Plastid osmotic stress activates cellular stress responses in Arabidopsis

Abstract

Little is known about cytoplasmic osmoregulatory mechanisms in plants, and even less is understood about how the osmotic properties of the cytoplasm and organelles are coordinately regulated. We have previously shown that Arabidopsis (Arabidopsis thaliana) plants lacking functional versions of the plastid-localized mechanosensitive ion channels Mechanosensitive Channel of Small Conductance-Like2 (MSL2) and MSL3 contain leaf epidermal plastids under hypoosmotic stress, even during normal growth and development. Here, we use the msl2 msl3 mutant as a model to investigate the cellular response to constitutive plastid osmotic stress. Under unstressed conditions, msl2 msl3 seedlings exhibited several hallmarks of drought or environmental osmotic stress, including solute accumulation, elevated levels of the compatible osmolyte proline (Pro), and accumulation of the stress hormone abscisic acid (ABA). Furthermore, msl2 msl3 mutants expressed Pro and ABA metabolism genes in a pattern normally seen under drought or osmotic stress. Pro accumulation in the msl2 msl3 mutant was suppressed by conditions that reduce plastid osmotic stress or inhibition of ABA biosynthesis. Finally, treatment of unstressed msl2 msl3 plants with exogenous ABA elicited a much greater Pro accumulation response than in the wild type, similar to that observed in plants under drought or osmotic stress. These results suggest that osmotic imbalance across the plastid envelope can elicit a response similar to that elicited by osmotic imbalance across the plasma membrane and provide evidence for the integration of the osmotic state of an organelle into that of the cell in which it resides.

Figure 1.

msl2 msl3 mutant seedlings contain large, round nongreen plastids. Representative images of nongreen plastids in the leaf epidermis of Arabidopsis seedlings expressing the fluorescent plastid marker RecARED (pseudocolored red). Col-0, Columbia-0. Bar = 10 μm.

Figure 2.

msl2 msl3 mutant seedlings accumulate high levels of solutes. Ψ_S of mutant and wild-type seedlings grown under control (A) or moderately low water potential (B) conditions. Data from three independent experiments are presented (n = 5–7). Error bars indicate sem. Significant difference from the wild type is indicated. *, P < 0.01 (Student’s t test). Col-0, Columbia-0.

Figure 3.

msl2 msl3 mutant seedlings accumulate Pro and exhibit altered expression of Pro metabolism genes. A, Pro content of mutant and wild-type seedlings. The average of three biological replicates (each performed in triplicate; n ≥ 25 seedlings) is presented. Col-0, Columbia-0; FW, fresh weight. B, The qRT-PCR analysis of Pro metabolism gene expression normalized to the expression of the internal reference gene ACTIN. The average of three biological replicates (each performed in triplicate; n ≥ 25 seedlings) is presented. A and B, Significant differences from the wild type are indicated. Error bars indicate sem. *, P < 0.01 (Student’s t test). WT, Wild type. C, Immunoblot of P5CS1 protein levels in wild-type and mutant seedlings; 1.5-fold dilutions of each protein extract were loaded, and the blot was detected with anti-P5CS1 (top). Both α-tubulin (middle) and Ponceau S staining of Rubisco (bottom) were used for loading comparison because of the appearance of decreased levels of α-tubulin in msl2 msl3 mutants. [See online article for color version of this figure.]

Figure 4.

Pro accumulation and leaf morphology defects in msl2 msl3 seedlings are suppressed by increased cytoplasmic osmolarity. A, Representative images of whole seedlings and confocal micrographs of nongreen plastids in seedlings grown on media supplemented with indicated concentrations of NaCl. Col-0, Columbia-0. Bars in seedling images = 5 mm; bars in plastid images = 10 μm. B, Pro content of seedlings grown on the indicated concentrations of NaCl. The average of three biological replicates (each performed in triplicate; n ≥ 25 seedlings) is presented. Significant differences from the wild type are indicated. *, P < 0.01 (Student’s t test). FW, Fresh weight. C, Effect of the pgm-1 allele on Pro content in the msl2 msl3 background. The average of two biological replicates (each performed in triplicate; n ≥ 25 seedlings) is presented. Statistical groups a and b were determined using one-way ANOVA and Tukey’s test (P < 0.01). B and C, Error bars indicate sem. D, Confocal laser scanning microscopy images of nongreen plastids of plants grown on the indicated concentrations of NaCl. Scale bar = 10 μm.

Figure 5.

msl2 msl3 mutants exhibit elevated levels of ABA and induced expression of ABA biosynthesis and ABA response genes. A, ABA levels in wild-type and mutant seedlings. The average of three biological replicates of ≥30 seedlings each is presented. Error bars indicate sd. Col-0, Columbia-0; FW, fresh weight. B, The qRT-PCR analysis of ABA biosynthesis and stress response gene expression. The average of three biological replicates (each performed in triplicate; n ≥ 25 seedlings) is presented. Error bars indicate sem. Significant differences from the wild type are indicated. *, P < 0.01 (Student’s t test). WT, Wild type.

Figure 6.

msl2 msl3 mutants exhibit hypersensitivity to ABA during germination in an osmotic stress-dependent manner. Germination rates of mutant and wild-type seed on MS (A) or MS supplemented with 1 μm ABA (B and C). Germination was scored based on radical emergence. The average of three biological replicates of ≥50 seeds each is presented. Error bars indicate sd. Significant differences from the wild type are indicated. Col-0, Columbia-0. *, P < 0.01 (Student’s t test).

Figure 7.

Pro accumulation in msl2 msl3 mutants partially requires ABA biosynthesis and is more responsive to application of exogenous ABA than the wild type. A, Pro levels in seedlings grown on media containing 10 μm FLU and/or 0.5 μm ABA. The average of two to three biological replicates (each performed in triplicate; n ≥ 25 seedlings) from three independent experiments is presented. Significant differences from untreated wild type or untreated msl2 msl3 are indicated. *, P < 0.01 (Student’s t test). B, Effect of the aba2-1 lesion on Pro accumulation in the msl2 msl3 mutant background. The average of two biological replicates (each performed in triplicate; n ≥ 25 seedlings) is presented. Statistical groups a and b were determined using one-way ANOVA and Tukey’s test (P < 0.01). Error bars indicate sem. Col-0, Columbia-0; FW, fresh weight.