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.

MdGRF11, a growth-regulating factor, participates in the regulation of flowering time and interacts with MdTFL1/MdFT1 in apple.

GROWTH-REGULATING FACTORs (GRFs) are plant-specific transcription factors that play important roles in regulating plant growth, development, and tolerance to stresses. However, there has been no in-depth research on the GRF genes and their roles in apple. A total of sixteen GRF genes were identified in the apple genome (GDDH13 V1.1), that expressed differentially in various tissues, in which the highest expression levels were observed particularly in shoot tips and apical buds. Among MdGRFs, the MdGRF11 was cloned and further investigated. Overexpression of the MdGRF11 in Arabidopsis plants promoted flowering, root elongation and leaf size. Further investigation indicated that MdGRF11 interacts with key flower genes FLOWERING LOCUS T (FT) and TERMINAL FLOWER1 (TFL1). The transient assays demonstrated that MdGRF11 represses the expression of knotted-like homeobox (MdKNOX19). Furthermore, MdTFL1 can compete with MdFT1 for complex formation with MdGRF11 to regulate the expression of MdKNOX19. Taken together, our results suggest that MdGRF11 protein is involved in fine-tuning of the floral transition possibly through interaction with the MdFT1 and MdTFL1 proteins.

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.

Genome-wide identification of the 14-3-3 gene family and its participation in floral transition by interacting with TFL1/FT in apple.

BACKGROUND: Apple (Malus domestica Borkh.) is a popular cultivated fruit crop with high economic value in China. Apple floral transition is an important process but liable to be affected by various environmental factors. The 14-3-3 proteins are involved in regulating diverse biological processes in plants, and some 14-3-3 members play vital roles in flowering. However, little information was available about the 14-3-3 members in apple. RESULTS: In the current study, we identified eighteen 14-3-3 gene family members from the apple genome database, designated MdGF14a to MdGF14r. The isoforms possess a conserved core region comprising nine antiparallel α-helices and divergent N and C termini. According to their structural and phylogenetic features, Md14-3-3 proteins could be classified into two major evolutionary branches, the epsilon (ɛ) group and the non-epsilon (non-ɛ) group. Moreover, expression profiles derived from transcriptome data and quantitative real-time reverse transcription PCR analysis showed diverse expression patterns of Md14-3-3 genes in various tissues and in response to different sugars and hormone treatments during the floral transition phase. Four Md14-3-3 isoforms (MdGF14a, MdGF14d, MdGF14i, and MdGF14j) exhibiting prominent transcriptional responses to sugars and hormones were selected for further investigation. Furthermore, yeast two-hybrid and bimolecular fluorescence complementation experiments showed that the four Md14-3-3 proteins interact with key floral integrators, MdTFL1 (TERMINAL FLOWER1) and MdFT (FLOWERING LOCUS T). Subcellular localization of four selected Md14-3-3 proteins demonstrated their localization in both the cytoplasm and nucleus. CONCLUSION: We identified the Md14-3-3 s family in apple comprehensively. Certain Md14-3-3 genes are expressed predominantly during the apple floral transition stage, and may participate in the regulation of flowering through association with flower control genes. Our results provide a preliminary framework for further investigation into the roles of Md14-3-3 s in floral transition.

Genome-wide identification and expression profiling of the YUCCA gene family in Malus domestica.

The plant hormone auxin is essential for plant growth and development. YUCCA proteins catalyse the rate-limiting step for endogenous auxin biosynthesis. In this study, we isolated 20 MdYUCCA genes from apple genome. MdYUCCA6a, MdYUCCA8a, and MdYUCCA10a were expressed in most organs and could support whole plant basal auxin synthesis. MdYUCCA4a, MdYUCCA10b, and MdYUCCA11a expression indicated roles for these genes in auxin biosynthesis in vegetative organs. MdYUCCA2b, MdYUCCA11b, and MdYUCCA11d were mainly expressed in flower organs. High temperature induced the expression of MdYUCCA4a, MdYUCCA6a, MdYUCCA8a, and MdYUCCA10a, and down-regulated the expression of MdYUCCA2b and MdYUCCA6b. Dual-luciferase assay indicated that MdPIF4 could trans-activate the MdYUCCA8a promoter. Overexpression of MdYUCCA8a increased IAA content, increased stem height, enhanced apical dominance, and led to silique malformation. These results provide a foundation for further investigation of the biological functions of apple MdYUCCAs.

Chronic cement dust load induce novel damages in foliage and buds of Malus domestica.

Cement industry-derived pollutants appear to play multiple roles in stimulating abiotic stress responses in plants. Cement dust deposition on agriculture fields can affect soils, photosynthesis, transpiration and respiration of plants. Here, we characterised the acute physiological responses of Malus × domestica leaves to different cement dust concentrations. The cement dust was sprinkled over plants daily for 2 months at 10 and 20 g/plant, with 0 g/plant serving as the control. Leaf physiological responses revealed significant increases in oxidative stress and antioxidant enzyme activity levels. Additionally, ascorbic acid, soluble sugar, free amino acid, and pigment levels decreased after exposure to cement dust. Macroscopic morphometric parameters, such as weight, dry matter content, and lengths and widths of leaves and buds, were significantly reduced in the cement-treated groups. A histological analysis of leaves and buds revealed decreased cellular areas, cellular damage, and abridged leaf thickness, while an ion leakage assay confirmed the negative effects on tissue integrity. These results provide evidence that cement dust is a hazardous pollutant that induces abiotic stress responses and has degradative effects on leaf health, pigment and biochemical metabolite levels, and anatomical features. Studies to determine the elemental residues of cement dust present in edible plant parts and the adverse impacts of their consumption on human health are strongly recommended.

Genome-Wide Identification of the MdKNOX Gene Family and Characterization of Its Transcriptional Regulation in Malus domestica.

Knotted1-like Homeobox (KNOX) proteins play important roles in regulating plant growth, development, and other biological processes. However, little information is available on the KNOX gene family in apple (Malus domestica Borkh.). In this study, 22 KNOX genes were identified in the apple genome. The gene structure, protein characteristics, and promoter region were characterized. The MdKNOX family members were divided into three classes based on their phylogenetic relationships. Quantitative real-time PCR analysis revealed that the majority of MdKNOX genes exhibited strongly preferential expression in buds and were significantly up-regulated during the flower induction period. The transcript levels of MdKNOX genes were responsive to treatments with flowering- and stress-related hormones. The putative upstream regulation factor MdGRF could directly bind to the promoter of MdKNOX15 and MdKNOX19, and inhibit their transcriptional activities, which were confirmed by yeast one-hybrid and dual-luciferase assays. The results provide an important foundation for future analysis of the regulation and functions of the MdKNOX gene family.

Cement dust induce stress and attenuates photosynthesis in Arachis hypogaea.

This study was conducted to investigate the changes in leaf physiological parameters to abiotic stress induced by different levels of cement dust. On day 15, Arachis hypogaea L. plants (sowing day was considered as day 0) were divided into six groups, and cement was sprinkled over plants with the help of hand pump, twice a week at T1 (5 g pot-1), T2 (8 g pot-1), T3 (10 g pot-1), T4 (15 g pot-1), T5 (20 g pot-1), and T0/control (0 g pot-1), until fruit maturity. Morphometric parameters such as root and shoot length, leaf area, and seed weight were significantly higher in T0, while the minimum was recorded in T5. Physiological analyses of leaves and roots revealed a remarkable reduction (p < 0.05) in sugar, amino acid, and protein contents, while the concentration of enzymatic antioxidants was increased in cement-treated plants. The concentration of abscisic acid in leaves was significantly higher in treatment groups as compared with control, while gibberellic acid concentration was low. Strikingly, cement dust decreases the level of leaf photosynthetic pigments, reduces stomatal conductance, and adversely affects photosynthesis. Leaf histological analysis revealed confirmatory evidence of stomatal closure, cell damage, reduced cell area, and abridged leaf thickness. Salient features of the present study provide useful evidence to estimate cement dust as a critical abiotic stress factor, which has adverse effects on photosynthesis, leaf anatomical features, stomatal functioning, and productivity. Our work opens new avenues for a deep portfolio of cement-based stress mediating pathophysiology in Arachis hypogaea.

Comparative RNA-Sequencing and DNA Methylation Analyses of Apple (Malus domestica Borkh.) Buds with Diverse Flowering Capabilities Reveal Novel Insights into the Regulatory Mechanisms of Flower Bud Formation.

In plants, DNA methylation (i.e. chromatin modification) is important for various biological processes, including growth, development and flowering. Because 'Fuji' apple trees are alternate bearing and have a long ripening period and poor-quality flower buds, we used bud types with diverse flowering capabilities to investigate the epigenetic regulatory mechanisms influencing flower bud formation. We examined the DNA methylation changes and the transcriptional responses in the selected apple bud types. We observed that in the apple genome, approximately 79.5%, 67.4% and 23.7% of the CG, CHG and CHH sequences are methylated, respectively. For each sequence context, differentially methylated regions exhibited distinct methylation patterns among the analyzed apple bud types. Global methylation and transcriptional analyses revealed that nonexpressed genes or genes expressed at low levels were highly methylated in the gene-body regions, suggesting that gene-body methylation is negatively correlated with gene expression. Moreover, genes with methylated promoters were more highly expressed than genes with unmethylated promoters, implying promoter methylation and gene expression are positively correlated. Additionally, flowering-related genes (e.g. SOC1, AP1 and SPLs) and some transcription factor genes (e.g. GATA, bHLH, bZIP and WOX) were highly expressed in spur buds (highest flowering rate), but were associated with low methylation levels in the gene-body regions. Our findings indicate a potential correlation between DNA methylation and gene expression in apple buds with diverse flowering capabilities, suggesting an epigenetic regulatory mechanism influences apple flower bud formation.

Transcriptomic analysis reveals the regulatory module of apple (Malus × domestica) floral transition in response to 6-BA.

BACKGROUND: Insufficient production of flower buds is an intractable problem in 'Fuji' apple orchards. Although cytokinin (CK) promotes flower bud formation in apple trees, little is known about the mechanisms regulating this phenomenon. RESULTS: In the present study, high-throughput RNA sequencing (RNA-Seq) of 'Nagafu No. 2' buds was conducted to characterize the transcriptional response to 6-BA treatment during key period of floral transition. A weighted gene co-expression network analysis (WGCNA) of the differentially expressed genes identified hormone signal transduction pathways, totaling 84 genes were highly correlated with the expression pattern of flowering-time genes. The up-regulation of CK signal components and a gibberellin (GA) signal repressor were found to contribute to the promotion of floral transition. In relative comparison to non-treated buds, a series of sugar metabolism- and signal- related genes were associated with relatively high levels of sucrose, fructose, and glucose during floral induction in the 6-BA treated buds. Several transcription factors (i.e. SPLs, SOC1, FD, and COL) that are involved in GA, aging, and photoperiod-regulated flowering pathways were also upregulated by the 6-BA treatment. In addition, potential transcription factors integrating CK signaling to trigger floral induction in apple were also assessed; including PHYTO-CHROME-INTERACTING FACTOR (PIF1,3), WUSCHEL-related homeobox (WOX3,13), and CK response regulators (ARR2). CONCLUSIONS: The present study provides insight into the response of flowering and development-related pathways and transcription factors to 6-BA during the period of floral transition in apple. It extends our knowledge of the fundamental mechanisms associated with CK-regulated floral transition in apple trees.

Expression of genes in the potential regulatory pathways controlling alternate bearing in 'Fuji' (Malus domestica Borkh.) apple trees during flower induction.

Most perennial fruit trees have an alternate bearing problem where a heavy fruit load is produced one year (ON year) but few flowers and fruits produced the next year (OFF year), resulting in a significant fluctuation in production. In the present study, comparative transcriptome analysis of terminal buds of apple (Malus domestica Borkh., cv. Nagafu No. 2) trees was conducted during the floral induction period in the ON and OFF years to identify the potential regulatory pathways controlling alternate bearing. A total of 1027 differentially expressed genes (DEGs), most of which were involved in secondary metabolism, sugar metabolism, plant hormone pathways, were identified. The analysis focused on differences in sugar content and hormone levels between the ON and OFF trees. Sucrose content, zeatin-riboside (ZR), and abscisic acid (ABA) levels were lower in ON-year buds than in OFF-year buds. ON buds also had elevated levels of gibberellins (GAs), with a higher expression of GA20 oxidase (GA20ox) and a significant lower level of RGA-like2 (RGL2). Expression analyses also revealed a significantly higher level of SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE genes (MdSPL1, MdSPL6 and MdSPL12) transcripts levels in buds of OFF trees at 45 days after full bloom (DAFB). LEAFY (LFY) expression increased significantly prior to flower induction in OFF buds. These findings provide new information of the role of hormones in alternate bearing, as well as other processes, and provide new insights into the molecular mechanisms regulating alternate bearing in perennial fruit trees.

Mediation of Flower Induction by Gibberellin and its Inhibitor Paclobutrazol: mRNA and miRNA Integration Comprises Complex Regulatory Cross-Talk in Apple.

Guaranteeing successful flowering is very important in economic plant species, especially apple (Malus domestica Borkh.), which is difficult to induce to flower. However, the gene expression and networks involved in flowering have not been totally characterized. Here, we employed mRNA and microRNA (miRNA) sequencing to understand the different responses to gibberellin- and its inhibitor paclobutrazol- (PAC) mediated flower induction. Significant opposite cytological and morphological changes were observed in treated terminal buds, which led to a reduced flowering rate under gibberellin and an increased flowering rate under PAC. We also found that the differentially expressed mRNAs, miRNAs and miRNA target genes participated in different biological networks including hormones, photosynthesis, redox state and other metabolic processes, which provided important clues to understand the complex networks involved in apple flower induction. Additionally, we subsequently focused on one important candidate, MdSPL3, which is one of 31 apple SPL gene family members and whose transcription was inhibited by gibberellin but promoted by PAC. Functional investigation showed that MdSPL3 was located in the nucleus, and ectopic MdSPL3 activated floral meristem identity genes, promoted the formation of floral primordia and led to an earlier flowering phenotype in Arabidopsis. Our research identified critical mRNA and miRNA responsive to gibberellin or PAC, and provided a candidate framework for flower induction. This carefully orchestrated regulatory cross-talk highlighted potential targets for developing regulatory techniques and genetic improvement of flower induction in apple.

Genome Identification of B-BOX Gene Family Members in Seven Rosaceae Species and Their Expression Analysis in Response to Flower Induction in Malus domestica.

BBX proteins play important roles in regulating plant growth and development including photomorphogenesis, photoperiodic regulation of flowering, and responses to biotic and abiotic stresses. At present, the genomes of seven Rosaceae fruit species have been fully sequenced. However, little is known about the BBX gene family and their evolutionary history in these Rosaceae species. Therefore, in this study total, 212 BBX genes were investigated from seven Rosaceae species (67 from Malus × domestica, 40 from Pyruscommunis, 22 from Rosa Chinesis, 20 from Prunuspersica, 21 from Fragariavesca, 22 from Prunusavium, and 20 from Rubusoccidentalis). The chemical properties, gene structures, and evolutionary relationships of the BBX genes were also studied. All the BBX genes were grouped into six subfamilies on the basis of their phylogenetic relationships and structural features. Analysis of gene structure, segmental and tandem duplication, gene phylogeny, and tissue-specific expression with the ArrayExpress database showed their diversification in function, quantity, and structure. The expression profiles of 19 MdBBX genes in different tissues were evaluated through qRT-PCR. These genes showed distinct transcription level among the tested tissues (bud, flower, fruit, stem, and leaf). Moreover, expression patterns of 19 MdBBX genes were examined during flowering induction time under flowering-related hormones and treatments (GA3, 6-BA, and sucrose). The expressions of the candidates BBX genes were affected and showed diverse expression profile. Furthermore, changes in response to these flowering-related hormones and treatment specifying their potential involvement in flowering induction. Based on these findings, BBX genes could be used as potential genetic markers for the growth and development of plants particularly in the area of functional analysis, and their involvement in flower induction in fruit plants.

Genome-wide analysis of carotenoid cleavage oxygenase genes and their responses to various phytohormones and abiotic stresses in apple (Malus domestica).

Carotenoid cleavage oxygenases (CCOs) are able to cleave carotenoids to produce apocarotenoids and their derivatives, which are important for plant growth and development. In this study, 21 apple CCO genes were identified and divided into six groups based on their phylogenetic relationships. We further characterized the apple CCO genes in terms of chromosomal distribution, structure and the presence of cis-elements in the promoter. We also predicted the cellular localization of the encoded proteins. An analysis of the synteny within the apple genome revealed that tandem, segmental, and whole-genome duplication events likely contributed to the expansion of the apple carotenoid oxygenase gene family. An additional integrated synteny analysis identified orthologous carotenoid oxygenase genes between apple and Arabidopsis thaliana, which served as references for the functional analysis of the apple CCO genes. The net photosynthetic rate, transpiration rate, and stomatal conductance of leaves decreased, while leaf stomatal density increased under drought and saline conditions. Tissue-specific gene expression analyses revealed diverse spatiotemporal expression patterns. Finally, hormone and abiotic stress treatments indicated that many apple CCO genes are responsive to various phytohormones as well as drought and salinity stresses. The genome-wide identification of apple CCO genes and the analyses of their expression patterns described herein may provide a solid foundation for future studies examining the regulation and functions of this gene family.