Mg-chelatase I subunit 1 and Mg-protoporphyrin IX methyltransferase affect the stomatal aperture in Arabidopsis thaliana

Abstract

To elucidate the molecular mechanisms of stomatal opening and closure, we performed a genetic screen using infrared thermography to isolate stomatal aperture mutants. We identified a mutant designated low temperature with open-stomata 1 (lost1), which exhibited reduced leaf temperature, wider stomatal aperture, and a pale green phenotype. Map-based analysis of the LOST1 locus revealed that the lost1 mutant resulted from a missense mutation in the Mg-chelatase I subunit 1 (CHLI1) gene, which encodes a subunit of the Mg-chelatase complex involved in chlorophyll synthesis. Transformation of the wild-type CHLI1 gene into lost1 complemented all lost1 phenotypes. Stomata in lost1 exhibited a partial ABA-insensitive phenotype similar to that of rtl1, a Mg-chelatase H subunit missense mutant. The Mg-protoporphyrin IX methyltransferase (CHLM) gene encodes a subsequent enzyme in the chlorophyll synthesis pathway. We examined stomatal movement in a CHLM knockdown mutant, chlm, and found that it also exhibited an ABA-insensitive phenotype. However, lost1 and chlm seedlings all showed normal expression of ABA-induced genes, such as RAB18 and RD29B, in response to ABA. These results suggest that the chlorophyll synthesis enzymes, Mg-chelatase complex and CHLM, specifically affect ABA signaling in the control of stomatal aperture and have no effect on ABA-induced gene expression.

Fig. 1

Leaf temperature and stomatal aperture in lost mutants. a Thermal image (right) and the corresponding bright-field image (left) of lost mutants and the control phot2 mutant. The images of the 3-week-old plants were taken under illuminated growth conditions. The scale bar indicates 2 cm. b Leaf temperatures of the lost and phot2 mutants were calculated from quantification of the thermal image in a. Values are means of 11–25 leaves with standard deviations (SDs). Asterisks indicate a significant statistical difference relative to the control phot2 by the t test (P < 0.01). The experiments were repeated on three occasions with similar results. c Stomatal aperture of the lost and phot2 mutants under illuminated growth conditions. Epidermal fragments were isolated from the plants at ZT5–9 and immediately subjected to measurement of stomatal aperture. Values are means of three independent experiments with standard errors (SEs) (n = 25). Asterisks indicate a significant statistical difference relative to the control phot2 by the t test (P < 0.01)

Fig. 2

A missense mutation in CHLI1 gene is responsible for the lost1 phenotype. a Typical phenotypes in control phot2, lost1, and two independent complementation lines gCHLI1/lost1 (#1 and #2). Plants were grown in soil under illuminated growth conditions for 5 weeks. The scale bar represents 1 cm. b Chlorophyll contents of phot2, lost1, and gCHLI1/lost1 (#1 and #2) rosette leaves. Chlorophyll contents were calculated based on fresh weight (FW) of leaves. Chlorophyll a and chlorophyll b contents are indicated by gray and white colors, respectively. Values are means of three independent experiments with SDs. Asterisks indicate a significant difference in total chlorophyll content relative to the control phot2 by the t test (*P < 0.01 and **P < 0.05). c Mapping analysis of the LOST1 locus. Numbers in parentheses indicate percentages of no recombination in 40 chromosomes. The LOST1 locus was close to CAPS marker G4539 and Mg-chelatase I subunit 1 (CHLI1). d Determination of the mutation in the lost1 mutant. The genomic structure of the CHLI1 gene on chromosome 4 is shown (upper). Black boxes and bars indicate exons and introns, respectively. Partial CHLI1 cDNA sequences and the deduced amino acid sequences from wild-type and lost1 are shown (lower). The lost1 mutant results from a G to A nucleotide substitution in the third exon. The nucleotide change causes the substitution of Lys for Arg at amino acid 219. Nucleotide and amino acid numbers are indicated in the right. Asterisks indicate the same nucleotide of the CHLI1 gene in wild type and lost1. e CHLI1 expression analyzed by RT–PCR in lost1 and the control phot2. Total RNA was extracted from 4-week-old seedlings. TUB2 was used as an internal standard for cDNA amounts. All PCRs were performed with 25 cycles. The experiments were repeated on three occasions with similar results. f Thermal image (right) and the corresponding bright-field image (left) of control phot2, lost1, and gCHLI1/lost1 (#1 and #2). The images were obtained and are presented as in Fig. 1a. g Leaf temperatures of control phot2, lost1, and gCHLI1/lost1 (#1 and #2). Leaf temperature was calculated from quantification of the infrared image f as described in Fig. 1b. Values are means of 12–16 leaves with SDs. Asterisk indicates a significant difference in leaf temperature relative to the control phot2 by the t test (P < 0.01). The experiments were repeated on three occasions with similar results. h Stomatal apertures under illuminated growth conditions in control phot2, lost1, and gCHLI1/lost1 (#1 and #2). Apertures were measured and presented as described in Fig. 1c. Values are means of 25 stomata measurements with SEs. Asterisks indicate a significant difference in stomatal aperture relative to lost1 by the t test (P < 0.01). The experiments were repeated on three occasions with similar results

Fig. 3

Stomatal phenotype in lost1 and rtl1 and expression of CHLI1, CHLH, and CHLM in guard cells. a Effect of ABA on light-induced stomatal opening in lost1, rtl1, and control phot2 mutants. Epidermal peels from dark-adapted plants in buffer containing 20 µM ABA or equal volumes of DMSO were irradiated with 10 µmol m⁻² s⁻¹ blue light and 50 µmol m⁻² s⁻¹ red light. Values are means of 25 stomata measurements with SEs. Asterisks indicate a significant difference in each treatment relative to phot2 by the t test (P < 0.01). The experiments were repeated on three occasions with similar results. b Expression of chlorophyll synthetic enzyme, CHLI1, CHLH, and CHLM, in wild-type (Col) guard cells. CHLI1, CHLH, and CHLM mRNA amounts were analyzed by RT–PCR. Total RNA was extracted from GCP isolated from 5-week-old wild type plants. TUB2 was used as an internal standard for cDNA amounts. All PCRs were performed with 35 cycles. Bands amplified by RT–PCR are indicated by an arrow. The experiments were repeated on three occasions with similar results

Fig. 4

ABA-induced gene expression in Mg-chelatase subunit mutants. Seedlings of lost1 and chli1 mutants were treated with 50 µM ABA or equal volumes of DMSO for 3 h. Relative mRNA levels of the ABA-induced RAB18 and RD29B genes were quantified by quantitative RT–PCR. Values are means of three independent experiments with SDs. Asterisks indicate a significant difference in each treatment relative to the control plants by the t test (P < 0.05). TUB2 was used as an internal standard for cDNA amounts

Fig. 5

The chlm mutant exhibited an ABA-insensitive phenotype for stomatal response. a Typical phenotypes of wild type (Col) and chlm plants. Plants were grown in soil under illuminated growth conditions for 5 weeks. The scale bar represents 1 cm. b Schematic structure of the CHLM gene and the locus of T-DNA insertion in the chlm mutant. Exons and introns are indicated by black boxes and bars, respectively. The T-DNA insertion was located at −41 bp in the 5′UTR of the CHLM gene. c Analysis of CHLM and TUB2 expression by RT–PCR in chlm and wild-type plants. Total RNA was extracted from 6-week-old seedlings. TUB2 was used as an internal standard for cDNA amounts. All PCRs were performed with 30 cycles. The experiments were repeated on three occasions with similar results. d Chlorophyll contents of chlm and wild-type rosette leaves. Chlorophyll contents were calculated based on fresh weight (FW) of leaves. Chlorophyll a and chlorophyll b contents are indicated by gray and white colors, respectively. Values are means of three independent experiments with SDs. Asterisk indicates a significant difference in total chlorophyll content relative to the wild type by the t test (P < 0.01). e Inhibition of light-induced stomatal opening by ABA in the chlm mutant. Stomatal apertures were measured and presented as described in Fig. 3a. Values are means of 25 stomatal measurements with SEs. Asterisks indicate a significant difference in each treatment relative to the wild type by the t test (P < 0.01). The experiments were repeated on three occasions with similar results. f ABA-induced gene expression in the chlm mutant. Relative mRNA levels of ABA-induced genes were quantified and presented as described in Fig. 4. Values are means of three independent experiments with SDs. Asterisks indicate a significant difference in each treatment relative to the wild type by the t test (P < 0.01). TUB2 was used as an internal standard for cDNA amounts