://dx.doi.org/10.1016/j.micron.2015.07.010 0968-4328/© 2015 Elsevier Ltd. All rights reserved.
tilisin (Pr1) activity, the virulence of subcultures decreased (Safavi, 2011). True morels (Morchella, phylum Ascomycota) are highly prized for their medicinal and nutritional values and intensively collected around the world by mycophiles (Kuo, 2005). In recent years, the artificial cultivation of Morchella mushrooms, especially those belong to Elata Clade, including M. elata, M. importuna, M. septimelata and M. sextelata, has been developed rapidly in China (Du et al., 2014). However, along with the rapid expansion of artificial cultivation, the prominent problem of spawn aging has been frustrating the morel farming. Application of the senesced spawns characterized by slim mycelia, reduced growth rate and untidy growth in cultivation may result in remarkable decrease of production. Unfortunately, aging in Morchella mushrooms has not been systematically studied. Programmed cell death (PCD) represents a highly conserved cellular suicide programme in multicellular organisms (Hamann et al., 2008). The main types of PCD of apoptosis, autophagy and necrosis (necroptosis) may be involved in fungal senescence (Bursch, 2001; Hamann et al., 2008; Pinan-Lucarré et al., 2007; Pollack
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et al., 2009; Sharon et al., 2009; Shlezinger et al., 2012; Sridharan and Upton, 2014 Sridharan and Upton, 2014). Apoptosis has been characterized ultrastructurally by cell shrinkage and chromatin condensation, and plasma membrane blebbing (Sharon et al., 2009). The major characteristics of apoptosis except membrane blebbing were observed by electron microscopy in vegetative incompatibility of N. crassa, which implied the possible involvement of apoptosis in heterokaryon incompatibility of the filamentous fungus (Jacobson et al., 1998). Moreover, the typical characteristics of autophagy of vacuolar enlargement and cytoplasmic vacuolization were induced to appear by starvation in Aspergillus fumigates (Richie et al., 2007) and A. oryzae (Pollack et al., 2008) and were also observed in vegetative incompatibility of P. anserina (PinanLucarré et al., 2003). In addition, the ultrastructural characteristics of swollen mitochondria lacking cristae and disruption of nuclear and mitochondrial membranes indicated a necrotic mode of cell death in senescence induced by kalilo plasmids of Neurospora cells (Bok et al., 2003). In this study, we achieved aging in M. elata through successive subculturing. The morphological and ultrastructural characteristics of cultures at different phase of senescence were studied. The results revealed the involvement of autophagy, apoptosis and necrosis in senescence of M. elata. The molecular mechanism of aging in Morchella spp. need further study. The systematic study of morel aging will promote the breeding and spawn production of Morchella mushrooms and thus beneficial for the development of morel farming. 2. Materials and methods 2.1. Fungal isolation The ascocarps of M. elata occurring on forest land were collected from Chengdu, Sichuan province of China. Strains were isolated from the stipe and pileus of a dry ascoma by tissue isolation (Pacumbaba and Pacumbaba, 1999) onto Complete Yeast extracts Medium (CYM) plates (glucose 20 g l−1 , yeast extracts 2 g l−1 , peptone 2 g l−1 , K2 HPO4 1 g l−1 , MgSO4 0.5 g l−1 , KH2 PO4 0.46 g l−1 , and agar 20 g l−1 ). Briefly, the stipe and pileus were cut with a flame-sterilized scalpel into segments of about 1 cm2 . Each segment was dipped into a 10 ml centrifuge tube with 5 ml sterile water and shaken for 60 s by hand. The shaking for wash was repeated four times. Then, the tissue segment was blotted with aseptic filter papers, cut into smaller pieces of about 1 mm2 with a sterilized scalpel. The small pieces of tissue were transferred with a flame-sterilized inoculating hoop onto CYM plates and incubated at 20 ◦ C for 3–5 days away from light. Newly-grown hyphae were transferred to CYM slants as pure cultures to be used in successive subculturing. The isolates designated as Ms (Ms04-1 and 04-2) were isolated from stipe and a serial of Mp isolates (Mp04-1, 04-2 and 04-3) were from pileus of the same sporocarp. The taxonomic status of all tested isolates was confirmed by internal transcribed spacers (ITS) sequence analysis (He and Liu, 2010) before further study. 2.2. Successive subculturing To obtain cultures of different ages, the mycelia were inoculated on one side of CYM plates directly and on autoclaved cellophane membranes placed on the surface of CYM plates. It should be noted that the volume of medium in a Petri dish must be fixed, i.e., 18 ml medium in a Petri dish of 9 cm in diameter. The mycelia grown on cellophane membranes were used for light microscopy and those from CYM plates without cellophane membranes were also used for colony observation and transmission electron microscopy (see
below). All cultures were incubated at 24 ◦ C in dark place. The growth front was marked every 12 h. Before the mycelia reaching the other side of a Petri dish, the hyphal tips excised from colony edges were transferred to fresh plates with growth tips toward the margin of medium and continued to cultivate until complete stop of mycelia growth. Each subculture was repeated three times with inocula of the same age and from the same plate. 2.3. Linear growth rate and total growth length The period of mycelia linear growth before cessation was recorded as its lifespan in hours. During this period, the growth rate was measured in millimeters per hour and presented as the mean value +SD. The total growth length was recorded as the sum of mean value of each batch of subculture before growth cessation. 2.4. Light microscopy The colony on the plate was observed and recorded directly. For microscopic observation, cellophane membranes with hyphal tips adhering were cut off and placed on glass slides. At least three slides for each sample were examined under bright field illumination on an optical microscope with DIC module (Zeiss Axio Observer Z1, Oberkochen, Germany). 2.5. Transmission electron microscopy The agar in the region of the hyphal tips was chopped into 1 mm blocks. After cleaning with distilled water, the blocks were fixed in 2.5% glutaraldehyde buffered with 0.1 M phosphate buffered saline (PBS, pH 7.0) for more than 4 h at 4 ◦ C, rinsed for 30 min with three changes of distilled water, and then post-fixed in 1% osmium tetroxide for 2 h at 4 ◦ C, followed by rinsing with PBS three times, dehydration in a graded ethanol series, and permutation with absolute acetone 2 times with 15 min each time. Fixed samples were embedded overnight at 37 ◦ C in a mixture of acetone and embedding liquid (SPI-PON 812 Kit, USA) (2:1, v/v), and in pure embedding liquid for 2 h. Subsequently, they were transferred into embedding mold and polymerized for 48 h at 65 ◦ C. Ultra-thin sections were prepared by Leica UC7 Ultramicrotome with a diamond knife. Prior to viewing with a transmission electron microscope (Hitachi HT-7700, Japan), ultra-thin sections were stained with 2% uranyl acetate for 25 min, followed by rinsing with 75% ethanol and distilled water, and stained again with 2% lead citrate for 15 min. 3. Results and discussion 3.1. Systemic senescence within a distinct time frame Both isolates from stipe and pileus of M. elata showed systemic senescence within a distinct time frame. The lifespan was different from 1536 h (Mp04-3, Fig. 1) to 2256 h (Mp04-2 and Ms04-2, Fig. 2). The subculture times were 12, 13, and 17 for Mp04-3, Mp04-1, and Ms04-1 (Fig. 1) and Mp04-2 and Ms04-2 (Fig. 2), respectively. The total growth length was from 71.6 cm (Mp04-1) to 110.7 cm (Mp042) (Table 1). In addition, there was no obvious correlation between sites (stipe or pileus) for isolation of fungal strains and physiological state of the isolates reflected by linear growth rate and total growth length (Figs. 1 and 2, Table 1). The lifespan can be roughly divided into two phases with respect to mycelia linear growth rate: juvenile phase and senescent phase. At the juvenile phase, the isolates grew fast at a almost identical linear growth rate. However, once entering the senescent phase, they grew at constant reduced rate until cessation within 12 days. The period of the senescent phase was definitely 240–288 h; while that of the juvenile phase was diverse relying on different isolates (Figs. 1 and 2). Therefore, the lifespan
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Fig. 1. Growth curve of isolate Mp04-1, Mp04-3, and Ms04-1 of Morchella elata successively subcultured on CYM plates.
Fig. 2. Growth curve of isolate Mp04-2 and Ms04-2 of Morchella elata successively subcultured on CYM plates.
Table 1 Total growth length of Morchella elata through successive subculturing. Isolate
Mp04-1
Mp04-2
Mp04-3
Ms04-1
Ms04-2
Total length (cm)
71.6
110.7
76.3
73.4
110.1
and total growth length mainly depended on the period of juvenile phase. In this study, several isolates of M. elata exihibited systemic senescence through successive subculturing, manifested in 12–17 subcultures and lifespan of 1536–2256 h. Successive subculturing has been used in animal and human cells for study of replicative senescence in vitro (Cristofalo et al., 2004). In filamentous
ascomycetes, senescence usually manifests in 5 30 subcultures made at weekly intervals. A strain that has not senesced in some 50 subcultures is commonly regarded as immortal (Maheshwari and Navaraj, 2008). In Neurospora, senescing strains have been found in populations of N. crassa, N. intermedia and N. tetrasperma (Griffiths and Bertrand, 1984; Maheshwari et al., 1994; Maas et al., 2005). The mean lifespan of wild-type strains of P. anserina is 25 days when
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grown on rich-medium at 27 ◦ C (Osiewacz, 2002; Rizet, 1953). While the replicative lifespan of Ascobolus immerses is 35–40 days (Barra et al., 2000). 3.2. Morphological characteristics During the juvenile phase, the isolates grew fast and evenly at the rate of 0.35–0.5 mm/h and the mycelium covered the whole plate only in 6 days. The culture produced white to light grayish colony with abundant fluffy aerial mycelium. Extended culture period to 7 days, the isolates began to secrete light brown pigments, giving the colony a characteristic brownish appearance (Fig. 3A). However, during the senescent phase, the culture produced pigments earlier (3–5 days after inoculation), first from the older mycelium near inocula and progressed to the periphery of the plate with aging (Fig. 3B). In the late phase of senescence, the formation of aerial hyphae decreased and the colony margin was plumose rather than even. The aerial hyphae were more slender than those from younger ones. Finally, the growth of a senescent culture stopped completely and the hyphae died at the front growth (Fig. 3C). During the juvenile phase, the leading hyphae grew vigorously. These hyphae were 12–17 m wide and were divided by septa into internodes. Primary branches grew out of the leading hyphae at angles less than 30◦ (with wider angles in rare cases) to form the primary hyphae. Short hyphae branched off at right angles from these primary branches, and in rare cases from secondary branches, to form the thinner tertiary branches (5–10 m) (Fig. 3D). In the early senescent phase, the leading hyphae grew sparsely. The angles between leading and primary hyphae were bigger (30–45◦ ) than those in the juvenile phase. The tertiary hyphae that branched off at right angles from primary and secondary branches were more abundant (Fig. 3E). In the late senescent phase, the primary branches grew out of the leading hyphae at bigger angles of 45–60◦ (at right angles in rare cases). The abundant tertiary hyphae were thinner (4–8 m). Most significantly, some apical hyphae collapsed in the final stage of senescence (Fig. 3F). After certain periods of rapid growth with almost constant rate, the isolates of M. elata entered the senescent stage characterized by slow down of mycelia growth, final death at the front growth and some phenotype changes. M. elata shared some morphological features with P. anserina during senescence: irregular growth prior to a complete growth arrest and final burst of apical hyphal cells from senescent cultures (Osiewacz, 2002; Rizet, 1953). However, increased pigmentation in senescent culture of P. anserina (Rizet, 1953) was not observed in M. elata. Alternatively, the time of pigmentation in senescent cultures of M. elata was advanced. Furthermore, the fact that the angles between the leading and primary hyphae increased constantly with aging, has not been found in other filamentous ascomycetes.
shrink away from the cell wall resulted in plasmolyzed cells. The nucleus and mitochondrium remained visible in some cells, but became irregularly shaped. Other cellular organelles were difficult to distinguish. The membrane did not always pull away uniformly and was often ruptured, causing leakage of the degraded cellular contents into the gap between the plasma membrane and cell wall. The membranes around some vacuoles were still intact, but more were obscure. In addition, many vacuoles appeared to be coalescing resulted in bigger ones. The lysosome containing recognizable remnants of autophagocytosed mitochondria and other cytoplasmic elements could be observed (Fig. 4C). Along with aging proceeded, the cells were extensively vacuolated and the organelles began to be degraded, causing most of the organelles in these cells unidentifiable. However, the cell wall was still intact and the lipid storage vesicles were easily distinguishable (Fig. 4D). In the final stage of senescence, the plasma membrane and all the cellular organelles were broken up and disappeared. The hyphal cell became an empty shell within some membrane fragments, although still remained its original shape for sustaining of the intact rigid cell wall. Rupture of the plasma membrane may lead to the release of cytoplasmic contents out of the cell, because the cell wall is not the main barrier for movement of many substances (Fig. 4E and F). The ultrastructural characteristics of M. elata in the final stage of senescence were similar to those of M. esculenta (Hervey et al., 1978). In the previous study, Hervey et al. found that the growth of subculture of GB groups of single ascospore isolates was determinate. Microscopic examination revealed that the cell wall of the apical cells of determinant cultures was still intact but devoid of both nuclei and cytoplasm. In senesced hyphal cells of M. elata, the characterized enlargement of vacuoles and the existence of organelle-sequestrated lysosomes imply the involvement of autophagy in senescence. Autophagy is a broad term for catabolic processes involving the lysosomal/vacuolar pathway (Pollack et al., 2009) and was identified as a longevity assurance mechanism in the aging model P. anserina (Knuppertz et al., 2014; Philipp et al., 2013). Moreover, some ultrastructural details of aging in M. elata corresponded with apoptotic events, for example, condensation of the nucleus and cytoplasm and cytoplasmolysis. However, the fragmentation of the cell contents into apoptotic bodies by membrane blebbing was not seen at a high frequency in this study, which is similar to the ultrastructural changes in dying cells of vegetative incompatibility in N. crassa (Jacobson et al., 1998). In addition, in the final stage of senescence, some apical hyphal cells from senescent cultures of M. elata burst with plasma membrane and all cellular organelles disrupted, which indicated a necrotic mode of cell death. This mode of cell death was also found in senescence of N. crassa and N. intermedia induced by kalilo plasmids (Bok et al., 2003). 3.4. The mechanism of aging in M. elata needs further study
3.3. Ultrastructural features The cytoplasm of juvenile cultures stained lightly and evenly. The nucleus and mitochondrium were easily distinguishable and the membranes were intact. Other organelles and structures that were visible in various cells included plasma membrane, endoplasmic reticulum and large amounts of small vacuoles with intact membranes (Fig. 4A and B). The septum that separate neighbor cells was obvious. Surprisingly, most septal pores were sealed by the woronin bodies. Two types of inclusion bodies lack of bounded membrane were distinguished and shared in juvenile hyphal cells. The near-spherical structures were lipid storage vesicles. However, the chemical composition and function of some rod-like crystal bodies that often appeared near the woronin body need further studies (Fig. 4B). During the senescent phase, the cytoplasm and nucleus were condensed and the plasma membrane appeared to
In this study, ultrastructural examination revealed the involvement of autophagy, apoptosis and necrosis in senescence of M. elata achieved by successive subculturing. In P. anserina, it is clear that oxidative stress, in particular as the result of reactive oxygen species (ROS) generation at the mitochondrial respiratory chain, plays a crucial role in healthy aging (Lorin et al., 2006). ROS is locally accumulated in the apical region of filamentous ascomycetes accompanied by the generation of adenosine triphosphate (ATP) (Semighini and Harris, 2008). Increased ROS levels during aging trigger mitochondrion-dependent PCD in senescent cultures and subsequently result in ATP depletion (Osiewacz, 2002). Intracellular ATP levels have a determining role in the interplay between apoptosis and necrosis. Depletion of intracellular ATP levels switches the energy requiring apoptotic cell death to necrosis (Skulachev, 2006). Furthermore, oxidative stress is a common stim-
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Fig. 3. Colony morphology (A–C) and light microscopy images (D–F) of isolate Ms04-1 (Morchella elata) cultures growing in 9 cm diameter Petri dish. (A) and (D), (B) and (E), (C) and (F) were cultures cultivated for 144 h, 1392 h and 1536 h, respectively, and were representative of those at the juvenile phase, in the early and final stage of senescence, respectively. The collapsed apical hyphae in the final stage of senescence (F) were marked with arrows.
Fig. 4. Transmission electron microscopy images of ultrathin sections of isolate Ms04-1 (Morchella elata) cultures. (A) and (B), (C) and (D), (E) and (F) were cultures cultivated for 144 h, 1392 h, and 1536 h, respectively, representing those at the juvenile phase, in the early and final stage of senescence, respectively. At the juvenile phase, most septal pores were sealed by the woronin bodies (WB) (Fig. 4B, arrow). In the early stage of senescence, the plasma membrane appeared to shrink away from the cell wall (Fig. 4C and D, arrows). In some apical hyphal cells, the lysosome containing recognizable remnants of autophagocytosed mitochondria and other cytoplasmic elements could be observed (Fig. 4C, lightning). In addition, the membrane apart from the cell wall often ruptured, causing leakage of the degraded cellular contents into the gap between the plasma membrane and cell wall (Fig. 4C, arrow head). C–crystal body, E–endoplasmic reticulum, L–lipid storage vesicle, M–mitochondrium, MF–membrane fragments, N–nuclear, S–septum, V–vacuole, W–cell wall. Scale bars = 0.5 m.
ulus for apoptosis, necrosis and autophagy (Nikoletopoulou et al., 2013). Under laboratory conditions, the vegetative growth and pseudosclerotial development of Morchella spp. are rapidly accomplished in several days (Figs. 1 and 2). Rapid growth and development are accompanied with increased ROS levels or oxidative stress (Georgiou et al., 2006; He et al., 2014; Király and Czövek, 2007), which may be the direct or indirect stimulus for apoptosis, necrosis and autophagy. Furthermore, the induction of autophagy in apical hyphal cells could contribute together with plugging of septal pores (Fig. 4) to the protection of neighboring cells from cell death (Pinan-Lucarré et al., 2005). Finally, it should be noted
that conventional microscopy is not always possible to distinguish autophagy, apoptosis and necrosis. The characterizations and molecular mechanisms of autophagy, apoptosis and necrosis in senescence of M. elata need further extensive study.
Acknowledgements This work was financially supported by the Science and Technology Innovatory Fund for Young Distinguished Scholars in Henan Province, China (No. 134100510017). We sincerely thank Mr. Ya Zhang for the provision of some information on morel cultivation
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