Fig 1.
Effects of KAI2 on drought resistance.
(A) Biomass of kai2-2 and WT plants under drought stress and well-watered control (n = 9 biological replicates). The different letters above the error bars indicate significant differences (P < 0.05) in all combinations according to a Tukey's honest significant difference test. (B) Biomass reduction of kai2-2 and WT plants under drought stress relative to respective well-watered control. Data represent the means and standard errors (n = 9 biological replicates). Asterisks indicate significant differences as determined by a Student’s t-test, ***P < 0.001. (C) Averaged pot weights of kai2-2 and WT plants during drought stress (n = 9 biological replicates). Black arrows indicate when water was added to the root growth area in the soil. Red arrow indicates when biomass was measured. (D-E) WT and kai2-2 mutant plants were grown on water-saturated soil for 8 days. Watering was then stopped for 7 days (D) and 14 days (E).
Fig 2.
Drought-associated traits of kai2-2 leaves.
(A-C) kai2-2 and WT plants were grown and exposed to drought. At indicated time points, (A) soil relative moisture contents (n = 10) and relative humidity, (B) leaf relative water content (RWC) (n = 4 biological replicates), and (C) electrolyte leakage (n = 4 biological replicates) were determined. (D) Electrolyte leakage (Left) of kai2-2 and WT plants at a similar RWC (Right) during drought treatment (n = 4 biological replicates). Data represent the means and standard errors. (E) Average stomatal density of rosette leaves (abaxial side) from 14-day-old plate-grown kai2-2 and WT plants. Error bars represent standard errors (n = 15 leaves/15 independent plants/genotype). (F) Representative guard cells of rosette leaves from 21-day-old plate-grown kai2-2 and WT plants treated with 0, 30 and 50 μM ABA. (G-H) Average width of stomatal aperture of rosette leaves from 21-day-old WT and kai2-2 plants in the presence or absence of ABA. Aperture width are shown in micrometers (G) or in percentage of the average aperture width obtained from absence of ABA treatment (H). Error bars represent standard errors (n = 5 plants; for each plant the average of nine stomatal measurements from a single leaf was calculated). (I) Leaf surface temperatures of well-watered WT and kai2-2 plants before (0 d) and after a 7-d drought period (7 d). Plants were 21-d-old at the start of water withholding. Common optical camera (Left) and thermal imaging camera (Right) were used to take pictures at the same time. Asterisks indicate significant differences between the genotypes under well-watered control or drought conditions as determined by a Student’s t-test, *P < 0.05; **P < 0.01; ***P < 0.001.
Fig 3.
KAI2 effects on ABA responses and metabolism.
(A) Cotyledon opening percentage of kai2-2 and WT seedlings in the absence or presence of different concentrations of exogenous ABA. Data represent the means and standard deviation of 3 independent experiments (n = 50 seeds/genotype/experiment). Asterisks indicate significant differences as determined by a Student’s t-test, *P < 0.05; **P < 0.01; ***P < 0.001. (B) Endogenous ABA contents in leaves of 24-d-old kai2-2 and WT plants under normal and dehydration conditions. Data represent the means and standard errors (n = 5 plants). (C) Expression of genes involved in ABA biosynthesis and catabolism in leaves of 24-day-old kai2-2 and WT plants under normal and dehydration conditions. Relative expression levels were normalized to a value of 1 in the WT grown under normal conditions. Data represent the means and standard errors (n = 5 biological replicates). The different letters above the error bars indicate significant differences (P < 0.05) in all combinations according to a Tukey's honest significant difference test.
Fig 4.
Transcriptome analysis of kai2-2 and WT plants under normal and dehydration conditions.
(A) Relative water content (RWC) of leaves from 24-d-old well-watered kai2-2 and WT plants exposed to dehydration treatment. Data represent the means and standard errors (n = 5 plants). Asterisks indicate significant differences according to a Student’s t-test, ***P < 0.001. Rosette leaf samples collected in 3 biological repeats at 0, 2 and 4 h (arrows) were used for microarray analysis. Room temperature and relative room humidity were recorded during the dehydration period. (B) Diagrams illustrating experimental design and comparisons between the treatments. The number of differentially expressed genes (DEGs) identified from various comparisons are noted in red (upregulated relative to control) or blue (downregulated relative to control). Data were obtained from the microarray analysis of 3 biological repeats. (C) Venn diagram analysis showing the overlapping and non-overlapping DEGs among the comparisons. M-C/W-C, kai2-2 well-watered control 0 h vs. WT well-watered control 0 h; M-D2/W-D2, kai2-2 dehydrated 2 h vs. WT dehydrated 2 h; M-D4/W-D4, kai2-2 dehydrated 4 h vs. WT dehydrated 4 h; M-D/W-D, M-D2/W-D2 and/or M-D4/W-D4; W-D2/W-C, WT dehydrated 2 h vs. WT well-watered control 0 h; W-D4/W-C, WT dehydrated 4 h vs. WT well-watered control 0 h; W-D/W-C, W-D2/W-C and/or W-D4/W-C; M-D2/M-C, kai2-2 dehydrated 2 h vs. kai2-2 well-watered control 0 h; M-D4/M-C, kai2-2 dehydrated 4 h vs. kai2-2 well-watered control 0 h; M-D/M-C, M-D2/M-C and/or M-D4/M-C.
Fig 5.
Anthocyanin production in kai2-2 and WT plants.
(A) kai2-2 and WT plants were grown for 5 weeks, and watering was withheld for 10 days. Inflorescences were cut from representative plants before photographing. (B) Anthocyanin content in kai2-2 and WT plants under well-watered and drought conditions. Data represent the means and standard errors (n = 4 plants). Asterisks indicate significant differences between the genotypes under drought conditions as determined by a Student’s t-test, *P < 0.05; **P < 0.01; ***P < 0.001.
Fig 6.
Cuticle permeability of kai2-2, 35S:KAI2 transgenic lines OE1 and OE2, and WT plants.
(A) Chlorophyll leaching from rosette leaves of 28-day-old kai2-2, OE1, OE2 and WT plants at different time periods. Data represent the means and standard errors (n = 3 plants/genotype). (B) Fold-change of overexpression levels of KAI2 gene in leaves of 14-day-old OE1 and OE2 plants in comparison with WT (n = 5 biological replicates). Asterisks indicate significant differences between the WT and other genotypes under well-watered condition as determined by a Student’s t-test, *P < 0.05; **P < 0.01; ***P < 0.001. (C) Toluidine blue staining patterns of rosette leaves of 28-day-old kai2-2, OE1, OE2 and WT plants. Red arrow indicates the fifth leaves, which were used for transmission electron microscope (TEM) analysis.(D) TEM images of the surface of the fifth leaves (adaxial side) derived from kai2-2, OE1, OE2 and WT plants. CW, cell wall. Blue arrows indicate cuticular layer (electron-dense, darker-staining layer) and green arrows indicate wax-rich cuticle proper (electron-translucent layer). (E) Thickness of cuticle of the fifth leaves (adaxial side) derived from kai2-2, OE1, OE2 and WT plants. Data represent the means and standard errors (n = 3 biological replicates). Different letters above the error bars indicate significant differences (P < 0.05) among the genotypes according to a Tukey's honest significant difference test.
Fig 7.
Models for functions of SLs and KARs/KAI2-ligand (KL) in plant growth, development and drought response.
(A) SL-regulation of shoot branching, senescence, secondary thickening and root development is mediated by SL-specific receptor D14. KAR-regulation (or hypothetical KL-regulation) of seed germination, hypocotyl elongation and leaf development is mediated by KAI2. MAX2 is the checkpoint for both SL and KAR/KL signaling pathways in plant growth and development. (B) SLs and KARs/KL regulate plant resistance to drought through D14-MAX2 and KAI2-MAX2 cascade, respectively. Question mark indicates the contribution of D14 to drought resistance was unknown until this study. (C) Biomass of kai2-2, d14-2, kai2-2 d14-2 and WT plants under well-watered control and drought stress (n = 9 biological replicates). (D) Biomass reduction of kai2-2, d14-2, kai2-2 d14-2 and WT plants under drought relative to respective well-watered control. Data represent the means and standard errors (n = 9 biological replicates). Different letters above the error bars indicate significant differences (P < 0.05) among the genotypes according to a Tukey's honest significant difference test. (E) Averaged pot weights of kai2-2, d14-2, kai2-2 d14-2 and WT plants during drought stress (n = 9 biological replicates). Black arrows indicate when water was added to the root growth area in the soil. Red arrow indicates when biomass was measured. (F) Plant phenotypes before harvest.
Fig 8.
Model illustrating functions of KAI2 in plant resistance to drought.
Karrikins (KARs) or a putative, endogenous KAI2 ligand (KL) activate KAI2 signaling, which promotes plant resistance to drought through several biochemical and physiological processes.