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.

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.