Photosynthesis in rice is increased by CRISPR/Cas9-mediated transformation of two truncated light-harvesting antenna

Abstract

Plants compete for light partly by over-producing chlorophyll in leaves. The resulting high light absorption is an effective strategy for out competing neighbors in mixed communities, but it prevents light transmission to lower leaves and limits photosynthesis in dense agricultural canopies. We used a CRISPR/Cas9-mediated approach to engineer rice plants with truncated light-harvesting antenna (TLA) via knockout mutations to individual antenna assembly component genes CpSRP43, CpSRP54a, and its paralog, CpSRP54b. We compared the photosynthetic contributions of these components in rice by studying the growth rates of whole plants, quantum yield of photosynthesis, chlorophyll density and distribution, and phenotypic abnormalities. Additionally, we investigated a Poales-specific duplication of CpSRP54. The Poales are an important family that includes staple crops such as rice, wheat, corn, millet, and sorghum. Mutations in any of these three genes involved in antenna assembly decreased chlorophyll content and light absorption and increased photosynthesis per photon absorbed (quantum yield). These results have significant implications for the improvement of high leaf-area-index crop monocultures.

Keywords: CRISPR/Cas9; chlorophyll; photosynthesis; rice; truncated light antenna.

Figure 1

TLA3 mutants have reduced height and chlorophyll content. (A) Measurements of plant height for three independent CRISPR/Cas9-generated TLA3 mutant lines as compared to Kitaake wild type (WT). (B) Measurements of chlorophyll content for these three mutants, as measured using a SPAD meter. (C) Representative images of the homozygous parental line of each of the three mutant lineages characterized in (A) and (B), demonstrating reduced stature and a pale green leaf phenotype. (D) Protein domain model of validated CRISPR/Cas9-mediated TLA3 mutations generated using two sgRNAs indicating the relative position and size in base pairs of large deletions within CpSRP43. Frameshift mutations occurred in the middle of chromodomain 1 in each line. CTP, Chloroplast targeting peptide.

Figure 2

A duplication of CpSRP54 occurred in the Poales lineage. (A) Protein domain model of CpSRP54 with CTP (chloroplast targeting peptide). (B) Phylogenetic tree of CpSRP54 showing a duplication event in the Poales lineage to produce CpSRP54a and CpSRP54b Created with BioRender.com.

Figure 3

CpSRP54a and CpSRP54b both contribute to leaf chlorophyll accumulation. (A, B, C) Representative images of several mutant TLA4 (A, B) and TLA4L (C) lineages demonstrate reduced plant height and a pale green leaf phenotype as compared to WT. TLA4L mutants display a particularly severe phenotype. (D) Plant height of representative TLA4 and TLA4L mutant individuals from independently CRISPR/Cas9-generated lineages. Blue bars represent TLA4 mutants, yellow bars represent TLA4L mutants, and gray bars represent null-segregants (n). Null-segregants show restored WT phenotypes. (E) Measurements of chlorophyll content for TLA4, TLA4L, and TLA4L(n) individuals as measured using a SPAD meter. Blue bars represent TLA4 mutants, yellow bars represent TLA4L mutants, and gray bars represent null-segregants (n). (F) Protein domain model with CTP (Chloroplast targeting peptide) of validated CRISPR/Cas9-mediated TLA4 mutations generated using two sgRNAs indicating the relative position and size in base pairs of indels within CpSRP54a. All three mutant lines have multiple indels.

Figure 4

TLA mutants have reduced chlorophyll content and height. (A) Plots of chlorophyll content, (y-axis) across time (x-axis) for WT and all mutant lines demonstrate that chlorophyll content increases over time for all lines. (B) Most mutants are reduced in height compared to the wild-type.

Figure 5

Chlorophyll density is positively correlated with leaf photon absorbance fraction among both wild-type (WT, green) and TLA mutant (black) lines. The high correlation (R² = 0.80) of this relationship implies similar chlorophyll structures and orientations between TLA mutant and WT lines.

Figure 6

Photosynthetic rates in TLA mutants. The photosynthetic rates for mutant lines are increased compared to the Kitaake wild-type (WT) when accounting for the photosynthetic rate per photon absorbed at 400 ppm CO₂ (A, B). The relationship is the same at 1200 ppm CO₂ (C, D). The photosynthetic rate of 2-TLA3 8-2-3-4 (pink) is often less than the photosynthetic rate Kitaake WT (green).