Embracing native diversity to enhance the maximum quantum efficiency of photosystem II in maize

Abstract

The sustainability of maize cultivation would benefit tremendously from early sowing, but is hampered by low temperatures during early development in temperate climates. We show that allelic variation within the gene encoding subunit M of the NADH-dehydrogenase-like (NDH) complex (ndhm1) in a European maize landrace affects several quantitative traits that are relevant during early development in cold climates through NDH-mediated cyclic electron transport around photosystem I, a process crucial for photosynthesis and photoprotection. Beginning with a genome-wide association study for maximum potential quantum yield of photosystem II in dark-adapted leaves (Fv/Fm), we capitalized on the large phenotypic effects of a hAT transposon insertion in ndhm1 on multiple quantitative traits (early plant height [EPH], Fv/Fm, chlorophyll content, and cold tolerance) caused by the reduced protein levels of NDHM and associated NDH components. Analysis of the ndhm1 native allelic series revealed a rare allele of ndhm1 that is associated with small albeit significant improvements of Fv/Fm, photosystem II efficiency in light-adapted leaves (ΦPSII), and EPH compared with common alleles. Our work showcases the extraction of favorable alleles from locally adapted landraces, offering an efficient strategy for broadening the genetic variation of elite germplasm by breeding or genome editing.

Figure 1.

Fine-mapping of a QTL associated with EPH and Fv/Fm in growth stage V4. A) Scheme for fine-mapping of a QTL for EPH and Fv/Fm on maize chromosome 2. Genomic fragments unique to KE0678 are colored red. B) Top: phenotypic segregation during vegetative growth of F_2:3 RILs linked to the QTL on chromosome 2. Bottom: HIF3A and HIF3B in a growth chamber experiment in optimal conditions. C) Genetic composition of key HIFs that enabled fine-mapping to a 314 kb genomic region. Vertical lines represent the genomic positions of KASP markers from Supplementary Table S5. Genomic fragments inherited from KE0678 are colored red. D, E) Phenotyping of key HIFs for EPH (D) and Fv/Fm (E) in a growth chamber. Bars show means ± SE (n = 3 to 6 plants) and dots observations from single plants. Significant differences (t-test) are marked with stars. *** P < 0.001, ** P < 0.01. Figure 1A was created with BioRender.com.

Figure 2.

Candidate gene analysis in fine-mapped region. A) Pairwise sequence alignment (sequence identity > 99%, length > 1 kb) of the fine-mapped region in KE0482 and KE0678. Black dots: Flanking markers of the fine-mapped region. Orange: alignment on forward strand. Green: alignment on reverse strand. B) Genes in fine-mapped region annotated in B73_AGPv4 reference genome. Red: Zm00001d002815 (ndhm1) is polymorphic between KE0482 and KE0678. C)  Ndhm1 alleles and their polymorphisms in comparison to B73 reference sequence of KE0482 and KE0678. Gray: 5′ and 3′ UTR, the 3′UTR is indicated by a triangle at the end. Black: coding sequence. In ndhm1A-2, a hAT transposon, flanked by 2 terminal inverted repeat (TIR) motifs (purple, orange) is inserted in the intron (red triangle). The 2 copies of ndhm1 that are located next to each other on chromosome 2 in Kemater lines are surrounded by a blue (KE0482) and red (KE0678) box, respectively. D) Transcripts derived from ndhm1A-1 and ndhm1A-2. E, F) Relative transcript (E) and protein (F) levels of ndhm1 in KE0482, KE0678, HIF1A, HIF1B, HIF3A, and HIF3B. Bars show means ± SE (n = 3 plants). Significant differences (t-test) are marked with stars. ***P < 0.001, **P < 0.01, . P < 0.1. RQ: relative quantity. Figure 2D was created with BioRender.com.

Figure 3.

Impact of a hAT transposon insertion in ndhm1 (ndhm1A-2) on NDH components and photosynthetic parameters. A) Proteome data of 6 genotypes (KE0482, KE0678, HIF1A, HIF1B, HIF3A, and HIF3B) in optimal conditions (n = 3 plants/genotype). Grouping was done by ndhm1 allele (ndhm1A-1 = KE0482 allele or ndhm1A-2 = KE0678 allele). One dot is one protein, filled dots are proteins taking part in CET. Cutoff for differential expression: absolute log2(FC: fold change) > 1; false discovery rate < 5%. Proteins that are differentially expressed and involved in CET are labeled in the volcano plots. B) Chlorophyll fluorescence induction curves of dark-adapted plants. The small boxes on the right side of the plots show a zoom in the F0-rise indicative for CET after turning off a weak AL. C) Relationship between assimilation rate (A) and intracellular CO₂ (C_i) concentration of KE0482, KE0678, HIF1A, HIF1B, HIF3A, and HIF3B. Adjusted means were calculated for the 2 different ndhm1 alleles considering the genomic background as random factor in a mixed linear model for each tested external CO₂ concentration separately. Dots are adjusted means ± SE. Significance of difference in assimilation rate was assessed by Wald tests for each external CO₂ concentration independently. *** P < 0.001, ** P < 0.01.

Figure 4.

Phenotypic analysis of a ndhm1 Mu-insertion mutant. A) Structure of ndhm1 allele of Flint line F7 and derived Mu-insertion mutant F-0123 from the BonnMu collection compared with B73_AGPv4 reference sequence. The position of the Mu element (red triangle) 24 bp upstream of the start codon was determined by Sanger sequencing. Gray: 5′ and 3′ UTR, the 3′ UTR is indicated by a triangle at the end. Black: coding sequence. B) Relative differences of ndhm1 transcript levels for parents of biparental mapping population (KE0482: n = 10; KE0678: n = 9) and F7 (n = 8) and F-0123 (n = 9). RQ: relative quantity. C, D) Phenotypic differences for target traits EPH (C) and Fv/Fm (D) between F7 (n = 10) and F-0123 (n = 14) and the parents of the biparental population (KE0482: n = 12; KE0678: n = 12). Bars show means ± SE. Significant differences (t-test) are marked with stars. *** P < 0.001, ** P < 0.01, * P < 0.05. E, F). Chlorophyll induction curves for F7 and F-0123, respectively. The small boxes on the right side of the plots show a zoom in the F0-rise indicative for CET after turning off a weak AL. A. u: arbitrary units; S: seconds.

Figure 5.

Analysis of allelic diversity of ndhm1 in Kemater DH lines. A to D) Phenotypic data from 471 Kemater lines with high-density genotypic and phenotypic data grouped by their haplotype allele of the lead haplotype from GWAS. Blue: haplotype of KE0482, which carries a second copy of ndhm1 (ndhm1B) and no transposon insertion in ndhm1 (ndhm1A-1). Red: haplotype of KE0678, which carries a second copy of ndhm1 (ndhm1B) and a transposon insertion in ndhm1 (ndhm1A-2). Each dot represents the adjusted mean of one Kemater line in up to 11 combinations of environments and years for EPH A), Fv/Fm B), chlorophyll content C) and final plant height D). Bars show means ± SE. Significant differences of alleles are indicated by letters (LSD, least significant differences; P < 0.05). E to H) Subset of 27 Kemater lines that was selected to be balanced for different ndhm1 haplotypes for in-depth molecular characterization. Lines were genotyped for the presence or absence of the transposon insertion in ndhm1A-2 and presence/absence of ndhm1B and phenotyped for their differences in ndhm1 transcript levels E) and for target traits EPH F), Fv/Fm G) and chlorophyll content H). Bars show means ± SE. Significant differences (LSD; P < 0.05) are indicated by letters. RQ: relative quantity.

Figure 6.

Association of lead haplotype alleles from a GWAS of KE0482 and KE0678 with a duplication of ndhm1. Normalized read-depth of high-coverage whole-genome sequencing data aligned against B73_AGPv4 reference genome grouped by allele of the haplotype with lowest P-value from GWAS. Each line represents a Kemater line, and each dot represents a single base in B73v4 reference. Hap1 and Hap2 are grouped together in the group “Other Haplotype”. Red: Normalized read depth > 2. Below the read depth graphs the relative positions of gene models of ndhm1-A1 and ndhm1-B are indicated. Gray: 5′ and 3′ UTR, the 3′ UTR is indicated by a triangle at the end. Black: coding sequence.

Figure 7.

Effect of ndhm1 allele after severe cold treatment on photosynthetic parameters and leaf proteome. A, B) Phenotypic differences of DH lines with different ndhm1 alleles in optimal conditions and 3 d after recovery from severe cold stress. One point is the mean of 3 to 6 biological replicates of a Kemater DH line. Bars show means ± SE. Significant differences (least significant differences; P < 0.05) are indicated by letters. C, D) Difference of protein levels depending on a cold treatment (4 °C/2 °C, 48 h) of genotypes with ndhm1A-1 (C, KE0482, HIF1A, HIF3A) or ndhm1A-2 (D, KE0678, HIF1B, HIF3B) alleles. One dot is one protein, filled dots are proteins with oxidoreductase activity (GO:0016701). Cutoff for differential expression: absolute log2(FC: fold change) > 1; false discovery rate < 5%. Differentially expressed proteins with oxidoreductase activity are labeled in the plots. G) Lesions on leaves of Kemater lines KE0482 and KE0678 after cold treatment.