Mingyu Han

Exogenous Salicylic Acid Alleviates Growth Inhibition and Oxidative Stress Induced by Hypoxia Stress in Malus robusta Rehd

Salicylic acid (SA) as a signal molecule mediates many biotic and environmental stress-induced physiologic responses in plants. In this study we investigated the role of SA in regulating growth and oxidative stress in Malus robusta Rehd under both normoxic and hypoxic conditions. Hypoxia stress inhibited plant growth and dramatically reduced biomass. Addition of SA significantly alleviated the plant growth inhibition. The amounts of superoxide radicals (O2−) and hydrogen peroxide (H2O2) significantly increased in leaves of the plants exposed to hypoxia stress and resulted in oxidative stress, which was indicated by accumulated concentration of malondialdehyde (MDA) and electrolyte leakage. Addition of SA significantly decreased the level of O2−, electrolyte leakage, and lipid peroxidation and enhanced the activities of superoxide dismutase (SOD), peroxidase (POD), and ascorbate peroxidase (APX) under hypoxia stress. As important antioxidants, ascorbate (AsA) and glutathione (GSH) contents in the plant leaves were slightly increased by SA treatment compared to hypoxia stress treatment alone. It was concluded that SA could alleviate the detrimental effects of hypoxia stress on plant growth and of oxidative stress by enhancing the antioxidant defense system in leaves of M. robusta Rehd.

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.

miRNA and Degradome Sequencing Reveal miRNA and Their Target Genes That May Mediate Shoot Growth in Spur Type Mutant “Yanfu 6”

The spur-type growth habit in apple trees is characterized by short internodes, increased number of fruiting spurs, and compact growth that promotes flowering and facilitates management practices, such as pruning. The molecular mechanisms responsible for regulating spur-type growth have not been elucidated. In the present study, miRNAs and the expression of their potential target genes were evaluated in shoot tips of “Nagafu 2” (CF) and spur-type bud mutation “Yanfu 6” (YF). A total of 700 mature miRNAs were identified, including 202 known apple miRNAs and 498 potential novel miRNA candidates. A comparison of miRNA expression in CF and YF revealed 135 differentially expressed genes, most of which were downregulated in YF. YF also had lower levels of GA, ZR, IAA, and ABA hormones, relative to CF. Exogenous applications of GA promoted YF shoot growth. Based on the obtained results, a regulatory network involving plant hormones, miRNA, and their potential target genes is proposed for the molecular mechanism regulating the growth of YF. miRNA164, miRNA166, miRNA171, and their potential targets, and associated plant hormones, appear to regulate shoot apical meristem (SAM) growth. miRNA159, miRNA167, miRNA396, and their potential targets, and associated plant hormones appear to regulate cell division and internode length. This study provides a foundation for further studies designed to elucidate the mechanism underlying spur-type apple architecture.

Identification and expression analysis of the IPT and CKX gene families during axillary bud outgrowth in apple (Malus domestica Borkh.)

Cytokinins (CKs) play a crucial role in promoting axillary bud outgrowth and targeting the control of CK metabolism can be used to enhance branching in plants. CK levels are maintained mainly by CK biosynthesis (isopentenyl transferase, IPT) and degradation (dehydrogenase, CKX) genes in plants. A systematic study of the IPT and CKX gene families in apple, however, has not been conducted. In the present study, 12 MdIPTs and 12 MdCKXs were identified in the apple genome. Systematic phylogenetic, structural, and synteny analyses were performed. Expression analysis of these genes in different tissues was also assessed. MdIPT and MdCKX genes exhibit distinct expression patterns in different tissues. The response of MdIPT, MdCKX, and MdPIN1 genes to various treatments (6-BA, decapitation and Lovastatin, an inhibitor of CKs synthesis) that impact branching were also investigated. Results indicated that most of the MdIPT and MdCKX, and MdPIN1 genes were upregulated by 6-BA and decapitation treatment, but inhibited by Lovastatin, a compound that effectively suppresses axillary bud outgrowth induced by decapitation. These findings suggest that cytokinin biosynthesis is required for the activation of bud break and the export of auxin from buds in apple tree with intact primary shoot apex or decapitated apple tree. MdCKX8 and MdCKX10, however, exhibited little response to decapitation, but were significantly up-regulated by 6-BA and Lovastatin, a finding that warrants further investigation in order to understand their function in bud-outgrowth.

Identification of Peach NAP Transcription Factor Genes and Characterization of their Expression in Vegetative and Reproductive Organs during Development and Senescence

The NAP (NAC-like, activated by AP3/P1) transcription factor belongs to a subfamily of the NAC transcription factor family, and is believed to have an important role in regulating plant growth and development. However, there is very little information about this subfamily in Rosaceous plants. We identified seven NAP genes in the peach genome. PpNAP2 was categorized in the NAP I group, and contained a conserved transcription activation region. The other PpNAP genes belonged to the NAP II group. The expression patterns of the PpNAP genes differed in various organs and developmental stages. PpNAP1 and PpNAP2 were highly expressed in mature and senescing flowers, but not in leaves, fruits, and flower buds. PpNAP3 and PpNAP5 were only expressed in leaves. The PpNAP4 expression level was high in mature and senescing fruits, while PpNAP6 and PpNAP7 expression was up-regulated in mature and senescent leaves and flowers. During the fruit development period, the PpNAP4 and PpNAP6 expression levels rapidly increased during the S1 and S4 stages, which suggests these genes are involved in the first exponential growth phase and fruit ripening. During the fruit ripening and softening period, the PpNAP1, PpNAP4, and PpNAP6 expression levels were high during the early storage period, which was accompanied by a rapid increase in ethylene production. PpNAP1, PpNAP4, and PpNAP6 expression slowly increased during the middle or late storage periods, and peaked at the end of the storage period. Additionally, abscisic acid (ABA)-treated fruits were softer and produced more ethylene than the controls. Furthermore, the PpNAP1, PpNAP4, and PpNAP6 expression levels were higher in ABA-treated fruits. These results suggest that PpNAP1, PpNAP4, and PpNAP6 are responsive to ABA and may regulate peach fruit ripening.

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.

Revealing critical mechanisms of BR-mediated apple nursery tree growth using iTRAQ-based proteomic analysis

Brassinosteroid is identified as an important hormone. However, information about brassinosteroid has not been fully elucidated, and few studies concerned its role in apple. The aim of this work was to study the role of brassinosteroid for apple tree growth. In our study, the effect of brassinosteroid on apple nursery tree was analyzed. The biomass, cell size and xylem content of apple nursery tree were obviously evaluated by brassinosteroid treatment; mineral elements contents, photosynthesis indexes, carbohydrate level and hormone contents were significantly high in brassinosteroid treated trees. To explore the molecular mechanisms of these phenotypic differences, iTRAQ-based quantitative proteomics were used to identify the expression profiles of proteins in apple nursery tree shoot tips in response to brassinosteroid at a key period (14days after brassinosteroid treatment). A total of 175 differentially expressed proteins were identified. They were mainly involved in chlorophyII biosynthesis, photosynthesis, carbohydrate metabolism, glycolysis, citric acid cycle, respiratory action, hormone signal, cell growth and ligin metabolism. The findings in this study indicate that brassinosteroid mediating apple nursery tree growth may be mainly through energy metabolism. Important biological processes identified here can be useful theoretical basis and provide new insights into the molecular mechanisms of brassinosteroid.nnnBIOLOGICAL SIGNIFICANCEnBrassinosteroid is very important for plant growth and development. However, the molecular mechanism of brassinosteroid mediating growth process is not perfectly clear in plant, especially in apple nursery tree. We used a combination of physiological and bioinformatics analysis to investigate the effects of brassinosteroid on apple nursery tree growth and development. The data reported here demonstrated that brassinosteroid regulates apple nursery tree growth mainly through energy metabolism. Therefore it can provide a theoretical basis from energy points for developing dwarfed or compact apple trees. This will benefit for low orchard management cost as well as early bearing, and high fruit yield as well as quality.

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.