Epigenomic mechanism regulating the quality and ripeness of apple fruit with differing harvest maturity.

Harvest maturity significantly affects the quality of apple fruit in post-harvest storage process. Although the regulatory mechanisms underlying fruit ripening have been studied, the associated epigenetic modifications remain unclear. Thus, we compared the DNA methylation changes and the transcriptional responses of mature fruit (MF) and immature fruit (NF). There were significant correlations between DNA methylation and gene expression. Moreover, the sugar contents (sucrose, glucose, and fructose) were higher in MF than in NF, whereas the opposite pattern was detected for the starch content. The expression-level differences were due to DNA methylations and ultimately resulted in diverse fruit textures and ripeness. Furthermore, the higher ethylene, auxin, and abscisic acid levels in MF than in NF, which influenced the fruit texture and ripening, were associated with multiple differentially expressed genes in hormone synthesis, signaling, and response pathways (ACS, ACO, ZEP, NCED, and ABA2) that were regulated by DNA methylations. Multiple transcription factor genes involved in regulating fruit ripening and quality via changes in DNA methylation were identified, including MIKCC-type MADS-box genes and fruit ripening-related genes (NAP, SPL, WRKY, and NAC genes). These findings reflect the diversity in the epigenetic regulation of gene expression and may be relevant for elucidating the epigenetic regulatory mechanism underlying the ripening and quality of apple fruit with differing harvest maturity.

FLOWERING LOCUS T1 and TERMINAL FLOWER1 regulatory networks mediate flowering initiation in apple.

Flower bud formation is a critical process that directly determines yield and fruit quality in fruit crops. Floral induction is modulated by the balance between 2 flowering-related proteins, FLOWERING LOCUS T (FT) and TERMINAL FLOWER1 (TFL1); however, the mechanisms underlying the establishment and maintenance of this dynamic balance remain largely elusive. Here, we showed that in apple (Malus × domestica Borkh.), MdFT1 is predominantly expressed in spur buds and exhibits an increase in expression coinciding with flower induction; in contrast, MdTFL1 exhibited downregulation in apices during flower induction, suggesting that MdTFL1 has a role in floral repression. Interestingly, both the MdFT1 and MdTFL1 transcripts are directly regulated by transcription factor basic HELIX-LOOP-HELIX48 (MdbHLH48), and overexpression of MdbHLH48 in Arabidopsis (Arabidopsis thaliana) and tomato (Solanum lycopersicum) results in accelerated flowering. Binding and activation analyses revealed that MdbHLH48 functions as a positive regulator of MdFT1 and a negative regulator of MdTFL1. Further studies established that both MdFT1 and MdTFL1 interact competitively with MdWRKY6 protein to facilitate and inhibit, respectively, MdWRKY6-mediated transcriptional activation of target gene APPLE FLORICAULA/LFY (AFL1, an apple LEAFY-like gene), ultimately regulating apple flower bud formation. These findings illustrate the fine-tuned regulation of flowering by the MdbHLH48-MdFT1/MdTFL1-MdWRKY6 module and provide insights into flower bud formation in apples.

Comparative Proteomic and Metabonomic Profiling of Buds with Different Flowering Capabilities Reveal Novel Regulatory Mechanisms of Flowering in Apple.

Flower bud formation in the apple tree life cycle is associated with multiple biological processes. To explore the physiological and molecular mechanisms underlying the protein and metabolite changes in buds with different flowering capabilities, axillary buds with no flowering (Ab), long-shoot buds with a low flowering rate (Lb), and spur buds with a higher flowering rate than the Lb (Sb) were analyzed using a Tandem Mass Tag™ proteomic technique in combination with nLC-MS/MS analyses. We identified 471 (88 up- and 383 down-regulated), 459 (176 up- and 283 down-regulated), and 548 (387 up- and 161 down-regulated) differentially expressed proteins in Sb vs. Lb, Sb vs. Ab, and Lb vs. Ab, respectively, that were involved in carbohydrate, amino acid and lipid transport, and metabolism. Additionally, 110 (91 increased and 19 decreased), 89 (71 increased and 18 decreased), and 99 (37 increased and 62 decreased) metabolites having significantly different levels were identified in Sb vs. Lb, Sb vs. Ab, and Lb vs. Ab, respectively. The identified metabolites were related to amino acids and their isoforms, sugars and polyols, and organic acids, and occurred at significantly greater levels in the Sbs than the other buds. Thus, flower bud formation is a complex process that involves various biochemical materials and signals, such as carbohydrates, amino acids and their isoforms, and organic acids.

MdNup62 involved in salt and osmotic stress tolerance in apple.

Abiotic stress of plants has serious consequences on the development of the apple industry. Nuclear pore complexes (NPCs) control nucleoplasmic transport and play an important role in the regulation of plant abiotic stress response. However, the effects of NPCs on apple salt and osmotic stress responses have not been reported yet. In this study, we analyzed the expression and function of NUCLEOPORIN 62 (MdNup62), a component of apple NPC. MdNup62 expression was significantly increased by salt and mannitol (simulated osmotic stress) treatment. The MdNup62-overexpressing (OE) Arabidopsis and tomato lines exhibited significantly reduced salt stress tolerance, and MdNup62-OE Arabidopsis lines exhibited reduced osmotic stress tolerance. We further studied the function of HEAT SHOCK FACTOR A1d (MdHSFA1d), the interacting protein of MdNup62, in salt and osmotic stress tolerance. In contrast to MdNup62, MdHSFA1d-OE Arabidopsis lines showed significantly enhanced tolerance to salt and osmotic stress. Our findings suggest a possible interaction of MdNup62 with MdHSFA1d in the mediation of nuclear and cytoplasmic transport and the regulation of apple salt and osmotic stress tolerance. These results contribute to the understanding of the salt and osmotic stress response mechanism in apple.

Chitinase-Like Protein PpCTL1 Contributes to Maintaining Fruit Firmness by Affecting Cellulose Biosynthesis during Peach Development.

The firmness of the flesh fruit is a very important feature in the eating process. Peach fruit is very hard during development, but its firmness slightly decreases in the later stages of development. While there has been extensive research on changes in cell wall polysaccharides during fruit ripening, little is known about the changes that occur during growth and development. In this study, we investigated the modifications in cell wall components throughout the development and ripening of peach fruit, as well as its impact on firmness. Our findings revealed a significant positive correlation between fruit firmness and cellulose content at development stage. However, the correlation was lost during the softening process, suggesting that cellulose might be responsible for the fruit firmness during development. Members of the chitinase-like protein (CTL) group are of interest because of their possible role in plant cell wall biosynthesis. Here, two CTL homologous genes, PpCTL1 and PpCTL2, were identified in peach. Spatial and temporal expression patterns of PpCTLs revealed that PpCTL1 exhibited high expression abundance in the fruit and followed a similar trend to cellulose during fruit growth. Furthermore, silencing PpCTL1 expression resulted in reduced cellulose content at 5 DAI (days after injection), this change that would have a negative effect on fruit firmness. Our results indicate that PpCTL1 plays an important role in cellulose biosynthesis and the maintenance of peach firmness during development.

The East Asian wild apples, Malus baccata (L.) Borkh and Malus hupehensis (Pamp.) Rehder., are additional contributors to the genomes of cultivated European and Chinese varieties.

The domestication process in long-lived plant perennials differs dramatically from that of annuals, with a huge amount of genetic exchange between crop and wild populations. Though apple is a major fruit crop grown worldwide, the contribution of wild apple species to the genetic makeup of the cultivated apple genome remains a topic of intense study. We used population genomics approaches to investigate the contributions of several wild apple species to European and Chinese rootstock and dessert genomes, with a focus on the extent of wild-crop gene flow. Population genetic structure inferences revealed that the East Asian wild apples, Malus baccata (L.) Borkh and M. hupehensis (Pamp.), form a single panmictic group, and that the European dessert and rootstock apples form a specific gene pool whereas the Chinese dessert and rootstock apples were a mixture of three wild gene pools, suggesting different evolutionary histories of European and Chinese apple varieties. Coalescent-based inferences and gene flow estimates indicated that M. baccata - M. hupehensis contributed to the genome of both European and Chinese cultivated apples through wild-to-crop introgressions, and not as an initial contributor as previously supposed. We also confirmed the contribution through wild-to-crop introgressions of Malus sylvestris Mill. to the cultivated apple genome. Apple tree domestication is therefore one example in woody perennials that involved gene flow from several wild species from multiple geographical areas. This study provides an example of a complex protracted process of domestication in long-lived plant perennials, and is a starting point for apple breeding programmes.

Cytokinin-responsive MdTCP17 interacts with MdWOX11 to repress adventitious root primordium formation in apple rootstocks.

Adventitious root (AR) formation plays an important role in vegetatively propagated plants. Cytokinin (CK) inhibits AR formation, but the molecular mechanisms driving this process remain unknown. In this study, we confirmed that CK content is related to AR formation and further revealed that a high auxin/CK ratio was beneficial to AR formation in apple (Malus domestica). A correlation between expression of CK-responsive TEOSINTE BRANCHED1, CYCLOIDEA, and PCF17 (MdTCP17) and AR formation in response to CK was identified, and overexpression of MdTCP17 in transgenic apple inhibited AR formation. Yeast two-hybrid, bimolecular fluorescence complementation, and co-immunoprecipitation assays revealed an interaction between MdTCP17 and WUSCHEL-RELATED HOMEOBOX11 (MdWOX11), and a significant correlation between the expression of MdWOX11 and AR ability. Overexpression of MdWOX11 promoted AR primordium formation in apple, while interference of MdWOX11 inhibited AR primordium production. Moreover, a positive correlation was found between MdWOX11 and LATERAL ORGAN BOUNDARIES DOMAIN29 (MdLBD29) expression, and yeast one-hybrid, dual luciferase reporter, and ChIP-qPCR assays verified the binding of MdWOX11 to the MdLBD29 promoter with a WOX-box element in the binding sequence. Furthermore, MdTCP17 reduced the binding of MdWOX11 and MdLBD29 promoters, and coexpression of MdTCP17 and MdWOX11 reduced MdLBD29 expression. Together, these results explain the function and molecular mechanism of MdTCP17-mediated CK inhibition of AR primordium formation, which could be used to improve apple rootstocks genetically.

Multi-omics analyses reveal MdMYB10 hypermethylation being responsible for a bud sport of apple fruit color.

Apple bud sports offer a rich resource for clonal selection of numerous elite cultivars. The accumulation of somatic mutations as plants develop may potentially impact the emergence of bud sports. Previous studies focused on somatic mutation in the essential genes associated with bud sports. However, the rate and function of genome-wide somatic mutations that accumulate when a bud sport arises remain unclear. In this study, we identified a branch from a 10-year-old tree of the apple cultivar 'Oregon Spur II' as a bud sport. The mutant branch showed reduced red coloration on fruit skin. Using this plant material, we assembled a high-quality haplotype reference genome consisting of 649.61 Mb sequences with a contig N50 value of 2.04 Mb. We then estimated the somatic mutation rate of the apple tree to be 4.56 × 10 -8 per base per year, and further identified 253 somatic single-nucleotide polymorphisms (SNPs), including five non-synonymous SNPs, between the original type and mutant samples. Transcriptome analyses showed that 69 differentially expressed genes between the original type and mutant fruit skin were highly correlated with anthocyanin content. DNA methylation in the promoter of five anthocyanin-associated genes was increased in the mutant compared with the original type as determined using DNA methylation profiling. Among the genetic and epigenetic factors that directly and indirectly influence anthocyanin content in the mutant apple fruit skin, the hypermethylated promoter of MdMYB10 is important. This study indicated that numerous somatic mutations accumulated at the emergence of a bud sport from a genome-wide perspective, some of which contribute to the low coloration of the bud sport.

Identification and expression analysis of the lipid phosphate phosphatases gene family reveal their involvement in abiotic stress response in kiwifruit.

Lipid phosphate phosphatases (LPPs) are a key enzyme in the production and degradation of phosphatidic acid (PA), which plays an important role in plant growth, development, stress resistance and plant hormone response. Thus far, little is known about the LPP family genes in kiwifruit (Actinidia spp.). According to this study, 7 members in the AcLPP family were identified from the whole genome of kiwifruit, the subcellular localization predictions were mainly on the plasma membrane. Chromosomal localization analysis showed that the AcLPP genes were unevenly distributed on 5 chromosomes, it was determined to have undergone strong purifying selection pressure. There were 5 duplicate gene pairs and all underwent segmental duplication events. The LPP genes of kiwifruit were conserved when compared with other plants, especially in terms of evolutionary relationships, conserved motifs, protein sequences, and gene structures. Cis-regulatory elements mainly included hormone response elements and abiotic response elements. Functional annotation of GO revealed that AcLPP genes were closely related to phosphatase/hydrolase activity, phosphorus metabolism and dephosphorylation. AcLPP genes family were predicted to be targets of miRNA. Transcript level analysis revealed that the AcLPP family played diverse functions in different tissues and during growth, development, and postharvest storage stages. qPCR analysis showed that the members of AcLPP gene family might be regulated by ETH, ABA, GA3, and IAA hormone signals. The family members were regulated by the stress of salt stress, osmotic stress, cold stress, and heat stress. These results would provide a basis and reference for studying the agricultural characteristics of kiwifruit and improving its stress resistance.

Transcriptome analysis reveals dual action of salicylic acid application in the induction of flowering in Malus domestica.

In the apple tree, insufficient flower bud production is an intractable challenge, and very little information is available in this field due to the fact that research done in this sector is very rare owing to its extended life cycles and low rate of genetic transformation. Here we display novel changes and events in spur buds of Malus × domestica trees after they were exposed to salicylic acid (SA) treatment during the flower induction period. We found a significant increase in morphological indexes, followed by a wider and well-defined shoot apical meristem in SA-treated spur buds. Additionally, we observed increased oxidative stress markers and enzymatic antioxidants in control-treated buds during the flower induction period, while non-enzymatic antioxidants were recorded higher in SA-treated buds. Maximum flowering was observed in SA-treated trees in the next year. Furthermore, ultra-high-performance liquid chromatography (u-HPLC) analysis displays that SA treatment enhances SA and indole acetic acid (IAA), while having an antagonistic effect on gibberellin (GA). At different time points, transcriptome analysis was conducted to analyze the transcriptional response of CK and SA treated buds. Pathway enrichment was detected in differentially expressed genes (DEGs). Agamous (AGL) and SQUAMOSA-promoter binding protein-like (SPL) family related flowering genes display a positive signal for the increased flowering in SA-treated trees, which confirms our findings. As far as we know, there is no report available on the response of spur buds to SA treatment during the flower induction period. This data provides a new theoretical reference for the management of apple tree flowering and also provides an essential basis for future analysis of the regulation and control of flowering in M. domestica.

MdNup62 interactions with MdHSFs involved in flowering and heat-stress tolerance in apple.

Because of global warming, the apple flowering period is occurring significantly earlier, increasing the probability and degree of freezing injury. Moreover, extreme hot weather has also seriously affected the development of apple industry. Nuclear pore complexes (NPCs) are main channels controlling nucleocytoplasmic transport, but their roles in regulating plant development and stress responses are still unknown. Here, we analysed the components of the apple NPC and found that MdNup62 interacts with MdNup54, forming the central NPC channel. MdNup62 was localized to the nuclear pore, and its expression was significantly up-regulated in 'Nagafu No. 2' tissue-cultured seedlings subjected to heat treatments. To determine MdNup62's function, we obtained MdNup62-overexpressed (OE) Arabidopsis and tomato lines that showed significantly reduced high-temperature resistance. Additionally, OE-MdNup62 Arabidopsis lines showed significantly earlier flowering compared with wild-type. Furthermore, we identified 62 putative MdNup62-interacting proteins and confirmed MdNup62 interactions with multiple MdHSFs. The OE-MdHSFA1d and OE-MdHSFA9b Arabidopsis lines also showed significantly earlier flowering phenotypes than wild-type, but had enhanced high-temperature resistance levels. Thus, MdNUP62 interacts with multiple MdHSFs during nucleocytoplasmic transport to regulate flowering and heat resistance in apple. The data provide a new theoretical reference for managing the impact of global warming on the apple industry.

MdNup54 Interactions With MdHSP70 Involved in Flowering in Apple.

Flowering-related problems in "Fuji" apple have severely restricted the development of China's apple industry. Nuclear pore complexes (NPCs) control nucleoplasmic transport and play an important role in the regulation of plant growth and development. However, the effects of NPCs on apple flowering have not been reported. Here, we analysed the expression and function of MdNup54, a component of apple NPC. MdNup54 expression was the highest in flower buds and maintained during 30-70 days after flowering. MdNup54-overexpressing (OE) Arabidopsis lines displayed significantly earlier flowering than that of the wild type. We further confirmed that MdNup54 interacts with MdHSP70, MdMYB11, and MdKNAT4/6. Consistent with these observations, flowering time of MdHSP70-OE Arabidopsis lines was also significantly earlier. Therefore, our findings suggest a possible interaction of MdNup54 with MdHSP70 to mediate its nuclear and cytoplasmic transport and to regulate apple flowering. The results enhance the understanding of the flowering mechanism in apple and propose a novel strategy to study nucleoporins.

PpePL1 and PpePL15 Are the Core Members of the Pectate Lyase Gene Family Involved in Peach Fruit Ripening and Softening.

Pectin is the major component in the primary cell wall and middle lamella, maintaining the physical stability and mechanical strength of the cell wall. Pectate lyase (PL), a cell wall modification enzyme, has a major influence on the structure of pectin. However, little information and no comprehensive analysis is available on the PL gene family in peach (Prunus persica L. Batsch). In this study, 20 PpePL genes were identified in peach. We characterized their physicochemical characteristics, sequence alignments, chromosomal locations, and gene structures. The PpePL family members were classified into five groups based on their phylogenetic relationships. Among those, PpePL1, 9, 10, 15, and 18 had the higher expression abundance in ripe fruit, and PpePL1, 15, and 18 were upregulated during storage. Detailed RT-qPCR analysis revealed that PpePL1 and PpePL15 were responsive to ETH treatment (1 g L-1 ethephon) with an abundant transcript accumulation, which suggested these genes were involved in peach ripening and softening. In addition, virus-induced gene silencing (VIGS) technology was used to identify the roles of PpePL1 and PpePL15. Compared to controls, the RNAi fruit maintained greater firmness in the early storage stage, increased acid-soluble pectin (ASP), and reduced water-soluble pectin (WSP). Moreover, transmission electron microscopy (TEM) showed that cell wall degradation was reduced in the fruit of RNAi-1 and RNAi-15, which indicated that softening of the RNAi fruit has been delayed. Our results indicated that PpePL1 and PpePL15 play an important role in peach softening by depolymerizing pectin and degrading cell wall.

Selection and Validation of Reliable Reference Genes for Gene Expression Studies in Different Genotypes and TRV-Infected Fruits of Peach (Prunus persica L. Batsch) during Ripening.

Real-time quantitative PCR (RT-qPCR) is a powerful tool to detect and quantify transcription abundance, and the stability of the reference gene determines its success. However, the most suitable reference gene for different genotypes and tobacco rattle virus (TRV) infected fruits was unclear in peach (Prunus persica L. Batsch). In this study, 10 reference genes were selected and gene expression was characterized by RT-qPCR across all samples, including different genotypes and TRV-infected fruits during ripening. Four statistical algorithms (geNorm, NormFinder, BestKeeper, and RefFinder) were used to calculate the stability of 10 reference genes. The geNorm analysis indicated that two suitable reference genes should be used for gene expression normalization. In general, the best combination of reference genes was CYP2 and Tua5 for TRV-infected fruits and CYP2 and Tub1 for different genotypes. In 18S, GADPH, and TEF2, there is an unacceptable variability of gene expression in all experimental conditions. Furthermore, to confirm the validity of the reference genes, the expression levels of PpACO1, PpEIN2, and PpPL were normalized at different fruit storage periods. In summary, our results provide guidelines for selecting reliable reference genes in different genotypes and TRV-infected fruits and lay the foundation for accurate evaluation of gene expression for RT-qPCR analysis in peach.

Transcriptome Analysis Reveals Multiple Genes and Complex Hormonal-Mediated Interactions with PEG during Adventitious Root Formation in Apple.

Adventitious root (AR) formation is a bottleneck for the mass propagation of apple rootstocks, and water stress severely restricts it. Different hormones and sugar signaling pathways in apple clones determine AR formation under water stress, but these are not entirely understood. To identify them, GL-3 stem cuttings were cultured on polyethylene glycol (PEG) treatment. The AR formation was dramatically decreased compared with the PEG-free control (CK) cuttings by increasing the endogenous contents of abscisic acid (ABA), zeatin riboside (ZR), and methyl jasmonate (JA-me) and reducing the indole-3-acetic acid (IAA) and gibberellic acid 3 (GA3) contents. We performed a transcriptomic analysis to identify the responses behind the phenotype. A total of 3204 differentially expressed genes (DEGs) were identified between CK and PEG, with 1702 upregulated and 1502 downregulated genes. Investigation revealed that approximately 312 DEGs were strongly enriched in hormone signaling, sugar metabolism, root development, and cell cycle-related pathways. Thus, they were selected for their possible involvement in adventitious rooting. However, the higher accumulation of ABA, ZR, and JA-me contents and the upregulation of their related genes, as well as the downregulation of sugar metabolism-related genes, lead to the inhibition of ARs. These results indicate that AR formation is a complicated biological process chiefly influenced by multiple hormonal signaling pathways and sugar metabolism. This is the first study to demonstrate how PEG inhibits AR formation in apple plants.

Modification of the effect of maturity variation on nondestructive detection of apple quality based on the compensation model.

In this study, the effect of maturity variation on the prediction of the soluble solids content (SSC) and firmness of apples was determined using visible and near-infrared spectroscopy. In 2018, 520 apples from six ripening stages were collected. The single maturity model and multi-maturity model of SSC and firmness were established using partial least-squares regression. Apples at the same and different maturity stages were used to verify the developed model. Whereas the single maturity model was affected by maturity variation, the multi-maturity model could accurately predict the SSC and firmness of apples at different maturity stages. The multi-maturity model developed based on six maturity calibration sets had the best predictive performance. The root mean square error of prediction (RMSEP) of SSC and firmness was 0.614-0.802 °Brix and 0.402-0.650 kg/cm2, respectively. The long-term performance of the optimal multi-maturity model was evaluated using validation sets. The predictive performance was decreased and the RMSEP increased when the model was used to predict the SSC and firmness of apples in different seasons. The predictive performance of the model was improved after slope/bias (S/B) correction, and the RMSEP of SSC and firmness decreased to 0.405-0.587°Brix and 0.518-0.628 kg/cm2 respectively. Overall, the multi-maturity model eliminated the effect of maturity variation, and the multi-maturity model coupled with S/B correction permitted the rapid and accurate detection of the SSC and firmness of apples at different maturity stages and in different seasons.

Regulation of Flowering Time by Improving Leaf Health Markers and Expansion by Salicylic Acid Treatment: A New Approach to Induce Flowering in Malus domestica.

In the external coincidence model, internal and external molecular signals, provided by the circadian clock and sunlight, respectively, are required to induce flowering. Salicylic acid (SA) applications during floral induction have multiple effects. In the current study, Malus × domestica plants were exposed to SA during the flower-induction stage to analyze the effect on various health markers and flowering. A total of 56 equal-sized Fuji/M9 trees that were about 7 years old were randomly divided into two groups. The first group (SA-treated) was sprayed with 4 mM SA solution, while the second group was sprayed with distilled water which served as control (CK). The SA applications increased various leaf pigments. Abiotic stress markers were increased in CK during the flower-induction stage. In the SA-treated group, non-enzymatic antioxidants increased, whereas in the control group, enzymatic antioxidants increased during the flower-induction stage. Histo-morphometric properties of leaves were significantly improved in the SA-treated group. The relative expression of the mRNA levels of MdMED80, -81, -3, and -41 were significantly increased in SA-treated leaves, leading to an early and increased flowering phenotype. Thus, SA increased leaf expansion and health-related marker levels, which lead to early induction of flowering in M. domestica. Overall, our work established a role for leaf health assessments in the regulation of flowering in M. domestica.

Identification of apple TFL1-interacting proteins uncovers an expanded flowering network.

KEY MESSAGE: MdTFL1, a floral repressor, forms protein complexes with several proteins and could compete with MdFT1 to regulate reproductive development in apple. Floral transition is a key developmental stage in the annual growth cycle of perennial fruit trees that directly determines the fruit development in the subsequent stage. FLOWERING LOCUS T (FT)/TERMINAL FLOWER1 (TFL1) family is known to play a vital regulatory role in plant growth and flowering. In apple, the two TFL1-like genes (MdTFL1-1 and MdTFL1-2) function as floral inhibitors; however, their mechanism of action is still largely unclear. This study aimed to functionally validate MdTFL1 and probe into its mechanism of action in apple. MdTFL1-1 and MdTFL1-2 were expressed mainly in stem and apical buds of vegetative shoots, with little expression in flower buds and young fruit. Expression of MdTFL1-1 and MdTFL1-2 rapidly decreased during floral induction. On the other hand, transgenic Arabidopsis, which ectopically expressed MdTFL1-1 or MdTFL1-2, flowered later than wild-type plants; demonstrating their in planta capability to function redundantly as flower repressors. Furthermore, we identified hundreds of novel interaction proteins of the two apple MdTFL1 proteins using yeast two-hybrid screens. Independent experiments for several proteins confirmed the yeast two-hybrid interactions. Among them, the transcription factor Nuclear Factor-Y subunit C (MdNF-YC2) functions as a promoter of flowering in Arabidopsis by activating LEAFY (LFY) and APETALA1 (AP1) expression. MdFT1 showed a similar interaction pattern as MdTFL1, implying a possible antagonistic action in the regulation of flowering. These newly identified TFL1-interacting proteins (TIPs) not only expand the floral regulatory network, but may also introduce new roles for TFL1 in plant development.

Transcriptome analysis reveals the inhibitory nature of high nitrate during adventitious roots formation in the apple rootstock.

In the process of vegetative propagation of apple rootstocks, the development of adventitious roots (ARs) has crucial importance. Nitrate is an essential nutrient necessary for plant growth; however, the inhibitory effect of high nitrate on ARs formation has not been explored. The physiological and molecular mechanisms underlying ARs inhibition were examined in this study. Stem cuttings of B9 apple rootstock were cultured on two nitrate treatments (T1 = 18.7 mM L-1 and T2 = 37.5 mM L-1 ), where T2 was identified as ARs inhibiting treatment. Morphological and anatomical observations advocating that high availability of nitrate inhibited AR formation by delaying the ARs initiation and emergence stages, where the root number was 287%, and the length was 604.6% lower than the T1 cuttings. Moreover, the contents of endogenous hormones were also elevated in response to T2 at most of the time points, which may cause a hormonal imbalance within the plant body and drive toward ARs inhibition. Furthermore, 3686 genes were differentially expressed by high-throughput sequencing. Out of these, 1797 genes were upregulated, and 1889 genes were downregulated. Approximately 238 genes related to nitrate, hormones, root development, and cell-cycle induction pathways were selected according to their potential to be involved in ARs regulation. This is the first study providing information regarding the inhibitory effect of high nitrate on ARs formation in apple rootstock.

Molecular mechanism of MdWUS2-MdTCP12 interaction in mediating cytokinin signaling to control axillary bud outgrowth.

Shoot branching is an important factor that influences the architecture of apple trees and cytokinin is known to promote axillary bud outgrowth. The cultivar 'Fuji', which is grown on ~75% of the apple-producing area in China, exhibits poor natural branching. The TEOSINTE BRANCHED1/CYCLOIDEA/PCF (TCP) family genes BRANCHED1/2 (BRC1/2) are involved in integrating diverse factors that function locally to inhibit shoot branching; however, the molecular mechanism underlying the cytokinin-mediated promotion of branching that involves the repression of BRC1/2 remains unclear. In this study, we found that apple WUSCHEL2 (MdWUS2), which interacts with the co-repressor TOPLESS-RELATED9 (MdTPR9), is activated by cytokinin and regulates branching by inhibiting the activity of MdTCP12 (a BRC2 homolog). Overexpressing MdWUS2 in Arabidopsis or Nicotiana benthamiana resulted in enhanced branching. Overexpression of MdTCP12 inhibited axillary bud outgrowth in Arabidopsis, indicating that it contributes to the regulation of branching. In addition, we found that MdWUS2 interacted with MdTCP12 in vivo and in vitro and suppressed the ability of MdTCP12 to activate the transcription of its target gene, HOMEOBOX PROTEIN 53b (MdHB53b). Our results therefore suggest that MdWUS2 is involved in the cytokinin-mediated inhibition of MdTCP12 that controls bud outgrowth, and hence provide new insights into the regulation of shoot branching by cytokinin.