The CRK5 and WRKY53 Are Conditional Regulators of Senescence and Stomatal Conductance in Arabidopsis

Abstract

In Arabidopsis thaliana, cysteine-rich receptor-like kinases (CRKs) constitute a large group of membrane-localized proteins which perceive external stimuli and transduce the signal into the cell. Previous reports based on their loss-of-function phenotypes and expression profile support their role in many developmental and stress-responsive pathways. Our study revealed that one member of this family, CRK5, acts as a negative regulator of leaf aging. Enrichment of the CRK5 promoter region in W-box cis-elements demonstrated that WRKY transcription factors control it. We observed significantly enhanced WRKY53 expression in crk5 and reversion of its early-senescence phenotype in the crk5 wrky53 line, suggesting a negative feedback loop between these proteins antagonistically regulating chlorophyll a and b contents. Yeast-two hybrid assay showed further that CRK5 interacts with several proteins involved in response to water deprivation or calcium signaling, while gas exchange analysis revealed a positive effect of CRK5 on water use efficiency. Consistent with that, the crk5 plants showed disturbed foliar temperature, stomatal conductance, transpiration, and increased susceptibility to osmotic stress. These traits were fully or partially reverted to wild-type phenotype in crk5 wrky53 double mutant. Obtained results suggest that WRKY53 and CRK5 are antagonistic regulators of chlorophyll synthesis/degradation, senescence, and stomatal conductance.

Keywords: development; foliar temperature; osmotic stress; receptor-like kinase; senescence; stomatal conductance; transcription factor; water-use efficiency.

Figure 1

RNA seq analysis of plants at different ages (four and six weeks old). The study involved four genotypes: Col-0, crk5 and two complementation lines oeCRK5crk5#1, oeCRK5crk5#2. Heat map shows the expression patterns of selected senescence marker genes (A) and WRKY transcription factors (B).

Figure 2

(A) Identification of interaction between CRK5 promoter and six WRKY senescence-related transcription factors (WRKY6, WRKY22, WRKY30, WRKY53, WRKY54, WRKY70). The analysis was performed by Matchmaker Gold Yeast One-Hybrid System. Single-deficiency (SD/–Leu) screening assay was performed to estimate yeast transformation efficiency, while the addition of 200 ng/mL aureobasidin allowed for identifying potential binding partners. The combination of p53-AbAi and pGADT7-p53 was used as a positive control. (B) Transient transactivation luciferase reporter assay. The study was based on the transient transformation of N. benthamiana leaves with two genetic constructs simultaneously: (1) prom35S-WRKY53 and prom35S-HMG At2g34450 (negative control without luciferase activity), (2) prom35S-HMG promCRK5-LUC, (3) prom35S-WRKY53 and promCRK5-LUC. Mean values of luciferase activity (RLU) were derived from 12 tobacco plants (n = 12). Dots represent individual observations. Asterisks indicate a significant difference according to the t-test at level p < 0.001 (***).

Figure 3

(A) Morphological phenotype of dark-induced senescence in the analyzed genotypes. Photographs were taken of plants growing under ambient light (120 μmol m⁻² s⁻¹) conditions and then transferred for four days to continuous darkness. White scale bars indicate 1 cm (B–E) Analysis of pigment content changes under continuous darkness conditions. The figures show the level of chlorophyll a (B), chlorophyll b (C), chlorophyll a/b ratio (D), and carotenoids (E). Mean values (±SD) were derived from 6 plants (n = 6). Dots represent individual observations. Asterisks indicate a significant difference according to the t-test at level p < 0.001 (***), p < 0.01 (**), and p < 0.05 (*).

Figure 4

(A) qRT-PCR analysis of expression of WRKY53 and CRK5 in the analyzed genotypes. Data show relative expression normalized to the wild type and represent average values ± SD. Asterisks indicate a significant difference according to the t-test at level p < 0.001 (***), p < 0.01 (**). (B) Proposed model describing mutual CRK5 and WRKY53 relationship. We suggest the existence of a negative feedback loop between these proteins.

Figure 5

Foliar gas exchange characteristics in variable intercellular CO₂ concentration. The analysis involved 4-week-old and 6-week-old plants and was performed on CIRAS-3 Portable Photosynthesis System, with PAR of 300 µmol m⁻² s⁻¹ and Cuvette Flow of 300 mL/min (A/Ci C3 ramp program). Individual charts represent (A) stomatal conductance, (B) CO₂ assimilation, (C) evapotranspiration, and (D) water use efficiency (WUE). Mean values (±SD) were derived from 8 plants (n = 8).

Figure 6

Analysis of foliar temperature under variable light conditions. (A) Thermograms showing whole rosettes of different Arabidopsis genotypes after 30 s high light (4000 μmol photons m⁻² s⁻¹). (B) Plot showing dynamic foliar temperature changes under variable light conditions in comparison to the background—white surface on which plants were placed during the measurement. The white surface reflected the light, and its temperature increased by only 1,5C during high light exposure. The program was set as follows: 30 s of ambient light (150 μmol photons m⁻² s⁻¹), followed by 60 s of high blue light (4000 μmol photons m⁻² s⁻¹), followed by 60 s of ambient light (150 μmol photons m⁻² s⁻¹). (C) Average leaf temperature changes after 30 s and 60 s of high light exposure. Data represent mean values of 4 different leaves from 5 plants (n = 20). Statistical analysis was performed according to a t-test at level p < 0.05. Letters A, B, C and a, b, c above the bars indicate homogenous groups.

Figure 7

(A) Morphological phenotype of plants treated with 450 mM NaCl for 14 days. (B) Relative cellular electrolyte leakage was measured after 14 days of salt treatment compared to non-treated plants. Dots represent individual observations. Mean values (±SD) are derived from 10 plants (n = 10). Asterisks indicate a significant difference relative to Col-0 according to the one-way ANOVA and Tukey HSD test at level p < 0.001 (***), p < 0.01 (**). (C) The dynamics of maximum quantum yield of photosystem II (Fv/Fm) and plant growth within the osmotic stress treatment. Data represent mean values of 10 plants (n = 10). The statistics for Figure 7C (p-values of Tukey HSD test compared to Col-0) were presented in Table S4 in the supplementary data.