The plant-specific gibberellic acid stimulated Arabidopsis (GASA) gene family is critical for plant development. However, little is known about these genes, particularly in fruit tree species. We identified 15 putative Arabidopsis thaliana GASA (AtGASA) and 26 apple GASA (MdGASA) genes. The identified genes were then characterized (e.g., chromosomal location, structure, and evolutionary relationships). All of the identified A. thaliana and apple GASA proteins included a conserved GASA domain and exhibited similar characteristics. Specifically, the MdGASA expression levels in various tissues and organs were analyzed based on an online gene expression profile and by qRT-PCR. These genes were more highly expressed in the leaves, buds, and fruits compared with the seeds, roots, and seedlings. MdGASA genes were also responsive to gibberellic acid (GA3) and abscisic acid treatments. Additionally, transcriptome sequencing results revealed seven potential flowering-related MdGASA genes. We analyzed the expression levels of these genes in response to flowering-related treatments (GA3, 6-benzylaminopurine, and sugar) and in apple varieties that differed in terms of flowering (‘Nagafu No. 2’ and ‘Yanfu No. 6’) during the flower induction period. These candidate MdGASA genes exhibited diverse expression patterns. The expression levels of six MdGASA genes were inhibited by GA3, while the expression of one gene was up-regulated. Additionally, there were expression-level differences induced by the 6-benzylaminopurine and sugar treatments during the flower induction stage, as well as in the different flowering varieties. This study represents the first comprehensive investigation of the A. thaliana and apple GASA gene families. Our data may provide useful clues for future studies and may support the hypotheses regarding the role of GASA proteins during the flower induction stage in fruit tree species.

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.

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. 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). 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.

Grafting can improve the agricultural traits of crop plants, especially fruit trees. However, the regulatory networks and differentially expressed microRNAs (miRNAs) related to grafting in apple remain unclear. Herein, we conducted high-throughput sequencing and identified differentially expressed miRNAs among self-rooted Fuji, self-rooted M9, and grafted Fuji/M9. We analyzed the flowering rate, leaf morphology, and nutrient and carbohydrate contents in the three materials. The flowering rate, element and carbohydrate contents, and expression levels of flowering genes were higher in Fuji/M9 than in Fuji. We detected 206 known miRNAs and 976 novel miRNAs in the three materials, and identified those that were up- or downregulated in response to grafting. miR156 was most abundant in Fuji, followed by Fuji/M9, and then self-rooted M9, while miR172 was most abundant in M9, followed by Fuji/M9, and then Fuji. These expression patterns suggest that that these miRNAs were related to grafting. A Gene Ontology (GO) analysis showed that the differentially expressed miRNAs controlled genes involved in various biological processes, including cellular biosynthesis and metabolism. The expression of differentially expressed miRNAs and flowering-related genes was verified by qRT-PCR. Altogether, this comprehensive analysis of miRNAs related to grafting provides valuable information for breeding and grafting of apple and other fruit trees.