Increased leaf photosynthesis caused by elevated stomatal conductance in a rice mutant deficient in SLAC1, a guard cell anion channel protein

Abstract

In rice (Oryza sativa L.), leaf photosynthesis is known to be highly correlated with stomatal conductance; however, it remains unclear whether stomatal conductance dominantly limits the photosynthetic rate. SLAC1 is a stomatal anion channel protein controlling stomatal closure in response to environmental [CO(2)]. In order to examine stomatal limitations to photosynthesis, a SLAC1-deficient mutant of rice was isolated and characterized. A TILLING screen of N-methyl-N-nitrosourea-derived mutant lines was conducted for the rice SLAC1 orthologue gene Os04g0674700, and four mutant lines containing mutations within the open reading frame were obtained. A second screen using an infrared thermography camera revealed that one of the mutants, named slac1, had a constitutive low leaf temperature phenotype. Measurement of leaf gas exchange showed that slac1 plants grown in the greenhouse had significantly higher stomatal conductance (g (s)), rates of photosynthesis (A), and ratios of internal [CO(2)] to ambient [CO(2)] (C (i)/C (a)) compared with wild-type plants, whereas there was no significant difference in the response of photosynthesis to internal [CO(2)] (A/C (i) curves). These observations demonstrate that in well-watered conditions, stomatal conductance is a major determinant of photosynthetic rate in rice.

Fig. 1.

Gene structure of rice SLAC1. (A) A phylogenetic tree of the rice and Arabidopsis SLAC1 protein families, Escherichia coli TehA, and Saccharomyces pombe Mae1. (B) Schematic drawing of the rice SLAC1 gene. Boxes represent exons and black lines indicate introns. Exons in black were represented in the cDNAs, and the grey exon segments at the 5’ and 3’ ends indicate untranslated regions. The sites of mutations screened by the TILLING assay are indicated below. The arrows indicate the positions of the PCR primers used for TILLING assay.

Fig. 2.

Amino acid sequence alignment of the rice (Os) and Arabidopsis (At) SLAC1 full-length sequences. Identical residues are shaded in black and similar residues are shown in grey (similarity threshold of 0.4 for shading). Grey bars below the sequences indicate helical segments predicted from comparison with Haemophilius influenzae TehA (Chen et al., 2010). Asterisks indicate the phosphorylation sites in the Arabidopsis SLAC1 protein reported by Vahisalu et al. (2010). The arrows indicate the location of the amino acid substitutions occurring in the mutant line 5S56 shown in Table 1.

Fig. 3.

Expression profiles of rice SLAC1. (A) Schematic illustration of a rice seedling with a fully expanded third leaf. L1, L2, L3, and L4 indicate the first, second, third, and fourth leaf, respectively. Developmental stages (P0–P6) are also indicated. The shoot base (SB) is a 5mm segment from the bottom of the shoot containing pre-emerged leaves at stages P0–P3. (B) Transcript levels of SLAC1, HT1, and STOMAGEN in the developing rice leaves determined by qRT-PCR. Total mRNAs were isolated from the SB and developing leaves at the P4 and P5 stages. All results were normalized to the level of UBQ10 mRNA, and relative values were calculated, 1.0 representing 6cm P4 leaves. Data are the means of three independent experiments performed in triplicate, and bars represent ±SD. (C) SLAC1 mRNA accumulation in the slac1 mutant and wild-type (WT) plants. Total mRNAs were isolated from P4 and P5 leaves, and transcript levels were determined by qRT-PCR and normalized to those of UBQ10. Relative values were calculated, 1.0 representing P4 leaves of wild-type plants. Data are the means of three independent experiments performed in triplicate, and bars represent ±SD.

Fig. 4.

Thermal images of the wild type (WT) and the slac1 mutant subjected to low (100 ppm) and high (700 ppm) atmospheric [CO₂]. The subtractive images show changes in leaf temperature in response to the transfer from low to high [CO₂].

Fig. 5.

Stomatal densities on the adaxial and abaxial surfaces of flag leaves of wild-type (WT) and slac1 mutant plants grown under 350 ppm or 700 ppm [CO₂]. Data represent mean values ±SD of six independent experiments conducted with different plants.

Fig. 6.

Leaf photosynthetic rate changes correlate with CO₂-induced stomatal conductance changes. (A) Time courses of stomatal conductance to water (g _s) and the photosynthetic rate (A) in response to changes in [CO₂] in the slac1 mutant and wild-type (WT) leaves. Each value represents the mean ±SD of three leaves from 8-week-old plants. (B) The average g _s and A at 350 ppm or 700 ppm [CO₂] calculated from data used in (A). Data represent mean values ±SD of three independent experiments conducted with different plants. Asterisks denote a significant difference between the mutant and wild-type plants using Student’s t-test (P < 0.001).

Fig. 7.

Photosynthetic rate at different intercellular [CO₂] (A/C _i curves) of leaves from the slac1 mutant (circles) and wild type (triangles). Open and filled arrows indicate the data obtained at atmospheric [CO₂] (C _a) of 400 ppm and 800 ppm, respectively. The inset graph shows C _i/C _a ratios of the slac1 mutant (filled bars) and wild-type plants (open bars) at 400 ppm and 800 ppm C _a. Each point represents the mean values ±SD of three independent experiments conducted with different plants.

Fig. 8.

Carbon and nitrogen content (A) and C/N ratios (B) in the wild-type (WT) and slac1 mutant leaves. Total carbon and nitrogen contents are expressed on a leaf dry mass basis. Bars represent mean values ±SD of three independent experiments (n=3).