Fine Mapping of Carbon Assimilation Rate 8, a Quantitative Trait Locus for Flag Leaf Nitrogen Content, Stomatal Conductance and Photosynthesis in Rice

Abstract

Increasing the rate of leaf photosynthesis is one important approach for increasing grain yield in rice (Oryza sativa). Exploiting the natural variation in CO₂ assimilation rate (A) between rice cultivars using quantitative genetics is one promising means to identify genes contributing to higher photosynthesis. In this study, we determined precise location of Carbon Assimilation Rate 8 (CAR8) by crossing a high-yielding indica cultivar with a Japanese commercial cultivar. Fine mapping suggested that CAR8 encodes a putative Heme Activator Protein 3 (OsHAP3) subunit of a CCAAT-box-binding transcription factor called OsHAP3H. Sequencing analysis revealed that the indica allele of CAR8 has a 1-bp deletion at 322 bp from the start codon, resulting in a truncated protein of 125 amino acids. In addition, CAR8 is identical to DTH8/Ghd8/LHD1, which was reported to control rice flowering date. The increase of A is largely due to an increase of RuBP regeneration rate via increased leaf nitrogen content, and partially explained by reduced stomatal limitation via increased stomatal conductance relative to A. This allele also increases hydraulic conductivity, which would promote higher stomatal conductance. This indicates that CAR8 affects multiple physiological aspects relating to photosynthesis. The detailed analysis of molecular functions of CAR8 would help to understand the association between photosynthesis and flowering and demonstrate specific genetic mechanisms that can be exploited to improve photosynthesis in rice and potentially other crops.

Keywords: Oryza sativa; RuBP regeneration; leaf nitrogen content; photosynthesis; quantitative trait locus; stomatal conductance.

Figure 1

Substitution mapping of CAR8 using homozygous recombinant lines (BC₅F₆). Molecular markers are shown from the short arm (left) to the long arm (right) of chromosome 8. White segments, homozygous for “Koshihikari” alleles; black segments, homozygous for “Habataki” alleles. Field-grown plants were used. CO₂ assimilation rate of flag leaves was measured at an ambient CO₂ concentration of 370 μmol mol⁻¹, a PPFD of 2000 μmol photons m⁻² s⁻¹, a leaf-to-air vapor pressure difference of 1.3–1.6 kPa, and an air temperature of 30°C. Black bars in graphs indicate significant difference from “Koshihikari” at the 5% level by Dunnett's test. Error bars indicate SD (n = 3).

Figure 2

Fine mapping of CAR8 using homozygous recombinant lines (BC₅F₇). Molecular markers are shown from the short arm (left) to the long arm (right) of chromosome 8. White segments, homozygous for “Koshihikari” alleles; black segments, homozygous for “Habataki” alleles. Field-grown plants were used. CO₂ assimilation rate of flag leaves was measured at an ambient CO₂ concentration of 370 μmol mol⁻¹, a PPFD of 2000 μmol photons m⁻² s⁻¹, a leaf-to-air vapor pressure difference of 1.3–1.6 kPa, and an air temperature of 30°C. Black bars in graphs indicate significant difference from “Koshihikari” at the 5% level by Dunnett's test. Error bars indicate SD (n = 3).

Figure 3

Map-based cloning of CAR8. (A) Fine mapping of CAR8. The number of recombinants between molecular markers is indicated below the each line. (B) Structure of CAR8. The exon is shown as a gray box. Vertical lines without labels represent single-base substitutions between “Koshihikari” and “Habataki.” Small open boxes represent deletions. (C) Alignment of CAR8 amino acid sequences.

Figure 4

Response of CO₂ assimilation rate of flag leaves at full heading to intercellular CO₂ concentration. Plants of “Koshihikari” (circles), NIL(CAR8) (triangles), and “Habataki” (squares) were grown outdoors in 12-L pots. Leaf gas exchange was measured at a PPFD of 2000 μmol photons m⁻² s⁻¹ and an air temperature of 30°C. CO₂ assimilation rate limited by RuBP carboxylation (solid line) and CO₂ assimilation rate limited by RuBP regeneration (dotted line) were shown. Curve fitting was described in the Materials and Methods Section. The straight lines represent the measurement at ambient CO₂ concentration of 370 μmol mol⁻¹. Error bars indicate SD (n = 6).

Figure 5

Stomatal density (A) and stomatal pore length (B) in flag leaves of field-grown plants at full heading. Error bars indicate SD (n = 3). Values followed by the same letters indicate no significant difference among rice lines at P < 0.05 by LSD test.