Caiping Zhao

Transcription Profiles Reveal Sugar and Hormone Signaling Pathways Mediating Flower Induction in Apple (Malus domestica Borkh.)

Flower induction in apple (Malus domestica Borkh.) is regulated by complex gene networks that involve multiple signal pathways to ensure flower bud formation in the next year, but the molecular determinants of apple flower induction are still unknown. In this research, transcriptomic profiles from differentiating buds allowed us to identify genes potentially involved in signaling pathways that mediate the regulatory mechanisms of flower induction. A hypothetical model for this regulatory mechanism was obtained by analysis of the available transcriptomic data, suggesting that sugar-, hormone- and flowering-related genes, as well as those involved in cell-cycle induction, participated in the apple flower induction process. Sugar levels and metabolism-related gene expression profiles revealed that sucrose is the initiation signal in flower induction. Complex hormone regulatory networks involved in cytokinin (CK), abscisic acid (ABA) and gibberellic acid pathways also induce apple flower formation. CK plays a key role in the regulation of cell formation and differentiation, and in affecting flowering-related gene expression levels during these processes. Meanwhile, ABA levels and ABA-related gene expression levels gradually increased, as did those of sugar metabolism-related genes, in developing buds, indicating that ABA signals regulate apple flower induction by participating in the sugar-mediated flowering pathway. Furthermore, changes in sugar and starch deposition levels in buds can be affected by ABA content and the expression of the genes involved in the ABA signaling pathway. Thus, multiple pathways, which are mainly mediated by crosstalk between sugar and hormone signals, regulate the molecular network involved in bud growth and flower induction in apple trees.

Genome-wide identification of vegetative phase transition-associated microRNAs and target predictions using degradome sequencing in Malus hupehensis

BackgroundA long juvenile period between germination and flowering is a common characteristic among fruit trees, including Malus hupehensis (Pamp.) Rehd., which is an apple rootstock widely used in China. microRNAs (miRNAs) play an important role in the regulation of phase transition and reproductive growth processes.ResultsM. hupehensis RNA libraries, one adult and one juvenile phase, were constructed using tree leaves and underwent high-throughput sequencing. We identified 42 known miRNA families and 172 novel miRNAs. We also identified 127 targets for 25 known miRNA families and 168 targets for 35 unique novel miRNAs using degradome sequencing. The identified miRNA targets were categorized into 58 biological processes, and the 123 targets of known miRNAs were associated with phase transition processes. The KEGG analysis revealed that these targets were involved in starch and sucrose metabolism, and plant hormone signal transduction. Expression profiling of miRNAs and their targets indicated multiple regulatory functions in the phase transition. The higher expression level of mdm-miR156 and lower expression level of mdm-miR172 in the juvenile phase leaves implied that these two small miRNAs regulated the phase transition. mdm-miR160 and miRNA393, which regulate genes involved in auxin signal transduction, could also be involved in controlling this process. The identification of known and novel miRNAs and their targets provides new information on this regulatory process in M. hupehensis, which will contribute to the understanding of miRNA functions during growth, phase transition and reproduction in woody fruit trees.ConclusionsThe combination of sRNA and degradome sequencing can be used to better illustrate the profiling of hormone-regulated miRNAs and miRNA targets involving complex regulatory networks, which will contribute to the understanding of miRNA functions during growth, phase transition and reproductive growth in perennial woody fruit trees.

Expression analysis of key auxin synthesis, transport, and metabolism genes in different young dwarfing apple trees

The control of scion vigor by dwarfing apple rootstocks is most convincingly elucidated by the shoot–root–shoot signaling of auxins and other hormones. To identify auxin and auxin-related genes that may play roles in the composite tree’s auxin metabolism and dwarfing mechanism, the concentrations of IAA and the expression level of key auxin synthesis, transport, and metabolism genes were measured in leaves, phloem, and roots from the dwarfing Fuji/M9 and the vigorous Fuji/MM106. The results showed that the indole-3-acetic acid (IAA) content was lower in the dwarfing Fuji/M9 than in the vigorous Fuji/MM106. The IAA content in the Fuji/M9 rootstock’s phloem was higher than that of its scion phloem. The expression level of MdYUCCA10a gene was significantly lower in the leaves and roots of Fuji/M9 than in that of the Fuji/MM106. The phloem and roots of the Fuji/M9 rootstock showed low expression levels of MdPIN1b and MdPIN8a. The auxin-conjugated genes MdGH3-5b and MdGH3-9a showed lower expression levels in the Fuji/M9 than in the Fuji/MM106. However, the Fuji/M9 showed higher levels of the auxin-conjugate hydrolase genes MdIAR3c and MdILL6c. The low expression level of auxin synthesis gene MdYUCCA10a in Fuji/M9 probably induced the low auxin level. The lower expression levels of auxin transport genes MdPIN1b and MdPIN8a in the M9 rootstock were suggested to probably contribute to auxin accumulation in Fuji/M9 rootstock phloem. The low amount of auxin transported from the shoots along with the root auxin synthesis deficiencies reduced the root growth and then decreased the supply of root-produced substances to the shoots in Fuji/M9.

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.

Optimizing planting density for production of high-quality apple nursery stock in China

In China, apple (Malus × domestica Borkh.) nursery stock is generally of low quality because of extremely high planting density. The objective of this study was to determine the optimum planting density of 2-year-old grafted apple trees. Tree growth (height, trunk diameter, leaf area index) increased as density decreased. Trees grown at high densities (14.3–50 plants/m2) were the shortest with the smallest trunk diameters and leaf areas, whereas trees grown at lower densities (4.8–10 plants/m2) were generally largest in terms of height, diameter and leaf area. Trees grown at lower densities tended to have higher bud dry weight, leaf dry weight, nitrogen content, total soluble sugar concentration and total non-structural carbohydrate content. Higher levels of these parameters were generally observed with tree densities at or below 10 plants/m2. Therefore we conclude that 10 plants/m2 is the optimum density for maximizing the number of trees produced per unit land area while maintaining tree quality of nursery stock.

Genome-wide identification and expression profiling analysis of brassinolide signal transduction genes regulating apple tree architecture

Brassinolide (BR) is crucial for regulating plant architecture. Apple dwarfing rootstocks are used to control apple tree size. However, information regarding the effects of BR on apple trees is limited. In addition, the molecular mechanism underlying the dwarfing of apple rootstocks is poorly understood. To elucidate the role of BR signal transduction genes in controlling apple tree architecture, five BR receptor kinase 1 (BRI1), nine BR-signaling kinase 1 (BSK1), two BRI1 KINASE INHIBITOR 1 (BKI1), and seven BR-insensitive 2 (BIN2) genes were analyzed. Bioinformatic analyses revealed that gene duplication events likely contributed to the expansion and evolution of the identified genes. Nine homologs between apple and Arabidopsis thaliana were also identified, and their expression patterns in different tissues were characterized. Exogenous BR treatments increased the primary shoot length and altered the expression of BR signal transduction genes (MdBRI1-5, MdBSK3-8, MdBKI1–2, MdBIN1–4, and MdBIN6/7). The scion of Fuji/Malling 9 (M.9) trees exhibited inhibited growth compared with that of Fuji/Fuji trees. The Fuji/M.9 trees had lower levels of the positive regulators of BR signaling (MdBRI1-5,MdBSK1, MdBSK4/7, and MdBSK6) and higher levels of the negative regulators (MdBIN5-7) compared with the Fuji/Fuji trees. Thus, the above-mentioned genes may help to regulate apple tree size in response to BR. In addition, MdBRI1–5, MdBSK1, MdBSK4/7, MdBSK6, and MdBIN5–7 have important roles in different grafting combinations. Our results may provide the basis for future analyses of BR signal transduction genes regarding their potential involvement in the regulation of plant architecture.

Identification and Expression Analysis of Polygalacturonase Family Members during Peach Fruit Softening

Polygalacturonase (PG) is an important hydrolytic enzyme involved in pectin degradation during fruit softening. However, the roles of PG family members in fruit softening remain unclear. We identified 45 PpPG genes in the peach genome which are clustered into six subclasses. PpPGs consist of four to nine exons and three to eight introns, and the exon/intron structure is basically conserved in all but subclass E. Only 16 PpPG genes were expressed in ripening fruit, and their expression profiles were analyzed during storage in two peach cultivars with different softening characteristics. Eight PGs (PpPG1, -10, -12, -13, -15, -23, -21, and -22) in fast-softening “Qian Jian Bai” (QJB) fruit and three PGs (PpPG15, -21, and -22) in slow-softening “Qin Wang” (QW) fruit exhibited softening-associated patterns; which also were affected by ethylene treatment. Our results suggest that the different softening characters in QW and QJB fruit is related to the amount of PG members. While keeping relatively lower levels during QW fruit softening, the expression of six PGs (PpPG1, -10, -12, -11, -14, and -35) rapidly induced by ethylene. PpPG24, -25 and -38 may not be involved in softening of peach fruit.

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. BIOLOGICAL SIGNIFICANCE Brassinosteroid 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.

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.