Apple Ripening Is Controlled by a NAC Transcription Factor

Abstract

Softening is a hallmark of ripening in fleshy fruits, and has both desirable and undesirable implications for texture and postharvest stability. Accordingly, the timing and extent of pre-harvest ripening and associated textural changes following harvest are key targets for improving fruit quality through breeding. Previously, we identified a large effect locus associated with harvest date and firmness in apple (Malus domestica) using genome-wide association studies (GWAS). Here, we present additional evidence that polymorphisms in or around a transcription factor gene, NAC18.1, may cause variation in these traits. First, we confirmed our previous findings with new phenotype and genotype data from ∼800 apple accessions. In this population, we compared a genetic marker within NAC18.1 to markers targeting three other firmness-related genes currently used by breeders (ACS1, ACO1, and PG1), and found that the NAC18.1 marker was the strongest predictor of both firmness at harvest and firmness after 3 months of cold storage. By sequencing NAC18.1 across 18 accessions, we revealed two predominant haplotypes containing the single nucleotide polymorphism (SNP) previously identified using GWAS, as well as dozens of additional SNPs and indels in both the coding and promoter sequences. NAC18.1 encodes a protein that is orthogolous to the NON-RIPENING (NOR) transcription factor, a regulator of ripening in tomato (Solanum lycopersicum). We introduced both NAC18.1 transgene haplotypes into the tomato nor mutant and showed that both haplotypes complement the nor ripening deficiency. Taken together, these results indicate that polymorphisms in NAC18.1 may underlie substantial variation in apple firmness through modulation of a conserved ripening program.

Keywords: NAC-domain transcription factor; apple; apple texture; fruit ripening; marker-assisted selection.

FIGURE 1

The utility of four genetic markers for predicting harvest date and firmness-related apple phenotypes. For each trait, the percent variance explained for each of the four markers is shown in cases where a significant effect was detected from a type 2 ANOVA. A type 2 ANOVA was also performed with the four genetic markers and harvest date as a co-factor (Supplementary Figure 3).

FIGURE 2

Genotypes of four texture-related genetic markers across the top 9 apple cultivars sold in the United States (U.S. Apple Association). The “desirable” allele for each marker is defined as the allele that has been reported to lead to firmer apple texture.

FIGURE 3

Polymorphisms within the NAC18.1 gene. Maximum likelihood phylogenetic tree of 24 NAC18.1 sequences from 18 apple cultivars and the reference genome (GDDH13 v1.1) sequences of NAC18.1 (Md03g1222600) and its closest homolog (Md11g1239900). Amino acid sequence alignment of the N-terminal region of NAC18.1, illustrating additional variation in the coding sequence in strong LD with the D5Y variant. The complete amino acid sequence alignment is provided in Supplementary Figure 6.

FIGURE 4

Transgenic complementation of the tomato nor mutant using NAC18.1 transgene restores ripening. (A) Mature fruit of the tomato nor mutant and four independent T1 transgenic lines constitutively expressing either of two alleles of the NAC18.1 transgene, NAC18.1^C and NAC18.1^A and isogenic WT control (cv. Ailsa Craig). (B) Quantitative colorimetry of the fruit surface of nor, the transgenic fruit and WT fruit. The a* component (green-red axis) is shown, with the mean ± SE superimposed in black over the raw values (N = 5) in a color approximating the external color of the fruit. Genotypes not sharing a letter (a-e) are statistically distinct by one-way ANOVA and Tukey’s HSD test (p < 0.05).