Apple replant disease (ARD) is a worldwide problem that threatens the industry. However, the genetic mechanism underlying plant disease resistance against ARD remains unclear. In this study, a negative regulatory microRNA in Malus domestica, mdm-miR397b \, and its direct target MdLAC7b (Laccase) was selected for examination based on our previous small RNA and degradome sequencing results. Overexpressing the mdm-miR397b-MdLAC7b module altered the lignin deposition and JA contents in apple roots, which also led to increased resistance to Fusarium solani. Additionally, Y1H library screening using mdm-miR397b promoter recombinants identified a transcription factor, MdERF61, that represses mdm-miR397b transcriptional activity by directly binding to two GCC-boxes in the mdm-miR397b promoter. In summary, our results suggest that the MdERF61-mdm-miR397b-MdLAC7b module plays a crucial role in apple resistance to F. solani and offers insights for enhancing plant resistance to soilborne diseases in apple.

Apple is a major fruit crop grown worldwide and provides humans with an essential diet and health benefits. One of the health benefits is related to the accumulation of fruit anthocyanin, which also provides fruit with an attractive red colour. It is known that an MdMYB10 allele containing a transposable element (TE) insertion in its promoter (termed Red-TE allele) underlies anthocyanin accumulation and red colouration in the fruit skin of cultivated apples. However, the distribution of this Red-TE allele in wider Malus germplasm accessions is not clear. In this study, we showed that MdMYB10 RNA in fruit skin was specifically expressed from the Red-TE allele by using allele-specific expression analysis of transcriptome data. Apple cultivars and hybrids with homozygous Red-TE alleles showed stronger red colour than those with heterozygous alleles after analysing 65 cultivars and 337 hybrids. Furthermore, both hetero- and homozygous plants growing in the same high-temperature conditions had different colourations. However, the Red-TE allele was not detected in 16 wild apple accessions showing red skin, indicating that the red skin colour of these wild apples was not conferred by the Red-TE allele. These findings provide guidance for selecting cultivars able to develop consistent red colouration under high growth temperature conditions and open the opportunity for identifying novel genetic variants underpinning fruit red colouration in wild apple species.

Fruit domestication has long aimed to reduce bitterness, yet the molecular mechanisms behind this trait remain only partially understood. Wild apples and pears naturally accumulate high levels of bitter proanthocyanidins (PAs), also known as condensed tannins. In this study, a convergent domestication process was identified in both fruits, involving the selection of weak alleles of MYB transcription factors that regulate PA biosynthesis. In apples, domestication targeted the MYB-Tannin-Tamer (MdMYBTT) gene. A 411-base pair transposable element inserted into the third exon of this gene in cultivated varieties produced a truncated, non-functional protein unable to activate the PA biosynthetic gene Anthocyanidin Reductase 1 (ANR1). The resulting mdmybtt allele led to reduced PA levels and was fixed in domesticated apples through positive selection. Likewise, in pears, a 57-base pair insertion in the promoter of the MYBPA1 gene suppressed its expression in cultivated varieties, limiting PA production. This insertion created the mybpa1 allele, which was similarly fixed during pear domestication. These findings highlight a shared evolutionary strategy to reduce fruit bitterness by selecting mutations that suppress PA synthesis. These findings offer valuable insights into the molecular basis of domestication and inform breeding efforts to optimize both flavor and nutritional quality.

Flavonols contribute significantly to both plant defense and human health. In apple (Malus domestica), flavonols accumulate at high levels in leaves and fruit skin but are present at low levels in the fruit flesh, the primary tissue consumed by humans. Enhancing flavonol content in the flesh requires a deeper understanding of the underlying molecular mechanisms. In this study, we investigated the expression patterns and functional roles of four apple flavonol synthase genes (MdFLS1, 2, 3, 4). MdFLS1, 2, 3 exhibited higher expression in fruit skin than in flesh, while MdFLS4 was not expressed in either tissue. Transient overexpression of each gene promoted flavonol accumulation in apple fruit flesh, indicating that all four genes encode functional flavonol synthases. This enzyme activity was further confirmed through stable overexpression in transgenic Arabidopsis and tomato plants. Notably, light exposure of apple fruit flesh enhanced the expression of MdFLS1 and MdFLS2 and increased flavonol accumulation. These findings advance our understanding of flavonol biosynthesis in apple.

Fruit firmness is a major determinant of apple quality and shelf life. It is known to be regulated by ethylene signalling, however, the function of auxin in it is not known. Here, we identified MdIAA14b, an auxin-responsive gene, as a key regulator of apple fruit firmness. MdIAA14b showed allele-specific expression (ASE) across multiple apple cultivars and developmental stages as revealed by RNA-seq analysis. Relative to the low expression allele, the promoter of the high expression allele contained two insertions of 9 bp and 1636 bp. The 9-bp insertion generated an ARF-binding site that enabled activation by the transcription factor MdARF1. The 1636-bp insert, identified as a transposable element (TE), disrupted and reduced the expression of a long non-coding RNA (lncRNA_iaa14b), which exhibited an inverse expression pattern relative to MdIAA14b. Apple cultivars homozygous for the high-expression allele displayed increased cell density, smaller cell size, and firmer fruit, whereas those homozygous for the low-expression allele had larger cells and softer fruit. MdIAA14b overexpression in tomato increased cell number and enhanced fruit firmness. Furthermore, MdIAA14b overexpression altered the expression of auxin-responsive genes, including members of ARF, SAUR, and GH3 families, which may relate to increased cell division. Together, these findings reveal that naturally occurring cis-regulatory variations control MdIAA14b expression and, consequently, apple fruit firmness. This work provides a molecular framework for improving fruit texture and highlights the role of cis-regulatory variation in allele-specific gene regulation in perennial fruit crops.