Mutation in Arabidopsis mitochondrial Pentatricopeptide repeat 40 gene affects tolerance to water deficit

Abstract

The Arabidopsis Pentatricopeptide repeat 40 (PPR40) insertion mutants have increased tolerance to water deficit compared to wild-type plants. Tolerance is likely the consequence of ABA hypersensitivity of the mutants. Plant growth and development depend on multiple environmental factors whose alterations can disrupt plant homeostasis and trigger complex molecular and physiological responses. Water deficit is one of the factors which can seriously restrict plant growth and viability. Mitochondria play an important role in cellular metabolism, energy production, and redox homeostasis. During drought and salinity stress, mitochondrial dysfunction can lead to ROS overproduction and oxidative stress, affecting plant growth and survival. Alternative oxidases (AOXs) and stabilization of mitochondrial electron transport chain help mitigate ROS damage. The mitochondrial Pentatricopeptide repeat 40 (PPR40) protein was implicated in stress regulation as ppr40 mutants were found to be hypersensitive to ABA and high salinity during germination. This study investigated the tolerance of the knockout ppr40-1 and knockdown ppr40-2 mutants to water deprivation. Our results show that these mutants display an enhanced tolerance to water deficit. The mutants had higher relative water content, reduced level of oxidative damage, and better photosynthetic parameters in water-limited conditions compared to wild-type plants. ppr40 mutants had considerable differences in metabolic profiles and expression of a number of stress-related genes, suggesting important metabolic reprogramming. Tolerance to water deficit was also manifested in higher survival rates and alleviated growth reduction when watering was suspended. Enhanced sensitivity to ABA and fast stomata closure was suggested to lead to improved capacity for water conservation in such environment. Overall, this study highlights the importance of mitochondrial functions and in particular PPR40 in plant responses to abiotic stress, particularly drought.

Keywords: Arabidopsis; Drought tolerance; Mitochondria; Mutant analysis; Oxidative stress; Pentatricopeptide repeat 40; Water deficit.

Fig. 1

The ppr40-1 and ppr40-2 mutants survive water limitation at higher rates than Arabidopsis Col-0 wild-type plants. Plants were cultured in plant phenotyping system. 3-week-old plants were deprived of water for 13 days and subsequently re-watered. a Experimental design. b-d Representative images of each genotype grown in a phenotyping tray. b Plants before watering was withdrawn (day 0). c Plants after 12 days of water stress. d Plants 6 days after re-watering. e Survival rates in a typical experiment. 80 plants were monitored in this experiment, scored in groups of 10. Percentage (%) of recovered plants is shown. Error bars indicate standard deviation (n = 8). Statistical significance was determined with t-test, * and ** indicate significant differences compared to Col-0 at P < 0.05 and P < 0.01 levels, respectively

Fig. 2

Growth of Arabidopsis plants in standard and water-deprived conditions, as revealed by phenotypic analysis. Plant growth was monitored by analysis of RGB images, obtained between 5 and 13 days after watering was suspended. Change of rosette size was determined by measuring color-segmented green area of 40 plants of each genotype. Control: uninterrupted watering; drought: water withdrawal. Statistical analysis used Kruskal–Wallis test, to compare the treatments and genotypes. Error bars represent the standard deviation of means green area of 40 plants from each genotype. Different letters (in red) indicate significant differences at P < 0.05.

Fig. 3

Change of plant morphology as displayed by slenderness of leaves (SOL) parameter. Plant growth was monitored by a phenotyping platform as described in Fig. 2. SOL was calculated from RGB images. Control: uninterrupted watering; drought: water withdrawal. Statistical analysis used Kruskal–Wallis test, to compare the treatments and genotypes. Error bars represent the standard deviation of means green area of 40 plants from each genotype. Different letters (in red) indicate significant differences at P < 0.05

Fig. 4

Effect of water deprivation on stomatal conductance and Relative Water Content (RWC). a Change of stomatal conductance of Arabidopsis Col-0 wild-type and ppr40-1 mutant plants. Four-week-old plants were deprived of water for 7 days, and stomatal conductance was measured at daily intervals. b RWC in Arabidopsis ppr40-1 and ppr40-2 mutants and Col-0 wild-type plants, subjected to water deprivation for 10 days. Error bars indicate standard deviation. Experiments were performed in a growth chamber and repeated three times. Statistical analysis used Two-way ANOVA, Tukey test. Different letters indicate significant differences at P < 0.05

Fig. 5

Proline accumulation and expression of P5CS1 and PDH1 genes in ppr40-1, ppr40-2 mutants, and wild-type (Arabidopsis Col-0) plants in response to water deprivation. Experiments were performed in a growth chamber. a Change in proline content of wild-type and mutant plants which were stressed by water withdrawal for up to 10 days. b Expression of P5CS1 and PDH1 genes in water-stressed and well-watered plants after 8 days of treatment. Relative expression is shown where 1 corresponds to transcript levels of non-treated Col-0 plants. Statistical analysis used Two-way ANOVA, Fisher’s LSD, and Tukey test. The error bars indicate the standard deviation of means of three biological replicates. Different letters indicate significant differences at P < 0.05

Fig. 6

Metabolic profiles of six selected metabolites in Col-0 wild-type and ppr40-1 mutant plants. Metabolite profiles were determined by LC/MS analysis in leaves of four-week-old plants, cultured in well-watered (control) and water-restricted (drought) conditions. Statistical analysis used Two-way ANOVA and error bars represent the standard deviation of means of 6 samples of each genotype

Fig. 7

The impact of water deprivation on the photosynthetic parameters of Arabidopsis Col-0 wild-type and ppr40 mutant plants. Chlorophyll fluorescence (ChlF) imaging was used to monitor changes of photosynthetic parameters of water-stressed and well-watered plants using the automatic plant phenotyping platform. Experimental design is shown in Fig. 1a. Change of the maximum quantum yield of photosystem II (Fv/Fm) is shown (averages of 40 plants). Control: uninterrupted watering; drought: water withdrawal. Statistical analysis used Kruskal–Wallis test, to compare the treatments and genotypes. Error bars represent the standard deviation of means Fv/Fm of 40 plants from each genotype. Different letters (in red) indicate significant differences at P < 0.05

Fig. 8

Quantitative analysis of electron transport rates (ETR(II) in ppr40-1 and ppr40-2 mutants, and in wild-type (Arabidopsis Col-0) plants. ETR(II) was measured in dark-adapted conditions using a pulse amplitude-modulated fluorometer (Maxi-PAM). ETR values were obtained from well-watered (control) and water-stressed plants deprived of water for 10 days (drought). Error bars indicate standard deviation (n = 3). Experiments were repeated three times. Statistical analysis used Two-way ANOVA, Tukey test. Different letters indicate significant differences at P < 0.05

Fig. 9

Oxidative damage in water-deprived plants. a Lipid peroxidation rates of Arabidopsis Col-0 wild-type and ppr40 mutant plants in well-watered and water-stressed conditions. Plants were cultured in growth chambers in standard conditions for 4 weeks; then watering was suspended for 10 days. Malondialdehyde (MDA) levels are shown in response to gradual stress. b Expression of the ROS-induced ZAT12 gene in drought-stressed plants after 8 days of treatment. Relative transcript levels are shown, where 1 corresponds to values measured in Col-0 plants under control conditions. Reference: Actin2 and UBC18 genes. Averages of three replicates are shown, with standard deviation indicated by the bars on the diagrams. Statistical analysis used Two-way ANOVA, Tukey test. Different letters indicate significant differences at P < 0.05

Fig. 10

Expression of selected stress-induced genes in water-limited and well-watered conditions. Plants were grown and treated as indicated in Fig. 8. Transcript levels of RD29A RAB18 (a), ABA-responsive element-binding factor 2 (ABF2), and ABF3 (b), and AOX1a, AOX1d (c) genes were measured by qRT-PCR using Actin2 and UBC18 as reference genes. Relative expression levels are shown, with a value of 1 corresponding to transcript levels of Col-0 in control plants. The data represent the average of three replicates, with standard deviation indicated by the bars on the diagrams. Statistical analysis used Two-way ANOVA, Tukey test. Different letters indicate significant differences at P < 0.05