Plant mitochondrial FMT and its mammalian homolog CLUH controls development and behavior in Arabidopsis and locomotion in mice

Abstract

Mitochondria in animals are associated with development, as well as physiological and pathological behaviors. Several conserved mitochondrial genes exist between plants and higher eukaryotes. Yet, the similarities in mitochondrial function between plant and animal species is poorly understood. Here, we show that FMT (FRIENDLY MITOCHONDRIA) from Arabidopsis thaliana, a highly conserved homolog of the mammalian CLUH (CLUSTERED MITOCHONDRIA) gene family encoding mitochondrial proteins associated with developmental alterations and adult physiological and pathological behaviors, affects whole plant morphology and development under both stressed and normal growth conditions. FMT was found to regulate mitochondrial morphology and dynamics, germination, and flowering time. It also affects leaf expansion growth, salt stress responses and hyponastic behavior, including changes in speed of hyponastic movements. Strikingly, Cluh^± heterozygous knockout mice also displayed altered locomotive movements, traveling for shorter distances and had slower average and maximum speeds in the open field test. These observations indicate that homologous mitochondrial genes may play similar roles and affect homologous functions in both plants and animals.

Keywords: Arabidopsis thaliana; CLUH; FMT; Hyponasty; Locomotion; Mice; Mitochondria.

Fig. 1

Expression pattern of FRIENDLY MITOCHONDRIA (FMT) in shoot apices, flowers and embryos of Arabidopsis Col-0 plants detected by RNA in situ hybridization. Longitudinal sections of vegetative meristem (A), inflorescence meristem (B), stage 4 flowers (C), stage 6 flowers (D) and stage 8 flowers (E), control probe in sense orientation in longitudinal sections of inflorescence meristem (F), embryos at various stages (G–I), early torpedo stage (G), late torpedo stage (H) and mature embryos (I). Arrow indicates FMT expression in stamen (St) and carpel (Ca) primordia. Scale bar = 100 µm

Fig. 2

Arabidopsis seeds transformed with pFASTG02-FMT show green fluorescence. A Transgenic seeds overexpressing FMT in the pFASTG02 vector show green fluorescence (right), while non-transformed WT seeds (left) do not show green fluorescence. B Same field of view as in A, viewed under bright field light. Scale bar = 100 µm

Fig. 3

FMT regulates mitochondrial morphology and dynamics. A–C TEM of mitochondria from columella cells. Each square represents a 4 µm × 4 µm area selected from a representative cell from each genotype: A WT, B fmt and C FMT-OE. D Number of mitochondria per cell from three plant lines. E Aspect ratio (AR) from three plant lines. F Mitochondrial area (µm²) from three plant lines. G Inner mitochondrial membrane (IMM) fusion events from three plant lines. H Outer mitochondrial membrane (OMM) fusion events from three plant lines. I Nearest neighbor value (R_n) between three plant lines. Statistical analysis indicates significant differences (****P ≤ 0.0001, ***P ≤ 0.001, *P ≤ 0.05) using one-way ANOVA. Bars and error bars represent mean and standard deviation, respectively. Scale bar = 2 µm

Fig. 4

FMT regulates germination timing and total germination. A Plants 4 days after germination (DAG), (i) WT, (ii) fmt and (iii) FMT-OE. B Quantification of days to germination. C Quantification of germination rate (in %). Scale bar = 1 cm. Statistical analysis indicates significant differences (****P ≤ 0.0001, **P ≤ 0.01) using one-way ANOVA. Bars and error bars represent mean and standard deviation, respectively

Fig. 5

FMT regulates root length and flowering timing under long-day conditions. A Root length at 7 DAG, (i) WT, (ii) fmt and (iii) FMT-OE. B Quantification of root length at 7 DAG. C Root length at 14 DAG in (i) WT, (ii) fmt and (iii) FMT-OE. D Quantification of root length at 14 DAG. E) Flowering time phenotype at 35 DAG, (i) WT, (ii) fmt, (iii) FMT-OE. F Quantification of days to flowering. Scale bar = 1 cm. Statistical analysis indicates significant differences (****P ≤ 0.0001, ***P ≤ 0.001, **P ≤ 0.01) using one-way ANOVA. Bars and error bars represent mean and standard deviation, respectively

Fig. 6

FMT affects the response to salt stress in Arabidopsis. A Spatio-temporal root stress browser of FMT expression in response to 140 mM NaCl over 48 h. Control (MS) expression at 1 and 48 h provided on the left and right root cross-sections, respectively. Gene expression level is directly compared to the highest signal record for the given gene (FMT) and assigned an accompanying color, ranging from yellow (no expression) to red (highest/absolute) expression. B Quantification of total germination (in %). C Days to germination. D Primary root length under control and salt-stress conditions in WT. E Primary root length under control and salt-stress conditions in fmt. F Primary root length under control and salt-stress conditions in FMT-OE. G Days to flowering under control and salt-stress conditions in WT. H Days to flowering under control and salt-stress conditions in fmt. I Days to flowering under control and salt-stress conditions in FMT-OE. Black bars indicate control conditions. Gray bars indicate exposure to 125 mM NaCl. Statistical analysis indicates significant differences (****P ≤ 0.0001, ***P ≤ 0.001, *P ≤ 0.05) using one-way ANOVA. Bars and error bars represent mean and standard deviation, respectively

Fig. 7

FMT regulates mitochondrial morphology and dynamics under salt stress. A–C TEM of mitochondria from columella cells. Each square represents a 4 µm × 4 µm area selected from a representative cell from each genotype: A WT, B fmt and C FMT-OE. D–F Number of mitochondria per cell in plants under salt stress compared to control conditions in WT, fmt, and FMT-OE, respectively. G–I Number of inner mitochondrial membrane fusion events in plants under salt stress compared to control conditions in WT, fmt, and FMT-OE, respectively. J–L Number of outer mitochondrial membrane fusion events in plants under salt stress compared to control conditions in WT, fmt, and FMT-OE, respectively. M–O Average nearest neighbor ratio in in plants under salt stress compared to control conditions in WT, fmt, and FMT-OE, respectively. Statistical analysis indicates significant differences (****P ≤ 0.0001, *P ≤ 0.05) using one-way ANOVA. Bars and error bars represent mean and standard deviation, respectively

Fig. 8

Salt treatment delays floral transition in Col-0 plants and affects FMT expression in the shoot apical meristem. A–H Emergence of flower primordia analyzed by toluidine blue staining from apices of Col-0 plants grown under long days and harvested at 0, 1, 2 and 3 days after transfer (DAT) to salt media. I–N RNA in situ hybridization using FMT as a probe on longitudinal sections of the apical meristem of shifted plants grown in normal and salt conditions, harvested at end of day (ED) at 0, 1 and 2 days after transfer (DAT). Asterisks and arrows indicate meristem summit and floral primordia, respectively. Scale bars = 100 µm

Fig. 9

fmt and FMT-OE mutants exhibit changes in spatio-temporal growth. A Rosette area increased over time (15–23 days after sowing, DAS) determined by 3D imaging. Yellow: WT, red: fmt, blue: FMT-OE. B Diurnal relative expansion rate (RER) averaged over eight sequential 24-h periods. C p values from pairwise Student’s t tests applied over a 50-min sliding window, where p values < 0.05 indicated significance. Lines and color-shaded areas represent mean and standard deviation, respectively. Plants grown in a 12 h photoperiod

Fig. 10

FMT regulates hyponastic growth in Arabidopsis. A Average hyponastic angle (HYP) over time (15–23 DAS). B Diurnal HYP averaged over eight sequential 24-h periods. C, E p values from pairwise Student’s t tests applied over a 50 min sliding window, where p values < 0.05 indicated significance. D Change in speed of hyponastic angle (HYP) averaged over eight sequential 24-h periods. Lines and color-shaded areas represent mean and standard deviation, respectively. Plants grown in a 12 h photoperiod

Fig. 11

CLUH affects mouse mobility and speed. A Comparison of WT and heterozygous (HET) Cluh^± mice in open field tests: A total distance travelled, B average speed, C maximum speed. D Schematic illustration of the homology between Arabidopsis and mice regarding the effect of altered clustering of mitochondria