MdARF3 switches the lateral root elongation to regulate dwarfing in apple plants.

Apple rootstock dwarfing and dense planting are common practices in apple farming. However, the dwarfing mechanisms are not understood. In our study, the expression of MdARF3 in the root system of dwarfing rootstock 'M9' was lower than in the vigorous rootstock from Malus micromalus due to the deletion of the WUSATAg element in the promoter of the 'M9' genotype. Notably, this deletion variation was significantly associated with dwarfing rootstocks. Subsequently, transgenic tobacco (Nicotiana tabacum) cv. Xanthi was generated with the ARF3 promoter from 'M9' and M. micromalus genotypes. The transgenic apple with 35S::MdARF3 was also obtained. The transgenic tobacco and apple with the highly expressed ARF3 had a longer root system and a higher plant height phenotype. Furthermore, the yeast one-hybrid, luciferase, electrophoretic mobility shift assays, and Chip-qPCR identified MdWOX4-1 in apples that interacted with the pMm-ARF3 promoter but not the pM9-ARF3 promoter. Notably, MdWOX4-1 significantly increased the transcriptional activity of MdARF3 and MdLBD16-2. However, MdARF3 significantly decreased the transcriptional activity of MdLBD16-2. Further analysis revealed that MdARF3 and MdLBD16-2 were temporally expressed during different stages of lateral root development. pMdLBD16-2 was mainly expressed during the early stage of lateral root development, which promoted lateral root production. On the contrary, pMmARF3 was expressed during the late stage of lateral root development to promote elongation. The findings in our study will shed light on the genetic causes of apple plant dwarfism and provide strategies for molecular breeding of dwarfing apple rootstocks.

miR164-MhNAC1 regulates apple root nitrogen uptake under low nitrogen stress.

Nitrogen is an essential nutrient for plant growth and serves as a signaling molecule to regulate gene expression inducing physiological, growth and developmental responses. An excess or deficiency of nitrogen may have adverse effects on plants. Studying nitrogen uptake will help us understand the molecular mechanisms of utilization for targeted molecular breeding. Here, we identified and functionally validated an NAC (NAM-ATAF1/2-CUC2) transcription factor based on the transcriptomes of two apple rootstocks with different nitrogen uptake efficiency. NAC1, a target gene of miR164, directly regulates the expression of the high-affinity nitrate transporter (MhNRT2.4) and citric acid transporter (MhMATE), affecting root nitrogen uptake. To examine the role of MhNAC1 in nitrogen uptake, we produced transgenic lines that overexpressed or silenced MhNAC1. Silencing MhNAC1 promoted nitrogen uptake and citric acid secretion in roots, and enhanced plant tolerance to low nitrogen conditions, while overexpression of MhNAC1 or silencing miR164 had the opposite effect. This study not only revealed the role of the miR164-MhNAC1 module in nitrogen uptake in apple rootstocks but also confirmed that citric acid secretion in roots affected nitrogen uptake, which provides a research basis for efficient nitrogen utilization and molecular breeding in apple.

Mitogen-activated protein kinase MxMPK3-2 mediated phosphorylation of MxZR3.1 participates in regulating iron homoeostasis in apple rootstocks.

The micronutrient iron plays a crucial role in the growth and development of plants, necessitating meticulous regulation for its absorption by plants. Prior research has demonstrated that the transcription factor MxZR3.1 restricts iron absorption in apple rootstocks; however, the precise mechanism by which MxZR3.1 contributes to the regulation of iron homoeostasis in apple rootstocks remains unexplored. Here, MxMPK3-2, a protein kinase, was discovered to interact with MxZR3.1. Y2H, bimolecular fluorescence complementation and pull down experiments were used to confirm the interaction. Phosphorylation and cell semi-degradation tests have shown that MxZR3.1 can be used as a substrate of MxMPK3-2, which leads to the MxZR3.1 protein being more stable. In addition, through tobacco transient transformation (LUC and GUS) experiments, it was confirmed that MxZR3.1 significantly inhibited the activity of the MxHA2 promoter, while MxMPK3-2 mediated phosphorylation at the Ser94 site of MxZR3.1 further inhibited the activity of the MxHA2 promoter. It is tightly controlled to absorb iron during normal growth and development of apple rootstocks due to the regulatory effect of the MxMPK3-2-MxZR3.1 module on MxHA2 transcription level. Consequently, this research has revealed the molecular basis of how the MxMPK3-2-MxZR3.1 module in apple rootstocks controls iron homoeostasis by regulating the MxHA2 promoter's activity.

Near-gapless and haplotype-resolved apple genomes provide insights into the genetic basis of rootstock-induced dwarfing.

Dwarfing rootstocks have transformed the production of cultivated apples; however, the genetic basis of rootstock-induced dwarfing remains largely unclear. We have assembled chromosome-level, near-gapless and haplotype-resolved genomes for the popular dwarfing rootstock 'M9', the semi-vigorous rootstock 'MM106' and 'Fuji', one of the most commonly grown apple cultivars. The apple orthologue of auxin response factor 3 (MdARF3) is in the Dw1 region of 'M9', the major locus for rootstock-induced dwarfing. Comparing 'M9' and 'MM106' genomes revealed a 9,723-bp allele-specific long terminal repeat retrotransposon/gypsy insertion, DwTE, located upstream of MdARF3. DwTE is cosegregated with the dwarfing trait in two segregating populations, suggesting its prospective utility in future dwarfing rootstock breeding. In addition, our pipeline discovered mobile mRNAs that may contribute to the development of dwarfed scion architecture. Our research provides valuable genomic resources and applicable methodology, which have the potential to accelerate breeding dwarfing rootstocks for apple and other perennial woody fruit trees.

Genetic variations in MdSAUR36 participate in the negative regulation of mesocarp cell division and fruit size in Malus species.

Final fruit size of apple (Malus domestica) cultivars is related to both mesocarp cell division and cell expansion during fruit growth, but it is unclear whether the cell division and/or cell enlargement determine most of the differences in fruit size between Malus species. In this study, by using an interspecific hybrid population between Malus asiatica "Zisai Pearl" and Malus domestica cultivar "Red Fuji," we found that the mesocarp cell number was the main causal factor of diversity in fruit size between Malus species. Rapid increase in mesocarp cell number occurred prior to 28 days after anthesis (DAA), while cell size increased gradually after 28 DAA until fruit ripening. Six candidate genes related to auxin signaling or cell cycle were predicted by combining the RNA-seq data and previous QTL data for fruit weight. Two InDels and 10 SNPs in the promoter of a small auxin upregulated RNA gene MdSAUR36 in Zisai Pearl led to a lower promoter activity than that of Red Fuji. One non-synonymous SNP G/T at 379 bp downstream of the ATG codon of MdSAUR36, which was heterozygous in Zisai Pearl, exerted significant genotype effects on fruit weight, length, and width. Transgenic apple calli by over-expressing or RNAi MdSAUR36 confirmed that MdSAUR36 participated in the negative regulation of mesocarp cell division and thus apple fruit size. These results could provide new insights in the molecular mechanism of small fruit size in Malus accession and be potentially used in molecular assisted breeding via interspecific hybridization. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-024-01441-4.

Pan-genome analysis of 13 Malus accessions reveals structural and sequence variations associated with fruit traits.

Structural variations (SVs) and copy number variations (CNVs) contribute to trait variations in fleshy-fruited species. Here, we assemble 10 genomes of genetically diverse Malus accessions, including the ever-green cultivar 'Granny Smith' and the widely cultivated cultivar 'Red Fuji'. Combining with three previously reported genomes, we assemble the pan-genome of Malus species and identify 20,220 CNVs and 317,393 SVs. We also observe CNVs that are positively correlated with expression levels of the genes they are associated with. Furthermore, we show that the noncoding RNA generated from a 209 bp insertion in the intron of mitogen-activated protein kinase homology encoding gene, MMK2, regulates the gene expression and affects fruit coloration. Moreover, we identify overlapping SVs associated with fruit quality and biotic resistance. This pan-genome uncovers possible contributions of CNVs to gene expression and highlights the role of SVs in apple domestication and economically important traits.

MdGRF11-MdARF19-2 module acts as a positive regulator of drought resistance in apple rootstock.

14-3-3 proteins play an important role in the response of plants to drought resistance. In this study, 14-3-3 protein MdGRF11 was cloned from Malus xiaojinensis, and its positive regulation of drought resistance was verified using Orin calli and M. xiaojinensis plants. The transcription factor MdARF19-2 was further screened for interaction with this protein in vitro and in vivo. We also conducted experiments using Orin calli and found that the overexpression of MdARF19-2 decreased the level of reactive oxygen species (ROS) and increased the activity of enzymes that scavenge ROS in plant materials. This indicates that MdARF19-2 is a positive regulator in the drought resistance of plants. The drought tolerance was further improved by the overexpression of both MdGRF11 and MdARF19-2 in the calli. In addition, we examined several genes related to ROS scavenging with auxin response factor binding elements in their promoters and found that their level of expression was regulated by the MdGRF11-MdARF19-2 module. In conclusion, the enhancement of plant drought resistance by MdGRF11 could be owing to its accumulation at the protein level in response to drought, which then combined with MdARF19-2, affecting the expression of MdARF19-2 downstream genes. Thus, it scavenges ROS, which ultimately improves the resistance of plant to drought stress.

Genome-wide analysis of the MdZR gene family revealed MdZR2.2-induced salt and drought stress tolerance in apple rootstock.

The DNL-type zinc finger protein constitutes a zinc ribbon protein (ZR) family, which belongs to a branch of zinc finger protein and plays an essential role in response to abiotic stress. Here, we identified six apple (Malus domestica) MdZR genes. Based on their phylogenetic relationship and gene structure, the MdZR genes were divided into three categories, including MdZR1, MdZR2, and MdZR3. Subcellular results showed that the MdZRs are located on the nuclear and membrane. The transcriptome data showed that MdZR2.2 is expressed in various tissues. The expression analysis results showed that MdZR2.2 was significantly upregulated under salt and drought treatments. Thus, we selected MdZR2.2 for further research. Overexpression of MdZR2.2 in apple callus improved their tolerance to drought and salt stress and ability to scavenge reactive oxygen species (ROS). In contrast, transgenic apple roots with silenced MdZR2.2 grew more poorly than the wild type when subjected to salt and drought stress, which reduced their ability to scavenge ROS. To our knowledge, this is the first study to analyze the MdZR protein family. This study identified a gene that responds to drought and salt stress. Our findings lay a foundation for a comprehensive analysis of the MdZR family members.

MxMPK6-2-mediated phosphorylation enhances the response of apple rootstocks to Fe deficiency by activating PM H+ -ATPase MxHA2.

Iron (Fe) deficiency significantly affects the growth and development, fruit yield and quality of apples. Apple roots respond to Fe deficiency stress by promoting H+ secretion, which acidifies the soil. In this study, the plasma membrane (PM) H+ -ATPase MxHA2 promoted H+ secretion and root acidification of apple rootstocks under Fe deficiency stress. H+ -ATPase MxHA2 is upregulated in Fe-efficient apple rootstock of Malus xiaojinensis at the transcription level. Fe deficiency also induced kinase MxMPK6-2, a positive regulator in Fe absorption that can interact with MxHA2. However, the mechanism involving these two factors under Fe deficiency stress is unclear. MxMPK6-2 overexpression in apple roots positively regulated PM H+ -ATPase activity, thus enhancing root acidification under Fe deficiency stress. Moreover, co-expression of MxMPK6-2 and MxHA2 in apple rootstocks further enhanced PM H+ -ATPase activity under Fe deficiency. MxMPK6-2 phosphorylated MxHA2 at the Ser909 site of C terminus, Thr320 and Thr412 sites of the Central loop region. Phosphorylation at the Ser909 and Thr320 promoted PM H+ -ATPase activity, while phosphorylation at Thr412 inhibited PM H+ -ATPase activity. MxMPK6-2 also phosphorylated the Fe deficiency-induced transcription factor MxbHLH104 at the Ser169 site, which then could bind to the promoter of MxHA2, thus enhancing MxHA2 upregulation. In conclusion, the MAP kinase MxMPK6-2-mediated phosphorylation directly and indirectly regulates PM H+ -ATPase MxHA2 activity at the protein post-translation and transcription levels, thus synergistically enhancing root acidification under Fe deficiency stress.

Kinase MxMPK4-1 and calmodulin-binding protein MxIQM3 enhance apple root acidification during Fe deficiency.

Iron (Fe) deficiency is a long-standing issue in plant mineral nutrition. Ca2+ signals and the mitogen-activated protein kinase (MAPK) cascade are frequently activated in parallel to perceive external cues, but their interplay under Fe deficiency stress remains largely unclear. Here, the kinase MxMPK4-1, which is induced during the response to Fe deficiency stress in apple rootstock Malus xiaojinensis, cooperates with IQ-motif containing protein3 (MxIQM3). MxIQM3 gene expression, protein abundance, and phosphorylation level increased under Fe deficiency stress. The overexpression of MxIQM3 in apple calli and rootstocks mitigated the Fe deficiency phenotype and improved stress tolerance, whereas RNA interference or silencing of MxIQM3 in apple calli and rootstocks, respectively, worsened the phenotype and reduced tolerance to Fe deficiency. MxMPK4-1 interacted with MxIQM3 and subsequently phosphorylated MxIQM3 at Ser393, and co-expression of MxMPK4-1 and MxIQM3 in apple calli and rootstocks enhanced Fe deficiency responses. Furthermore, MxIQM3 interacted with the central-loop region of the plasma membrane (PM) H+-ATPase MxHA2. Phospho-mimicking mutation of MxIQM3 at Ser393 inhibited binding to MxHA2, but phospho-abolishing mutation promoted interaction with both the central-loop and C terminus of MxHA2, demonstrating phosphorylation of MxIQM3 caused dissociation from MxHA2 and therefore increased H+ secretion. Moreover, Ca2+/MxCAM7 (Calmodulin7) regulated the MxMPK4-1-MxIQM3 module in response to Fe deficiency stress. Overall, our results demonstrate that MxMPK4-1-MxIQM3 forms a functional complex and positively regulates PM H+-ATPase activity in Fe deficiency responses, revealing a versatile mechanism of Ca2+/MxCAM7 signaling and MAPK cascade under Fe deficiency stress.

Multi-omics analysis reveals the mechanism of bHLH130 responding to low-nitrogen stress of apple rootstock.

Nitrogen is critical for plant growth and development. With the increase of nitrogen fertilizer application, nitrogen use efficiency decreases, resulting in wasted resources. In apple (Malus domestica) rootstocks, the potential molecular mechanism for improving nitrogen uptake efficiency to alleviate low-nitrogen stress remains unclear. We utilized multi-omics approaches to investigate the mechanism of nitrogen uptake in two apple rootstocks with different responses to nitrogen stress, Malus hupehensis and Malus sieversii. Under low-nitrogen stress, Malus sieversii showed higher efficiency in nitrogen uptake. Multi-omics analysis revealed substantial differences in the expression of genes involved in flavonoid and lignin synthesis pathways between the two materials, which were related to the corresponding metabolites. We discovered that basic helix-loop-helix 130 (bHLH130) transcription factor was highly negatively associated with the flavonoid biosynthetic pathway. bHLH130 may directly bind to the chalcone synthase gene (CHS) promoter and inhibit its expression. Overexpressing CHS increased flavonoid accumulation and nitrogen uptake. Inhibiting bHLH130 increased flavonoid biosynthesis while decreasing lignin accumulation, thus improving nitrogen uptake efficiency. These findings revealed the molecular mechanism by which bHLH130 regulates flavonoid and lignin biosyntheses in apple rootstocks under low-nitrogen stress.

Genome-wide identification of WOX family members in nine Rosaceae species and a functional analysis of MdWOX13-1 in drought resistance.

WUSCHEL-related homeobox (WOX) transcription factors (TFs) are important in plant development processes and evolutionary novelties. In this study, a genome-wide comprehensive analysis of WOX genes from nine Rosaceae species was carried out, and their potential roles in Malus were subsequently investigated. 125 WOXs in 9 Rosaceae species were identified and classified into three clades, i.e., the ancient, intermediate, and WUS clades. Prunus. domestica contained the most intra-genomic collinearity among the nine Rosaceae species. Additionally, the cis-elements in WOX gene family members were compared and classified into three categories, including phytohormone-responsive, plant growth and development, and abiotic and biotic stresses. Overexpression (OE) of MdWOX13-1 also increased the callus weight and enhanced ROS scavenging against drought stress. Furthermore, via yeast-one hybrid assay and LUC analyses, MdWOX13-1 could directly bind to the MdMnSOD promoter. Therefore, our results will facilitate further study of the WOX genes' function in the Rosaceae family.

Strigolactone regulates adventitious root formation via the MdSMXL7-MdWRKY6-MdBRC1 signaling cascade in apple.

Propagation through stem cuttings is a popular method worldwide for species such as fruit tree rootstocks and forest trees. Adventitious root (AR) formation from stem cuttings is crucial for effective and successful clonal propagation of apple rootstocks. Strigolactones (SLs) are newly identified hormones involved in AR formation. However, the regulatory mechanisms underpinning this process remain elusive. In the present study, weighted gene co-expression network analysis, as well as rooting assays using stable transgenic apple materials, revealed that MdBRC1 served as a key gene in the inhibition of AR formation by SLs. We have demonstrated that MdSMXL7 and MdWRKY6 synergistically regulated MdBRC1 expression, depending on the interactions of MdSMXL7 and MdWRKY6 at the protein level downstream of SLs as well as the direct promoter binding on MdBRC1 by MdWRKY6. Furthermore, biochemical studies and genetic analysis revealed that MdBRC1 inhibited AR formation by triggering the expression of MdGH3.1 in a transcriptional activation pathway. Finally, the present study not only proposes a component, MdWRKY6, that enables MdSMXL7 to regulate MdBRC1 during the process of SL-controlled AR formation in apple, but also provides prospective target genes to enhance AR formation capacity using CRISPR (i.e. clustered regularly interspaced short palindromic repeats) technology, particularly in woody plants.

Grape polysaccharides: compositional changes in grapes and wines, possible effects on wine organoleptic properties, and practical control during winemaking.

Polysaccharides present in grapes interact with wine sensory-active compounds (polyphenols and volatile compounds) via different mechanisms and can affect wine organoleptic qualities such as astringency, color and aroma. Studies on the role that grape polysaccharides play in wines are reviewed in this paper. First, the composition of grape polysaccharides and their changes during grape ripening, winemaking and aging are introduced. Second, different interaction mechanisms of grape polysaccharides and wine sensory-active compounds (flavanols, anthocyanins and volatiles) are introduced, and the possible effects on wine astringency, color and aroma caused by these interactions are illustrated. Finally, the control of the grape polysaccharide content in practice is discussed, including classical winemaking methods (applying different maceration enzymes, temperature control, co-fermentation, blending), modern vinification technologies (pulsed electric field, ultrasound treatment), and the development of new grape polysaccharide products.

Strigolactones in Plants and Their Interaction with the Ecological Microbiome in Response to Abiotic Stress.

Phytohormones play an essential role in enhancing plant tolerance by responding to abiotic stresses, such as nutrient deficiency, drought, high temperature, and light stress. Strigolactones (SLs) are carotenoid derivatives that occur naturally in plants and are defined as novel phytohormones that regulate plant metabolism, growth, and development. Strigolactone assists plants in the acquisition of defensive characteristics against drought stress by initiating physiological responses and mediating the interaction with soil microorganisms. Nutrient deficiency is an important abiotic stress factor, hence, plants perform many strategies to survive against nutrient deficiency, such as enhancing the efficiency of nutrient uptake and forming beneficial relationships with microorganisms. Strigolactone attracts various microorganisms and provides the roots with essential elements, including nitrogen and phosphorus. Among these advantageous microorganisms are arbuscular mycorrhiza fungi (AMF), which regulate plant metabolic activities through phosphorus providing in roots. Bacterial nodulations are also nitrogen-fixing microorganisms found in plant roots. This symbiotic relationship is maintained as the plant provides organic molecules, produced in the leaves, that the bacteria could otherwise not independently generate. Related stresses, such as light stress and high-temperature stress, could be affected directly or indirectly by strigolactone. However, the messengers of these processes are unknown. The most prominent connector messengers have been identified upon the discovery of SLs and the understanding of their hormonal effect. In addition to attracting microorganisms, these groups of phytohormones affect photosynthesis, bridge other phytohormones, induce metabolic compounds. In this article, we highlighted the brief information available on SLs as a phytohormone group regarding their common related effects. In addition, we reviewed the status and described the application of SLs and plant response to abiotic stresses. This allowed us to comprehend plants' communication with the ecological microbiome as well as the strategies plants use to survive under various stresses. Furthermore, we identify and classify the SLs that play a role in stress resistance since many ecological microbiomes are unexplained.

A bulked segregant analysis tool for out-crossing species (BSATOS) and QTL-based genomics-assisted prediction of complex traits in apple.

INTRODUCTION: Genomic heterozygosity, self-incompatibility, and rich-in somatic mutations hinder the molecular breeding efficiency of outcrossing plants. OBJECTIVES: We attempted to develop an efficient integrated strategy to identify quantitative trait loci (QTLs) and trait-associated genes, to develop gene markers, and to construct genomics-assisted prediction (GAP) modes. METHODS: A novel protocol, bulked segregant analysis tool for out-crossing species (BSATOS), is presented here, which is characterized by taking full advantage of all segregation patterns (including AB × AB markers) and haplotype information. To verify the effectiveness of the protocol in dealing with the complex traits of outbreeding species, three apple cross populations with 9,654 individuals were adopted. RESULTS: By using BSATOS, 90, 60, and 77 significant QTLs were identified successfully and candidate genes were predicted for apple fruit weight (FW), fruit ripening date (FRD), and fruit soluble solid content (SSC), respectively. The gene-based markers were developed and genotyped for 1,396 individuals in a training population, including 145 Malus accessions and 1,251 F1 plants of the three full-sib families. GAP models were trained using marker genotype effect estimates of the training population. The prediction accuracy was 0.7658, 0.6455, and 0.3758 for FW, FRD, and SSC, respectively. CONCLUSION: The BSATOS and GAP models provided a convenient and efficient methodology for candidate gene mining and molecular breeding in out-crossing plant species. The BSATOS pipeline can be freely downloaded from: https://github.com/maypoleflyn/BSATOS.

Transcription Factor IAA27 Positively Regulates P Uptake through Promoted Adventitious Root Development in Apple Plants.

Phosphate (P) deficiency severely limits the growth and production of plants. Adventitious root development plays an essential role in responding to low phosphorus stress for apple plants. However, the molecular mechanisms regulating adventitious root growth and development in response to low phosphorus stress have remained elusive. In this study, a mutation (C-T) in the coding region of the apple AUXIN/INDOLE-3-ACETIC ACID 27 (IAA27) gene was identified. MdIAA27T-overexpressing transgenic apple improved the tolerance to phosphorus deficiency, which grew longer and denser adventitious roots and presented higher phosphorous content than the control plants under low phosphorus conditions, while the overexpression of MdIAA27C displayed the opposite trend. Moreover, the heterologous overexpression of MdIAA27 in tobacco yielded the same results, supporting the aforementioned findings. In vitro and in vivo assays showed that MdIAA27 directly interacted with AUXIN RESPONSE FACTOR (ARF8), ARF26 and ARF27, which regulated Small Auxin-Up RNA 76 (MdSAUR76) and lateral organ boundaries domain 16 (MdLBD16) transcription. The mutation in IAA27 resulted in altered interaction modes, which in turn promoted the release of positive ARFs to upregulate SAUR76 and LBD16 expression in low phosphorus conditions. Altogether, our studies provide insights into how the allelic variation of IAA27 affects adventitious root development in response to low phosphorus stress.

A single QTL harboring multiple genetic variations leads to complicated phenotypic segregation in apple flesh firmness and crispness.

KEY MESSAGE: Within a QTL, the genetic recombination and interactions among five and two functional variations at MdbHLH25 and MdWDR5A caused much complicated phenotype segregation in apple FFR and FCR. The storability of climacteric fruit like apple is a quantitative trait. We previously identified 62 quantitative trait loci (QTLs) associating flesh firmness retainability (FFR) and flesh crispness retainability (FCR), but only a few functional genetic variations were identified and validated. The genetic variation network controlling fruit storability is far to be understood and diagnostic markers are needed for molecular breeding. We previously identified overlapped QTLs F16.1/H16.2 for FFR and FCR using an F1 population derived from 'Zisai Pearl' × 'Red Fuji'. In this study, five and two single-nucleotide polymorphisms (SNPs) were identified on the candidate genes MdbHLH25 and MdWDR5A within the QTL region. The SNP1 A allele at MdbHLH25 promoter reduced the expression and SNP2 T allele and/or SNP4/5 GT alleles at the exons attenuated the function of MdbHLH25 by downregulating the expression of the target genes MdACS1, which in turn led to a reduction in ethylene production and maintenance of higher flesh crispness. The SNPs did not alter the protein-protein interaction between MdbHLH25 and MdWDR5A. The joint effect of SNP genotype combinations by the SNPs on MdbHLH25 (SNP1, SNP2, and SNP4) and MdWDR5A (SNPi and SNPii) led to a much broad spectrum of phenotypic segregation in FFR and FCR. Together, the dissection of these genetic variations contributes to understanding the complicated effects of a QTL and provides good potential for marker development in molecular breeding.

Genome-wide identification of apple PPI genes and a functional analysis of the response of MxPPI1 to Fe deficiency stress.

Iron (Fe) deficiency affects plant growth and development. The proton pump interactor (PPI) in plants responds to multiple abiotic stresses, although it has not been well characterized under Fe deficiency stress. In this study, we systematically identified and analyzed the PPI gene family in apple. Three PPI candidate genes were found, and they contained 318-1349 amino acids and 3-7 introns. Under Fe deficiency stress, we analyzed the expression of all the PPI genes in roots of apple rootstock Malus xiaojinensis. Expression of the gene MD11G1247800, designated PPI1, is obviously induced by Fe deficiency treatment in M. xiaojinensis. We first cloned MxPPI1 from M. xiaojinensis and determined its subcellular localization, which indicated that it is localized in the cell membrane and nucleus in tobacco. We found that the level of expression of the MxPPI1 protein increased significantly under Fe deficiency stress in apple calli. Moreover, overexpressing MxPPI1 in apple calli enhanced the activities of ferric chelate reductase and H+-ATPase, H+ secretion, MxHA2 gene expression and total Fe content when compared with the wild type calli. We further found that MxPPI1 interacted with MxHA2 using bimolecular fluorescence complementation and luciferase complementation assays. Overall, we demonstrated that MxPPI1 interacts with MxHA2 to enhance the activity of H+-ATPase to regulate Fe absorption in M. xiaojinensis.