Mg-chelatase H subunit affects ABA signaling in stomatal guard cells, but is not an ABA receptor in Arabidopsis thaliana

Abstract

Mg-chelatase H subunit (CHLH) is a multifunctional protein involved in chlorophyll synthesis, plastid-to-nucleus retrograde signaling, and ABA perception. However, whether CHLH acts as an actual ABA receptor remains controversial. Here we present evidence that CHLH affects ABA signaling in stomatal guard cells but is not itself an ABA receptor. We screened ethyl methanesulfonate-treated Arabidopsis thaliana plants with a focus on stomatal aperture-dependent water loss in detached leaves and isolated a rapid transpiration in detached leaves 1 (rtl1) mutant that we identified as a novel missense mutant of CHLH. The rtl1 and CHLH RNAi plants showed phenotypes in which stomatal movements were insensitive to ABA, while the rtl1 phenotype showed normal sensitivity to ABA with respect to seed germination and root growth. ABA-binding analyses using (3)H-labeled ABA revealed that recombinant CHLH did not bind ABA, but recombinant pyrabactin resistance 1, a reliable ABA receptor used as a control, showed specific binding. Moreover, we found that the rtl1 mutant showed ABA-induced stomatal closure when a high concentration of extracellular Ca(2+) was present and that a knockout mutant of Mg-chelatase I subunit (chli1) showed the same ABA-insensitive phenotype as rtl1. These results suggest that the Mg-chelatase complex as a whole affects the ABA-signaling pathway for stomatal movements.

Fig. 1

Characterization of the rtl1 mutant. a Kinetics of the fresh weight change in the detached rosette leaves from 4-week-old wild-type (WT) (open circles) and rtl1 (closed circles) plants. The relative weights of leaves are presented as a percentage of the initial weight, which was the weight of each rosette leaf immediately after detachment from the plants, with the standard deviation (SD). b Four-week-old WT and rtl1 plants. Bar 10 mm. c Effect of ABA on light-induced stomatal opening in WT and rtl1 plants. Epidermal tissues from dark-adapted plants (shaded) were incubated under light (blue light at 10 μmol m⁻² s⁻¹ superimposed on background red light at 50 μmol m⁻² s⁻¹) for 2.5 h with (black) or without (white) 20 μM ABA. Data represent the mean with SD. d ABA-induced stomatal closing in WT and rtl1 plants. Pre-illuminated epidermal tissues were incubated under light (same as above) for 2.5 h with (black) or without (white) 20 μM ABA. Data represent the mean with SD. All experiments were repeated three times on different occasions with similar results

Fig. 2

CHLH missense mutation is responsible for the rtl1 phenotype. a Mapping analysis of the RTL1 locus. The RTL1 locus was close to SSLP marker nga151 and Mg-chelatase H subunit (CHLH). b Schematic representation of the structure of the CHLH gene (upper). Black boxes and lines represent exons and introns, respectively. The position of the amino acid substitution (L690 to F) in rtl1 is indicated. The partial sequences of CHLH cDNA and the deduced amino acid in wild-type (WT) and rtl1 are shown (lower). A single nucleotide substitution (C2068 to T) is shown by a box. The amino acid and nucleotide numbers are indicated on the right. c Typical phenotypes in the WT, rtl1, and two independent gCHLH/rtl1 complementation lines (#1 and #2). Plants were grown on soil for 4 weeks. Bar 10 mm. d Chlorophyll contents in rosette leaves of 4-week-old WT, rtl1, and gCHLH/rtl1 lines (#1 and #2). The bars show the contents of chl a (white) and chl b (black). e ABA-induced stomatal closing in WT, rtl1, and gCHLH/rtl1 lines (#1 and #2). Conditions were the same as shown in Fig. 1d. Data represent the mean with SD. f ABA-induced stomatal closing in the known chlh mutants, cch and gun5. Col and pOCA are the background plants of the cch and gun5-1 mutation, respectively. Conditions were the same as shown in Fig. 1d. Data represent the mean with SD. All experiments were repeated three times on different occasions with similar results

Fig. 3

Phenotypes in wild-type (WT), rtl1, and CHLH RNAi lines. a RT-PCR analysis of CHLH in WT, rtl1, and two CHLH RNAi lines (#1 and #2). Total RNA was isolated from rosette leaves from 4-week-old plants. PCRs were performed with 26 cycles. TUB2 was amplified as a control. b Immunoblot analysis of CHLH protein in WT, rtl1, and CHLH RNAi lines. Fifty micrograms of protein extracted from rosette leaves was loaded on each lane. The 14-3-3 proteins were detected using anti-14-3-3 antibody as a control. c Four-week-old WT, rtl1, and CHLH RNAi plants. Bar 10 mm. d Chlorophyll content of rosette leaves from 4-week-old WT, rtl1, and CHLH RNAi plants. The bars show the content of chl a (white) and chl b (black). e Effect of ABA on stomatal closing in WT, rtl1, and CHLH RNAi plants. Conditions were the same as in Fig. 1d. Data represent the mean with SD. All experiments repeated three times on different occasions gave similar results

Fig. 4

ABA-binding assay of the recombinant CHLH protein. a, b Coomassie Brilliant Blue staining of the recombinant His-tagged CHLH, PYR1, and ABI1 proteins used for the ABA-binding assay. Asterisks indicate degradation products of CHLH. The sizes of the molecular weight markers are shown on the left. c Determination of ABA-binding activity of the recombinant proteins by the filter method. Two micromolar recombinant protein and 50 nM ³H-labeled ABA were incubated in the absence (black) or presence of 1,000-fold unlabeled ABA (white) for 1 h at 25°C. Data represent the mean of three independent experiments with SD

Fig. 5

Effect of a high concentration of Ca²⁺ on the ABA-induced stomatal closing in rtl1 mutant. Stomatal apertures of WT and rtl1 were measured after incubation for 2.5 h in the presence of 0.1 and 5 mM Ca²⁺ with or without 20 μM ABA. Other conditions were the same as in Fig. 1d. Data represent the mean with SD. Experiments repeated three times on different occasions gave similar results

Fig. 6

Characterization of the T-DNA insertion mutant for Mg-chelatase I subunit 1 (chli1). a Schematic representation of the structure of the CHLI gene and the location of the T-DNA insertion site. Black boxes and lines represent exons and introns, respectively. The T-DNA was inserted into the third exon of the CHLI1 genomic DNA. b CHLI1 expression verification by RT-PCR in the chli1 mutant (upper panel). TUB2 was used as a control (lower panel). c Four-week-old plants, Col and chli1, grown under a 16-h photoperiod. Bar 10 mm. d Effect of ABA on stomatal closing in Col and chli1 plants. Stomatal apertures were measured after incubation for 2.5 h in the basal buffer with (black) or without (white) 20 μM ABA. Other conditions were the same as in Fig. 1d. Data represent the mean with SD. Experiments repeated three times on different occasions gave similar results