Complete chloroplast genome of the Malus baccata var. gracilis provides insights into the evolution and phylogeny of Malus species.

Malus baccata (L.) var. gracilis (Rehd.) has high ornamental value and breeding significance, and comparative chloroplast genome analysis was applied to facilitate genetic breeding for desired traits and resistance and provide insight into the phylogeny of this genus. Using data from whole-genome sequencing, a tetrameric chloroplast genome with a length of 159,992 bp and a total GC content of 36.56% was constructed. The M. baccata var. gracilis chloroplast genome consists of a large single-copy sequence (88,100 bp), a short single-copy region (19,186 bp), and two inverted repeat regions, IRa (26,353 bp) and IRb (26,353 bp). This chloroplast genome contains 112 annotated genes, including 79 protein-coding genes (nine multicopy), 29 tRNA genes (eight multicopy), and four rRNA genes (all multicopy). Calculating the relative synonymous codon usage revealed a total of 32 high-frequency codons, and the codons exhibited a biased usage pattern towards A/U as the ending nucleotide. Interspecific sequence comparison and boundary analysis revealed significant sequence variation in the vast single-copy region, as well as generally similar expansion and contraction of the SSC and IR regions for 10 analyzed Malus species. M. baccata var. gracilis and Malus hupehensis were grouped together into one branch based on phylogenetic analysis of chloroplast genome sequences. The chloroplast genome of Malus species provides an important foundation for species identification, genetic diversity analysis, and Malus chloroplast genetic engineering. Additionally, the results can facilitate the use of pendant traits to improve apple tree shape.

Apple-arbuscular mycorrhizal symbiosis confers resistance to Fusarium solani by inducing defense response and elevating nitrogen absorption.

Fusarium solani exerts detrimental effects on plant growth, which is one of the reasons for the incidence of apple replant disease. Arbuscular mycorrhizal fungi (AMF) enhance plant resistance to Fusarium wilt; however, the mechanism remains poorly understood. Therefore, the present study investigated the symbiosis between apple and AMF and explored the physiology, especially nitrate metabolism, antioxidant defense, and photosynthetic performance, when infected by F. solani. The experiment was carried out with four treatments, namely -AMF - F. solani, -AMF + F. solani, -AMF + F. solani, and + AMF + F. solani. In this study, the -AMF + F. solani treatment increased the activity of enzymes associated with nitrogen metabolism, such as the nitrate and nitrite reductases, in the apple root system. The +AMF + F. solani treatment showed higher antioxidant enzyme activities than the -AMF + F. solani by F. solani infection. The apple seedlings of the +AMF + F. solani treatment decreased reactive oxygen accumulation and reduced the oxidative damages triggered by F. solani infection. The improvement in antioxidant capacity due to the +AMF + F. solani treatment was closely associated with the upregulation of genes related to the antioxidant system. The F. solani infection greatly damaged the photosynthetic process, while the +AMF + F. solani treatment significantly improved it compared to the -AMF + F. solani treatment. In conclusion, the study demonstrated that the apple-AMF symbiosis plays an active role in regulating the resistance against F. solani infection by enhancing defense response and nitrogen metabolism.

Multi-omics analysis reveals the biosynthesis of flavonoids during the browning process of Malus sieversii explants.

Malus sieversii is a precious apple germplasm resource. Browning of explants is one of the most important factors limiting the survival rate of plant tissue culture. In order to explore the molecular mechanism of the browning degree of different strains of Malus sieversii, we compared the dynamic changes of Malus sieversii and Malus robusta Rehd. during the whole browning process using a multi-group method. A total of 44 048 differentially expressed genes (DEGs) were identified by transcriptome analysis on the DNBSEQ-T7 sequencing platform. KEGG enrichment analysis showed that the DEGs were significantly enriched in the flavonoid biosynthesis pathway. In addition, metabonomic analysis showed that (-)-epicatechin, astragalin, chrysin, irigenin, isoquercitrin, naringenin, neobavaisoflavone and prunin exhibited different degrees of free radical scavenging ability in the tissue culture browning process, and their accumulation in different varieties led to differences in the browning degree among varieties. Comprehensive transcriptome and metabonomics analysis of the data related to flavonoid biosynthesis showed that PAL, 4CL, F3H, CYP73A, CHS, CHI, ANS, DFR and PGT1 were the key genes for flavonoid accumulation during browning. In addition, WGCNA analysis revealed a strong correlation between the known flavonoid structure genes and the selected transcriptional genes. Protein interaction predictions demonstrated that 19 transcription factors (7 MYBs and 12 bHLHs) and 8 flavonoid structural genes had targeted relationships. The results show that the interspecific differential expression of flavonoid genes is the key influencing factor of the difference in browning degree between Malus sieversii and Malus robusta Rehd., providing a theoretical basis for further study on the regulation of flavonoid biosynthesis.

Auxin responsive factor MdARF17 promotes ethylene synthesis in apple fruits by activating MdERF003 expression.

KEY MESSAGE: Auxin (AUX) promotion of apple fruit ripening is ethylene-dependent, and AUX-MdARF17-MdERF003 plays a role in AUX-promoted ethylene synthesis in apple. Phytohormones play important roles in plant growth and fleshy fruit ripening, and the phytohormone auxin (AUX) can either promote or inhibit the ripening of fleshy fruits. Although AUX can influence ethylene (ETH) synthesis in apple (Malus domestica) fruits by affecting ETH system II, this mechanism remains to be explored. Here, we identified an ETH response factor (ERF) family transcription factor, MdERF003, whose expression could be activated by naphthalene acetic acid. The transient silencing of MdERF003 inhibited ETH synthesis in fruits, and MdERF003 could bind to the MdACS1 promoter. To explore the upstream target genes of MdERF003, we screened the MdARF family members by yeast one-hybrid assays of the MdERF003 promoter, and found that the transcription factor MdARF17, which showed AUX-promoted expression, could bind to the MdERF003 promoter and promote its expression. Finally, we silenced MdERF003 in apple fruits overexpressing MdARF17 and found that MdERF003 plays a role in MdARF17-promoted ETH synthesis in apple. Thus, AUX-MdARF17-MdERF003 promotes ETH synthesis in apple fruits.

Transcription factor MdbZIP44 targets the promoter of MdPPO2 to regulate browning in Malus domestica Borkh.

Apple (Malus domestica Borkh.) is among the most widely planted and economically valuable horticultural crops globally. Over time, the apple fruit's cut surface undergoes browning, and the degree of browning varies among different apple varieties. Browning not only affects the appearance of fruits but also adversely affects their taste and flavor. In the present study, we observed browning in different apple varieties over time and analyzed the expression of genes in the polyphenol oxidase gene family. The results indicated a strong correlation between the browning degree of the fruit and the relative expression of the polyphenol oxidase gene MdPPO2. With the MdPPO2 promoter as bait, the basic leucine zipper (bZIP) transcription factor MdbZIP44 was identified using the yeast single-hybrid screening method. Further investigation revealed that the overexpression of MdbZIP44 in 'Orin' callus could enhance the expression of MdPPO2 and promote browning of the callus. However, knocking out MdbZIP44 resulted in a callus with no apparent browning phenotype. In addition, our results confirmed the interaction between MdbZIP44 and MdbZIP11. In conclusion, the results indicated that MdbZIP44 can induce apple fruit browning by activating the MdPPO2 promoter. The results provide a theoretical basis for further clarifying the browning mechanism of apple fruit.

A Functional InDel in the WRKY10 Promoter Controls the Degree of Flesh Red Pigmentation in Apple.

MYB transcription factors have been linked to anthocyanin synthesis and various color phenotypes in plants. In apple, MYB10 confers a red-flesh phenotype due to a minisatellite insertion in its R6 promoter, but R6:MYB10 genotypes exhibit various degrees of red pigmentation in the flesh, suggesting the involvement of other genetic factors. Here, it is shown that MdWRKY10, a transcription factor identified via DNA pull-down trapping, binds to the promoter of MdMYB10 and activates its transcription. MdWRKY10 specifically interacts with the WDR protein MdTTG1 to join the apple MYB-bHLH-WDR (MBW) complex, which significantly enhances its transcriptional activation activity. A 163-bp InDel detected in the promoter region of the alleles of MdWRKY10 in a hybrid population of identical heterozygous genotypes regarding R6 by structural variation analysis, contains a typical W-box element that MdWRKY10 binds to for transactivation. This leads to increased transcript levels of MdWRKY10 and MdMYB10 and enhanced anthocyanin synthesis in the flesh, largely accounting for the various degrees of flesh red pigmentation in the R6 background. These findings reveal a novel regulatory role of the WRKY-containing protein complex in the formation of red flesh apple phenotypes and provide broader insights into the molecular mechanism governing anthocyanin synthesis in plants.