Sheng Fan

Identification, Classification, and Expression Analysis of GRAS Gene Family in Malus domestica

GRAS genes encode plant-specific transcription factors that play important roles in plant growth and development. However, little is known about the GRAS gene family in apple. In this study, 127 GRAS genes were identified in the apple (Malus domestica Borkh.) genome and named MdGRAS1 to MdGRAS127 according to their chromosomal locations. The chemical characteristics, gene structures and evolutionary relationships of the MdGRAS genes were investigated. The 127 MdGRAS genes could be grouped into eight subfamilies based on their structural features and phylogenetic relationships. Further analysis of gene structures, segmental and tandem duplication, gene phylogeny and tissue-specific expression with ArrayExpress database indicated their diversification in quantity, structure and function. We further examined the expression pattern of MdGRAS genes during apple flower induction with transcriptome sequencing. Eight higher MdGRAS (MdGRAS6, 26, 28, 44, 53, 64, 107, and 122) genes were surfaced. Further quantitative reverse transcription PCR indicated that the candidate eight genes showed distinct expression patterns among different tissues (leaves, stems, flowers, buds, and fruits). The transcription levels of eight genes were also investigated with various flowering related treatments (GA3, 6-BA, and sucrose) and different flowering varieties (Yanfu No. 6 and Nagafu No. 2). They all were affected by flowering-related circumstance and showed different expression level. Changes in response to these hormone or sugar related treatments indicated their potential involvement during apple flower induction. Taken together, our results provide rich resources for studying GRAS genes and their potential clues in genetic improvement of apple flowering, which enriches biological theories of GRAS genes in apple and their involvement in flower induction of fruit trees.

Phylogenetic analysis of IDD gene family and characterization of its expression in response to flower induction in Malus

Although INDETERMINATE DOMAIN (IDD) genes encoding specific plant transcription factors have important roles in plant growth and development, little is known about apple IDD (MdIDD) genes and their potential functions in the flower induction. In this study, we identified 20 putative IDD genes in apple and named them according to their chromosomal locations. All identified MdIDD genes shared a conserved IDD domain. A phylogenetic analysis separated MdIDDs and other plant IDD genes into four groups. Bioinformatic analysis of chemical characteristics, gene structure, and prediction of protein–protein interactions demonstrated the functional and structural diversity of MdIDD genes. To further uncover their potential functions, we performed analysis of tandem, synteny, and gene duplications, which indicated several paired homologs of IDD genes between apple and Arabidopsis. Additionally, genome duplications also promoted the expansion and evolution of the MdIDD genes. Quantitative real-time PCR revealed that all the MdIDD genes showed distinct expression levels in five different tissues (stems, leaves, buds, flowers, and fruits). Furthermore, the expression levels of candidate MdIDD genes were also investigated in response to various circumstances, including GA treatment (decreased the flowering rate), sugar treatment (increased the flowering rate), alternate-bearing conditions, and two varieties with different-flowering intensities. Parts of them were affected by exogenous treatments and showed different expression patterns. Additionally, changes in response to alternate-bearing and different-flowering varieties of apple trees indicated that they were also responsive to flower induction. Taken together, our comprehensive analysis provided valuable information for further analysis of IDD genes aiming at flower induction.

Identification and expression analysis of cytokinin response-regulator genes during floral induction in apple (Malus domestica Borkh)

Although cytokinin response-regulators (RRs) are key components in cytokinin (CK) signal transduction pathway that modulate diverse developmental processes in plants, little is known about apple RR genes and their potential function in floral induction. In the present study, 34 putative RR genes were identified in apple and divided into three types: 19 A-type MdRRA, 11 B-type MdRRB, and 8 pseudo-response regulators (MdPRR). The expression pattern of all of the MdRRB genes, two MdRRA genes, and flowering-time genes were investigated in response to CK during floral induction in two different apple varieties. Results indicated that two MdRRA genes were immediately induced by the 6-BA treatment, while none of the MdRRB was. However the MdRRB genes were expressed at higher levels in 6-BA-treated buds and ‘Yanfu No. 6’ buds at most sampling time points. Moreover, the expression of MdSOC1a and MdSOC1b were also affected by the 6-BA treatment. Promoter regions of MdSOC1a and MdSOC1b were identified and analyzed; indicating that extended ARR1-binding sequences are located in the MdSOC1a and MdSOC1b. These results suggest that some B-type MdRR genes (MdRRB9 and MdRRB11; which were grouped into a cluster with ARR1) and flowering-time genes may be involved in the positive effect of CK on apple floral induction.

Genome-wide identification, evolution, and expression analysis of GATA transcription factors in apple (Malus × domestica Borkh.)

Plant GATA transcription factors are type-IV zinc-finger proteins that play important regulatory roles in plant growth and development. In this study, we identified 35 GATA genes classified into four groups in the whole genome sequence of Malus domestica. A physiochemical property analysis indicated that GATA proteins are largely unstable hydrophilic proteins. An analysis of conserved protein motifs uncovered three highly conserved motifs, in addition to the GATA motif, in all MdGATA proteins. These three motifs, CCT, TIFY, and ASXH, were found to occur in specific GATA groups and may be related to GATA gene function. We identified 10 pairs of putative paralogs, indicating that MdGATA genes have mainly undergone whole genome duplication. Eighteen orthologous gene pairs were also identified between Arabidopsis thaliana and M. domestica. Furthermore, many light-responsive cis-elements were found in MdGATA gene promoters. Tissue-specific expression analysis performed by quantitative real-time reverse transcription PCR showed that MdGATA genes were preferentially expressed in flowers, leaves, and buds. Apple seedlings maintained in darkness for 7days exhibited a moderate decline in chlorophyll content along with significant down-regulation of most MdGATA genes, suggesting that MdGATA genes may be involved in light-responsive development and chlorophyll-level regulation. The distinctly higher expression levels observed for many MdGATA genes during three stages of floral induction also indicate that MdGATA genes may play a role in the apple flowering transition. The results presented here lay the foundation for further investigation of MdGATA gene family putative functions and improvement of apple yields.

Comprehensive analysis of GASA family members in the Malus domestica genome: identification, characterization, and their expressions in response to apple flower induction

BackgroundThe 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.ResultsWe 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.ConclusionThis 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.

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.

Genome-wide identification, characterization and expression analysis of long non-coding RNAs in different tissues of apple

Recently, the long non-coding RNAs (lncRNAs), which play important roles in various complex biological processes, have received more attention in plants. However, little information is available on lncRNAs in woody fruit trees and their potential regulatory roles remain poorly understood, especially in apple. Here, a total of 1726 high-confidence lncRNAs (hc-lncRNAs) were identified in different tissues including young fruits, shoot tips, stem phloem and root tips using high-throughput sequencing. These lncRNAs are distributed across all 17 apple chromosomes, and >85% come from intergenic regions. The apple lncRNAs have longer transcript lengths and greater exon numbers than protein-coding genes. Additionally, among the 1726 hc-lncRNA, 850 are predicted to have target genes. These target genes are involved in many processes including hormone signaling, sugar metabolism, and the cell cycle and stress responses. Furthermore, 57, 74, 168 and 78 lncRNAs specifically expressed in root tips, shoot tips, young fruits and stem phloem were analyzed using the COG (cluster of orthologous group)and GO (gene ontology) databases. Young fruits contain the most unique lncRNAs, which are involved in biological processes such as energy production and conversion, carbohydrate transport and metabolism, posttranslational modification and protein turnover. Quantitative real-time PCR (qRT-PCR) is employed to confirm the different expression levels among the four tissues. Moreover, the expression levels of eight fruit-related lncRNAs are investigated during different fruit development stages, which indicates they have important roles in fruit ripening and sugar metabolism. Overall, our genome-wide research on lncRNAs in different apple tissues provides valuable clues and information that can help elucidate the potential roles of lncRNAs in the growth and development of apple, as well as in other 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.

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.

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 Pyrus communis, 22 from Rosa Chinesis, 20 from Prunus persica, 21 from Fragaria vesca, 22 from Prunus avium, and 20 from Rubus occidentalis). 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.