- Email: [email protected]
New Beginnings: Mitochondrial Renewal by Massive Mitochondrial Fusion
TRPLSC 1565 No. of Pages 3
Spotlight
Regenerang protoplasts – Nicoana
New Beginnings: Mitochondrial Renewal by Massive Mitochondrial Fusion Ray J. Rose1,* and David W. McCurdy1,* Massive mitochondrial fusion (MMF) in germinating arabidopsis seeds, together with earlier studies, suggests a significant role for MMF in the life cycle of flowering plants. MMF is likely to facilitate nucleoid transmission, mitochondrial DNA (mtDNA) recombination, and the homogenization of mitochondrial components, thus providing a type of quality control for mitochondrial populations in new generations.
(A)
(B) Shoot apical meristem – Arabidopsis
n
(C)
n
(D) Germinang seeds – Arabidopsis
2 hS
TR
The existence of mitochondrial fusion in plant cells was inferred based on cytological observations as well as novel mtDNA restriction patterns following protoplast fusions [1]. However, the study by Ari(E) (F) mura et al. [2] using the photoconvertible fluorescent protein Kaede was the first definitive demonstration of mitochondrial Figure 1. Massive Mitochondrial Fusion in Three Different Contexts. (A) Freshly isolated tobacco fusion. Frequent and transient fusion of mesophyll protoplasts contain discrete round/oval mitochondria that can be clumped into irregular aggrethe red and green populations of mito- gates. (B) Mitochondria fused into long, tubular structures 4 h after culture. (C) Interphase cell from chondria generated by Kaede was Arabidopsis stem showing round/oval-shaped mitochondria (green). (D) Prometaphase cell from SAM with followed by fission[67_TD$IF][, and these cycles of individual elongated mitochondria (green) or mitochondria fused into a massive cage-like structure (blue). (E) Mitochondria are discrete and numerous in an embryonic Arabidopsis cotyledon cell 2 h after imbibition. (F) fusion and fission were suggested to Mitochondria show tubuloreticular morphology in an embryonic cotyledon cell at the testa rupture (TR) contribute to overcoming the heterogene- stage. Bars: 10 mm in (A,B), 2 mm in (C,D), and 5 mm in (E,F). Images in (A,B) are from Sheahan et al. [5] and ity in mtDNA levels and mitochondrial reproduced with permission from John Wiley & Sons; images in (C,D) are from Seguí-Simarro and Staehelin nucleoids, which are undetectable in [9]; images in (E,F) are from Paszkiewicz et al. [7] and reproduced with permission from the American Society of Plant Biologists. some plant mitochondria [2–4]. In investigating the organelle dynamics of single isolated tobacco protoplasts capable of regeneration into whole plants, within 4 h of culture mitochondria were observed to assume long tubular forms up to 16 mm in length (Figure 1A,B) [5,6]. This process was termed MMF and was followed by fission into smaller, round/
oval-shaped mitochondria [5]. MMF was confirmed by fusing protoplasts expressing GFP targeted to mitochondria with another group of protoplasts in which mitochondria were stained red with MitoTracker [6]. In freshly isolated tobacco protoplasts, 25% of mitochondria did
not contain visible nucleoids, but following MMF and fission nucleoids were observed in all mitochondria in the population [6]. This result suggested the possibility that mature mesophyll cells becoming totipotent by protoplast isolation and culture undergo MMF as a type of
Trends in Plant Science, Month Year, Vol. xx, No. yy
1
TRPLSC 1565 No. of Pages 3
quality control before producing a new generation asexually. MMF was not observed in subsequent divisions of the tobacco protoplasts, with mitochondria remaining small and numerous [6]. MMF was also observed in arabidopsis and Medicago truncatula protoplasts but, importantly, MMF was not observed in protoplasts derived from already dedifferentiated tobacco BY-2 cells, suggesting an association of MMF with dedifferentiation and reprogramming [6]. The studies by Sheahan et al. [5,6] demonstrating MMF were made using isolated protoplasts that were potentially totipotent and thus may not necessarily have significance in the life cycle of normal sexually reproducing plants. However, two studies, one recently by Paszkiewicz et al. [7] examining mitochondria in germinating seeds and an earlier study by SeguíSimarro et al. [8] looking at mitochondrial morphologies in shoot apical meristem (SAM) cells, document similar examples of mitochondrial fusion on a large scale. We propose that these examples represent a common phenomenon of MMF as first reported in cultured protoplasts [5,6], and thus MMF has a significant role in the plant life cycle. Using 3D electron microscopy, SeguίSimarro et al. [8] showed that, in interphase cells of the SAM and leaf primordium (LP), a large reticulate mitochondrion is formed that wraps around the nucleus, and this structure forms alongside a population of smaller, more peripheral mitochondria (Figure 1C,D). During mitosis and cytokinesis, most mitochondria fuse to form a cagelike mitochondrion encompassing the spindle/phragmoplast cytoskeletal arrays (Figure 1D). In the subsequent interphase, about 60% of the cage-like mitochondrion fragments into smaller, physically discrete mitochondria [8]. The authors suggested that this characteristic fusion/fission process facilitates the mixing of mitochondrial components and high rates of recombination of mtDNA. This mitochondrial cycle was observed
2
Trends in Plant Science, Month Year, Vol. xx, No. yy
only in the meristematic cells of the SAM and LP and not in root meristem or other cell types. Seguí-Simarro and Staehelin in a subsequent publication [9] pointed out that cells of the SAM give rise to the floral meristem and female gametes that house the maternally inherited mitochondria. They suggested that the cage-like mitochondria in the SAM would allow homogenization of protein content, prevent undesired mutations, and minimize the chance of a given mitochondrion containing no mtDNA. The recent paper by Paszkiewicz et al. [7] demonstrated the occurrence of extensive mitochondrial fusion in the context of germination, where autotrophic growth initiates the establishment of a new generation. The study closely followed mitochondria from dry seed to seed imbibition and germination. In dry seeds the mitochondria are rudimentary but carry mtDNA, and have been termed promitochondria. As germination proceeds following imbibition, extensive mitochondrial fusion, as visualized by mito-GFP, and membrane biogenesis occur to form a perinuclear ‘tubuloreticular’ structure by late germination at the testa rupture stage (Figure 1E,F). As is the case for the SAM, following formation of this tubuloreticular structure, fragmentation into numerous smaller and discrete mitochondria occurs [7]. Following this fragmentation event in the germinating seed cotyledons, however, they reported that not all mitochondria contain nucleoids. This observation is consistent with somatic cells having a subpopulation of mitochondria without detectable nucleoids, as previously observed in several studies [2,3,6]. However, as shown by Arimura et al. [2], frequent transient fusion and fission of mitochondrial pairs occurs in such cells. We propose that the formation of cage-like reticulate mitochondria in meristematic cells [8] and of tubuloreticular mitochondria in germinating seeds [7]
both represent forms of MMF as reported by Sheahan et al. [5,6] and that this process therefore is a characteristic of totipotent plant cells as well as cells involved in sexual reproduction and germination. This observation suggests that MMF contributes to a quality control process in the production of the next generation of mitochondria. This may be achieved by MMF providing an enhanced opportunity for intramolecular recombination, a characteristic feature of plant mitochondria. Mitochondrial recombination can contribute to quality control by homologous DNA replication repair and mtDNA heteroplasmy, which also results from recombination [7,10]. MMF also leads to mixing and unification of the mitochondrial compartment, which contributes to the maintenance of functional mitochondria [11,12]. The MMF process also influences nucleoid maintenance. In protoplasts preparing for division, MMF results in all mitochondria having observable nucleoids [6], and it was suggested by Seguí-Simarro and Staehelin [9] that this process would have a similar effect in the shoot meristem and subsequent gamete formation, but in this example nucleoid distribution was not measured. Consistent with nucleoid maintenance as a consequence of MMF in the SAM, more than 90% of promitochondria in seed embryos were found to have observable nucleoids [7]. However, after MMF during germination only 67% of mitochondria have detectable nucleoids, thus re-establishing the heterogeneity of nucleoids seen in somatic cells [2–4,6]. Since nucleoid heterogeneity is largely overcome in mature cells by transient fusion and fission between mitochondrial pairs [2], a more important consequence of MMF therefore might be the exchange of proteins and maximizing mtDNA repair and recombination. Presumably at least some mitochondria ultimately fail to be ‘renewed’ by the MMF/fission processes and this could be resolved by autophagy. Paszkiewicz et al. [7] addressed this point and demonstrated upregulation of
TRPLSC 1565 No. of Pages 3
mitophagy early in the germination process. 1
School of Environmental and Life Sciences, The University of Newcastle, Newcastle, NSW 2308, Australia *Correspondence:
[email protected] (R.J. Rose) and [email protected] (D.W. McCurdy). http://dx.doi.org/10.1016/j.tplants.2017.06.005 References 1. Belliard, G. et al. (1979) Mitochondrial recombination in cytoplasmic hybrids of Nicotiana tabacum by protoplast fusion. Nature 281, 401–403 2. Arimura, S. et al. (2004) Frequent fusion and fission of plant mitochondria with unequal nucleoid distribution. Proc. Natl. Acad. Sci. U. S. A. 18, 7805–7808
3. Takanashi, H. et al. (2006) Different amounts of DNA in each mitochondrion in rice root. Genes Genet. Syst. 81, 215–218 4. Preuten, T. et al. (2010) Fewer genes than organelles: extremely low and variable gene copy numbers in mitochondria of somatic plant cells. Plant J. 64, 948–959 5. Sheahan, M.B. et al. (2004) Organelle inheritance in plant cell division: the actin cytoskeleton is required for unbiased inheritance of chloroplasts, mitochondria and endoplasmic reticulum in dividing protoplasts. Plant J. 37, 379–390 6. Sheahan, M.B. et al. (2005) Mitochondria as a connected population: ensuring continuity of the mitochondrial genome during plant cell dedifferentiation through massive mitochondrial fusion. Plant J. 44, 744–755 7. Paszkiewicz, G. et al. (2017) Arabidopsis seed mitochondria are bioenergetically active immediately upon imbibition and specialize via biogenesis in preparation for autotrophic growth. Plant Cell 29, 109–128
8. Seguí-Simarro, J.M. et al. (2008) The mitochondrial cycle of Arabidopsis shoot apical meristem and leaf primordium meristematic cells is defined by a perinuclear tentaculate/cage-like mitochondrion. Plant Physiol. 148, 1380–1393 9. Seguí-Simarro, J.M. and Staehelin, L.A. (2009) Mitochondrial reticulation in shoot apical meristem cells of Arabidopsis provides a mechanism for homogenization of mtDNA prior to gamete formation. Plant Signal. Behav. 4, 168–1671 10. Gualberto, J.M. and Newton, K.J. (2017) Plant mitochondrial genomes: dynamics and mechanisms of mutation. Annu. Rev. Plant Biol. 68, 17.1–17.28 11. El-Zawily, A.M. et al. (2014) FRIENDLY regulates mitochondrial distribution, fusion and quality control in Arabidopsis. Plant Physiol. 166, 808–828 12. Møller, I.M. (2016) What is hot in plant mitochondria? Physiol. Plant. 157, 256–263
Trends in Plant Science, Month Year, Vol. xx, No. yy
3
Spotlight
Regenerang protoplasts – Nicoana
New Beginnings: Mitochondrial Renewal by Massive Mitochondrial Fusion Ray J. Rose1,* and David W. McCurdy1,* Massive mitochondrial fusion (MMF) in germinating arabidopsis seeds, together with earlier studies, suggests a significant role for MMF in the life cycle of flowering plants. MMF is likely to facilitate nucleoid transmission, mitochondrial DNA (mtDNA) recombination, and the homogenization of mitochondrial components, thus providing a type of quality control for mitochondrial populations in new generations.
(A)
(B) Shoot apical meristem – Arabidopsis
n
(C)
n
(D) Germinang seeds – Arabidopsis
2 hS
TR
The existence of mitochondrial fusion in plant cells was inferred based on cytological observations as well as novel mtDNA restriction patterns following protoplast fusions [1]. However, the study by Ari(E) (F) mura et al. [2] using the photoconvertible fluorescent protein Kaede was the first definitive demonstration of mitochondrial Figure 1. Massive Mitochondrial Fusion in Three Different Contexts. (A) Freshly isolated tobacco fusion. Frequent and transient fusion of mesophyll protoplasts contain discrete round/oval mitochondria that can be clumped into irregular aggrethe red and green populations of mito- gates. (B) Mitochondria fused into long, tubular structures 4 h after culture. (C) Interphase cell from chondria generated by Kaede was Arabidopsis stem showing round/oval-shaped mitochondria (green). (D) Prometaphase cell from SAM with followed by fission[67_TD$IF][, and these cycles of individual elongated mitochondria (green) or mitochondria fused into a massive cage-like structure (blue). (E) Mitochondria are discrete and numerous in an embryonic Arabidopsis cotyledon cell 2 h after imbibition. (F) fusion and fission were suggested to Mitochondria show tubuloreticular morphology in an embryonic cotyledon cell at the testa rupture (TR) contribute to overcoming the heterogene- stage. Bars: 10 mm in (A,B), 2 mm in (C,D), and 5 mm in (E,F). Images in (A,B) are from Sheahan et al. [5] and ity in mtDNA levels and mitochondrial reproduced with permission from John Wiley & Sons; images in (C,D) are from Seguí-Simarro and Staehelin nucleoids, which are undetectable in [9]; images in (E,F) are from Paszkiewicz et al. [7] and reproduced with permission from the American Society of Plant Biologists. some plant mitochondria [2–4]. In investigating the organelle dynamics of single isolated tobacco protoplasts capable of regeneration into whole plants, within 4 h of culture mitochondria were observed to assume long tubular forms up to 16 mm in length (Figure 1A,B) [5,6]. This process was termed MMF and was followed by fission into smaller, round/
oval-shaped mitochondria [5]. MMF was confirmed by fusing protoplasts expressing GFP targeted to mitochondria with another group of protoplasts in which mitochondria were stained red with MitoTracker [6]. In freshly isolated tobacco protoplasts, 25% of mitochondria did
not contain visible nucleoids, but following MMF and fission nucleoids were observed in all mitochondria in the population [6]. This result suggested the possibility that mature mesophyll cells becoming totipotent by protoplast isolation and culture undergo MMF as a type of
Trends in Plant Science, Month Year, Vol. xx, No. yy
1
TRPLSC 1565 No. of Pages 3
quality control before producing a new generation asexually. MMF was not observed in subsequent divisions of the tobacco protoplasts, with mitochondria remaining small and numerous [6]. MMF was also observed in arabidopsis and Medicago truncatula protoplasts but, importantly, MMF was not observed in protoplasts derived from already dedifferentiated tobacco BY-2 cells, suggesting an association of MMF with dedifferentiation and reprogramming [6]. The studies by Sheahan et al. [5,6] demonstrating MMF were made using isolated protoplasts that were potentially totipotent and thus may not necessarily have significance in the life cycle of normal sexually reproducing plants. However, two studies, one recently by Paszkiewicz et al. [7] examining mitochondria in germinating seeds and an earlier study by SeguíSimarro et al. [8] looking at mitochondrial morphologies in shoot apical meristem (SAM) cells, document similar examples of mitochondrial fusion on a large scale. We propose that these examples represent a common phenomenon of MMF as first reported in cultured protoplasts [5,6], and thus MMF has a significant role in the plant life cycle. Using 3D electron microscopy, SeguίSimarro et al. [8] showed that, in interphase cells of the SAM and leaf primordium (LP), a large reticulate mitochondrion is formed that wraps around the nucleus, and this structure forms alongside a population of smaller, more peripheral mitochondria (Figure 1C,D). During mitosis and cytokinesis, most mitochondria fuse to form a cagelike mitochondrion encompassing the spindle/phragmoplast cytoskeletal arrays (Figure 1D). In the subsequent interphase, about 60% of the cage-like mitochondrion fragments into smaller, physically discrete mitochondria [8]. The authors suggested that this characteristic fusion/fission process facilitates the mixing of mitochondrial components and high rates of recombination of mtDNA. This mitochondrial cycle was observed
2
Trends in Plant Science, Month Year, Vol. xx, No. yy
only in the meristematic cells of the SAM and LP and not in root meristem or other cell types. Seguí-Simarro and Staehelin in a subsequent publication [9] pointed out that cells of the SAM give rise to the floral meristem and female gametes that house the maternally inherited mitochondria. They suggested that the cage-like mitochondria in the SAM would allow homogenization of protein content, prevent undesired mutations, and minimize the chance of a given mitochondrion containing no mtDNA. The recent paper by Paszkiewicz et al. [7] demonstrated the occurrence of extensive mitochondrial fusion in the context of germination, where autotrophic growth initiates the establishment of a new generation. The study closely followed mitochondria from dry seed to seed imbibition and germination. In dry seeds the mitochondria are rudimentary but carry mtDNA, and have been termed promitochondria. As germination proceeds following imbibition, extensive mitochondrial fusion, as visualized by mito-GFP, and membrane biogenesis occur to form a perinuclear ‘tubuloreticular’ structure by late germination at the testa rupture stage (Figure 1E,F). As is the case for the SAM, following formation of this tubuloreticular structure, fragmentation into numerous smaller and discrete mitochondria occurs [7]. Following this fragmentation event in the germinating seed cotyledons, however, they reported that not all mitochondria contain nucleoids. This observation is consistent with somatic cells having a subpopulation of mitochondria without detectable nucleoids, as previously observed in several studies [2,3,6]. However, as shown by Arimura et al. [2], frequent transient fusion and fission of mitochondrial pairs occurs in such cells. We propose that the formation of cage-like reticulate mitochondria in meristematic cells [8] and of tubuloreticular mitochondria in germinating seeds [7]
both represent forms of MMF as reported by Sheahan et al. [5,6] and that this process therefore is a characteristic of totipotent plant cells as well as cells involved in sexual reproduction and germination. This observation suggests that MMF contributes to a quality control process in the production of the next generation of mitochondria. This may be achieved by MMF providing an enhanced opportunity for intramolecular recombination, a characteristic feature of plant mitochondria. Mitochondrial recombination can contribute to quality control by homologous DNA replication repair and mtDNA heteroplasmy, which also results from recombination [7,10]. MMF also leads to mixing and unification of the mitochondrial compartment, which contributes to the maintenance of functional mitochondria [11,12]. The MMF process also influences nucleoid maintenance. In protoplasts preparing for division, MMF results in all mitochondria having observable nucleoids [6], and it was suggested by Seguí-Simarro and Staehelin [9] that this process would have a similar effect in the shoot meristem and subsequent gamete formation, but in this example nucleoid distribution was not measured. Consistent with nucleoid maintenance as a consequence of MMF in the SAM, more than 90% of promitochondria in seed embryos were found to have observable nucleoids [7]. However, after MMF during germination only 67% of mitochondria have detectable nucleoids, thus re-establishing the heterogeneity of nucleoids seen in somatic cells [2–4,6]. Since nucleoid heterogeneity is largely overcome in mature cells by transient fusion and fission between mitochondrial pairs [2], a more important consequence of MMF therefore might be the exchange of proteins and maximizing mtDNA repair and recombination. Presumably at least some mitochondria ultimately fail to be ‘renewed’ by the MMF/fission processes and this could be resolved by autophagy. Paszkiewicz et al. [7] addressed this point and demonstrated upregulation of
TRPLSC 1565 No. of Pages 3
mitophagy early in the germination process. 1
School of Environmental and Life Sciences, The University of Newcastle, Newcastle, NSW 2308, Australia *Correspondence:
[email protected] (R.J. Rose) and [email protected] (D.W. McCurdy). http://dx.doi.org/10.1016/j.tplants.2017.06.005 References 1. Belliard, G. et al. (1979) Mitochondrial recombination in cytoplasmic hybrids of Nicotiana tabacum by protoplast fusion. Nature 281, 401–403 2. Arimura, S. et al. (2004) Frequent fusion and fission of plant mitochondria with unequal nucleoid distribution. Proc. Natl. Acad. Sci. U. S. A. 18, 7805–7808
3. Takanashi, H. et al. (2006) Different amounts of DNA in each mitochondrion in rice root. Genes Genet. Syst. 81, 215–218 4. Preuten, T. et al. (2010) Fewer genes than organelles: extremely low and variable gene copy numbers in mitochondria of somatic plant cells. Plant J. 64, 948–959 5. Sheahan, M.B. et al. (2004) Organelle inheritance in plant cell division: the actin cytoskeleton is required for unbiased inheritance of chloroplasts, mitochondria and endoplasmic reticulum in dividing protoplasts. Plant J. 37, 379–390 6. Sheahan, M.B. et al. (2005) Mitochondria as a connected population: ensuring continuity of the mitochondrial genome during plant cell dedifferentiation through massive mitochondrial fusion. Plant J. 44, 744–755 7. Paszkiewicz, G. et al. (2017) Arabidopsis seed mitochondria are bioenergetically active immediately upon imbibition and specialize via biogenesis in preparation for autotrophic growth. Plant Cell 29, 109–128
8. Seguí-Simarro, J.M. et al. (2008) The mitochondrial cycle of Arabidopsis shoot apical meristem and leaf primordium meristematic cells is defined by a perinuclear tentaculate/cage-like mitochondrion. Plant Physiol. 148, 1380–1393 9. Seguí-Simarro, J.M. and Staehelin, L.A. (2009) Mitochondrial reticulation in shoot apical meristem cells of Arabidopsis provides a mechanism for homogenization of mtDNA prior to gamete formation. Plant Signal. Behav. 4, 168–1671 10. Gualberto, J.M. and Newton, K.J. (2017) Plant mitochondrial genomes: dynamics and mechanisms of mutation. Annu. Rev. Plant Biol. 68, 17.1–17.28 11. El-Zawily, A.M. et al. (2014) FRIENDLY regulates mitochondrial distribution, fusion and quality control in Arabidopsis. Plant Physiol. 166, 808–828 12. Møller, I.M. (2016) What is hot in plant mitochondria? Physiol. Plant. 157, 256–263
Trends in Plant Science, Month Year, Vol. xx, No. yy
3