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. 2020 Nov 19;21(22):8753.
doi: 10.3390/ijms21228753.

Fusion of Mitochondria to 3-D Networks, Autophagy and Increased Organelle Contacts are Important Subcellular Hallmarks during Cold Stress in Plants

Philip Steiner  1 , Othmar Buchner  1 , Ancuela Andosch  1 , Gerhard Wanner  2 , Gilbert Neuner  3 , Ursula Lütz-Meindl  1
Affiliations

Fusion of Mitochondria to 3-D Networks, Autophagy and Increased Organelle Contacts are Important Subcellular Hallmarks during Cold Stress in Plants

Philip Steiner et al. Int J Mol Sci. .

Abstract

Low temperature stress has a severe impact on the distribution, physiology, and survival of plants in their natural habitats. While numerous studies have focused on the physiological and molecular adjustments to low temperatures, this study provides evidence that cold induced physiological responses coincide with distinct ultrastructural alterations. Three plants from different evolutionary levels and habitats were investigated: The freshwater alga Micrasterias denticulata, the aquatic plant Lemna sp., and the nival plant Ranunculus glacialis. Ultrastructural alterations during low temperature stress were determined by the employment of 2-D transmission electron microscopy and 3-D reconstructions from focused ion beam-scanning electron microscopic series. With decreasing temperatures, increasing numbers of organelle contacts and particularly the fusion of mitochondria to 3-dimensional networks were observed. We assume that the increase or at least maintenance of respiration during low temperature stress is likely to be based on these mitochondrial interconnections. Moreover, it is shown that autophagy and degeneration processes accompany freezing stress in Lemna and R. glacialis. This might be an essential mechanism to recycle damaged cytoplasmic constituents to maintain the cellular metabolism during freezing stress.

Keywords: FIB-SEM; Lemna sp.; Micrasterias denticulata; Ranunculus glacialis; TEM; electron microscopy; freezing stress; organelle networks; ultrastructure.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
TEM micrographs of mitochondria in Micrasterias denticulata cells during cold stress in comparison to controls at +20 °C. (a) Control at +20 °C with round, solitary mitochondrion and single mucilage vesicles. (b) +4 °C, 24 h chilling treatment with elongated mitochondria and slightly bloated endoplasmic reticulum. (c) +4 °C, 3 weeks treatment with elongation, aggregation, and fusion of mitochondria (arrows) and protrusion of mitochondria into mucilage vesicles (asterisks). (d) −2 °C freezing (without ice) treatment—elongation and aggregation of mitochondria visible. (e) −2 °C, extracellularly frozen cell with elongation and aggregation of mitochondria and protrusion of mitochondria into mucilage vesicle (asterisk). (f) Higher magnification of −2 °C, extracellularly frozen cell shows attachment and fusion of outer mitochondrial membrane (arrows) as well as aggregation to mitochondrial network. m: mitochondria, mv: mucilage vesicles, v: vacuole, cw: cell wall, er: endoplasmic reticulum.
Figure 2
Figure 2
3-D FIB-SEM reconstructions of organelles of Micrasterias denticulata during low temperature stress in comparison to controls at +20 °C. (a) Control at +20 °C with single unfused mitochondria (b) −2 °C freezing (without ice) treatment. Mitochondria aggregated and fused to single mitochondrial clusters. (c) −2 °C, extracellularly frozen cell, with large mitochondrial network. (d) −2 °C, extracellularly frozen cell, with mitochondrial network in contact with mucilage vesicles. (e) Higher magnification of −2 °C, extracellularly frozen cell depicts protrusion of mitochondria into transparent mucilage vesicles. purple: mitochondria, green: chloroplast, (transparent) brown: mucilage vesicles, transparent red with blue crystal: peroxisomes, yellow: starch grains, blue: cell wall with cell pores.
Figure 3
Figure 3
(a) Schematic depiction of the analyzed area in Micrasterias. (b) Mitochondrial fusions and contacts (%) in Micrasterias denticulata during extracellular freezing stress at −2 °C in comparison to controls at +20 °C determined by analysis of TEM micrographs (n = 10). Mean values were not significantly different (p = 0.069; t-test). Boxes indicate the median (horizontal line inside the box) and the 25th and the 75th percentile (bottom and top border). Whiskers indicate maxima and minima and extend maximum to 1.5 times box-height.
Figure 4
Figure 4
TEM micrographs of Lemna sp. during cold stress in comparison to controls at +20 °C. (a) +20 °C control of Lemna sp., with single, round mitochondrion. (b) +4 °C, 24 h chilled Lemna sp. depicting mitochondrial fusion (arrow). (c) +4 °C, 3 weeks chilled Lemna sp. with mitochondrial aggregation, fusion (arrow) and bloated ER. (d) −2 °C, extracellularly frozen Lemna sp. with signs of degradation (chloroplast envelope dissolved; autophagic structures), mitochondrial elongation and increased number of multi vesicular bodies. at: autophagic structures, m: mitochondria, mvb: multi vesicular body, er: endoplasmic reticulum, chl: chloroplast, sg: starch grain, d: dictyosome, n: nucleus.
Figure 5
Figure 5
TEM micrographs of palisade parenchyma cells of R. glacialis during and after cold stress in comparison to controls at +10 °C. (a) Control of R. glacialis at +10 °C with single mitochondrion. (b) −5 °C extracellularly frozen R. glacialis with aggregated mitochondria, bloated ER and numerous autophagic structures. (c) R. glacialis at +10 °C, 15 min after −5 °C extracellular freezing stress. Mitochondrial fusion and aggregation (arrow), minor bloating of ER, and multi vesicular bodies are clearly visible. (d) Recovery of R. glacialis at +10 °C, 24 h after −5 °C extracellular freezing stress. Minor structural alterations of mitochondria and ER still visible. No autophagic structures were observed. at: autophagic structures, m: mitochondria, mvb: multi vesicular body, er: endoplasmic reticulum, chl: chloroplast.
Figure 6
Figure 6
TEM micrographs of Micrasterias denticulata, Lemna sp. and palisade parenchyma cells of Ranunculus glacialis during freezing stress in comparison to controls at +20 °C. (a) Micrasterias control at +20 °C with regular thylakoid structure of chloroplast and distinct dictyosome shape and number of cisternae. (b) Micrasterias during freezing stress at −2 °C with degrading dictyosomes and bloated thylakoids. (c) Micrasterias during freezing stress at −2 °C with protrusion of peroxisome into mucilage vesicle (arrow). (d) Lemna control at +20 °C with regular thylakoid structure of chloroplast, distinct size and shape of dictyosome and solitary distributed, round mitochondria. (e) Lemna during −2 °C freezing stress with bloated thylakoids, enlarged ER and autophagic structures. (f) R. glacialis control at +10 °C with dictyosome in division and regular thylakoid structure. (g) R. glacialis control at +10 °C with regular dictyosome, regular thylakoid and ER structure. (h) R. glacialis during freezing stress at −5 °C with degraded thylakoids and enlarged ER structure. at: autophagic structures, chl: chloroplast, d: dictyosome, er: endoplasmic reticulum, p: peroxisome, m: mitochondria, mv: mucilage vesicles.
Figure 7
Figure 7
Photosynthetic oxygen production (apparent photosynthesis, open boxplots) and dark respiration (grey boxplots) of (a) Micrasterias denticulata and (b) Lemna sp. after 1 h, 24 h, and 3 weeks of chilling stress at +4 °C. Each boxplot relates to 3 independent biological replicates (n = 3). Different letters (a, b) indicate significant differences between means (p < 0.05). Letters with superscript numbers (a’, b’) are related to the dark respiration rate Rd. (One-way ANOVA followed by Duncan’s and Games Howell’s test). Boxes indicate the median (horizontal line inside the box) and the 25th and the 75th percentile (bottom and top border). Whiskers indicate maxima and minima and extend maximum to 1.5 times box-height.
Figure 8
Figure 8
Gas exchange measurements during a freezing and thawing (recovery) experiment on R. glacialis leaves. The Boxplots show the dark respiration rate (Rd) in dependence on the diffusive conductance rate (GH20). The mean value is increased but without statistical significance (p > 0.05; repeated measures ANOVA with Bonferroni-correction). Boxes indicate the median (horizontal line inside the box) and the 25th and the 75th percentile (bottom and top border). Whiskers indicate maxima and minima and extend maximum to 1.5 times box-height.
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