Proteome Analyses Using iTRAQ Labeling Reveal Critical Mechanisms in Alternate Bearing <i>Malus prunifolia</i>

Alternate bearing (AB) trees, including Malus prunifolia, are characterized by alternating cycles of heavy (ON tree) and low (OFF tree) fruit loads. The mechanisms regulating the AB phenomenon have not been fully characterized. We completed an iTRAQ-based investigation of M. prunifolia to identify the proteome and metabolic differences between the leaves of ON and OFF trees. We identified 667 differentially expressed proteins, and they influenced multiple biochemical pathways, including photosynthesis, carbohydrate metabolism, glycolysis, protein processing, redox activities, and secondary metabolism. Bioinformatics analyses indicated photosynthesis was the most significant biological process affecting the AB. We observed that 47 photosynthetic proteins affecting photosystem I and II reaction centers, cytochrome b6/f complex, electron transport, and light-harvesting chlorophyll were less abundant in ON tree leaves than in OFF tree leaves. Additionally, physiological analyses validated the potential metabolic activities. Nitrogen and phosphorus contents were significantly higher in ON tree leaves, while potassium levels were lower. Starch content, ZR, GA4+7 levels, and flower control gene expression levels (i.e., MdFT1, MdLFY, MdAP1, and MdSPL9) were lower in ON tree leaves than in OFF tree leaves, suggesting they affected the AB phenotype. Our findings help further investigate on the photosynthesis as well as other processes in AB. Those identified DEPs and important biological processes can be useful theoretical basis and provide new insights into the molecular mechanisms regulating AB in perennial woody plants

Proteome Analyses Using iTRAQ Labeling Reveal Critical Mechanisms in Alternate Bearing <i>Malus prunifolia</i>

Alternate bearing (AB) trees, including <i>Malus prunifolia</i>, are characterized by alternating cycles of heavy (ON tree) and low (OFF tree) fruit loads. The mechanisms regulating the AB phenomenon have not been fully characterized. We completed an iTRAQ-based investigation of <i>M. prunifolia</i> to identify the proteome and metabolic differences between the leaves of ON and OFF trees. We identified 667 differentially expressed proteins, and they influenced multiple biochemical pathways, including photosynthesis, carbohydrate metabolism, glycolysis, protein processing, redox activities, and secondary metabolism. Bioinformatics analyses indicated photosynthesis was the most significant biological process affecting the AB. We observed that 47 photosynthetic proteins affecting photosystem I and II reaction centers, cytochrome b6/f complex, electron transport, and light-harvesting chlorophyll were less abundant in ON tree leaves than in OFF tree leaves. Additionally, physiological analyses validated the potential metabolic activities. Nitrogen and phosphorus contents were significantly higher in ON tree leaves, while potassium levels were lower. Starch content, ZR, GA₄₊₇ levels, and flower control gene expression levels (i.e., <i>MdFT1</i>, <i>MdLFY</i>, <i>MdAP1</i>, and <i>MdSPL9</i>) were lower in ON tree leaves than in OFF tree leaves, suggesting they affected the AB phenotype. Our findings help further investigate on the photosynthesis as well as other processes in AB. Those identified DEPs and important biological processes can be useful theoretical basis and provide new insights into the molecular mechanisms regulating AB in perennial woody plants

Table_1_Genome-Wide Identification of the MdKNOX Gene Family and Characterization of Its Transcriptional Regulation in Malus domestica.docx

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.</p

Additional file 2: of Transcriptomic analysis reveals the regulatory module of apple (Malus × domestica) floral transition in response to 6-BA

Figure S1 Pearson correlation between sample replicates. C means control, B means 6-BA treatment. 0, 1, 2, 3 present the order of sampling time point. (PDF 292 kb

Data_Sheet_1_PpePL1 and PpePL15 Are the Core Members of the Pectate Lyase Gene Family Involved in Peach Fruit Ripening and Softening.DOCX

Pectin is the major component in the primary cell wall and middle lamella, maintaining the physical stability and mechanical strength of the cell wall. Pectate lyase (PL), a cell wall modification enzyme, has a major influence on the structure of pectin. However, little information and no comprehensive analysis is available on the PL gene family in peach (Prunus persica L. Batsch). In this study, 20 PpePL genes were identified in peach. We characterized their physicochemical characteristics, sequence alignments, chromosomal locations, and gene structures. The PpePL family members were classified into five groups based on their phylogenetic relationships. Among those, PpePL1, 9, 10, 15, and 18 had the higher expression abundance in ripe fruit, and PpePL1, 15, and 18 were upregulated during storage. Detailed RT-qPCR analysis revealed that PpePL1 and PpePL15 were responsive to ETH treatment (1 g L−1 ethephon) with an abundant transcript accumulation, which suggested these genes were involved in peach ripening and softening. In addition, virus-induced gene silencing (VIGS) technology was used to identify the roles of PpePL1 and PpePL15. Compared to controls, the RNAi fruit maintained greater firmness in the early storage stage, increased acid-soluble pectin (ASP), and reduced water-soluble pectin (WSP). Moreover, transmission electron microscopy (TEM) showed that cell wall degradation was reduced in the fruit of RNAi-1 and RNAi-15, which indicated that softening of the RNAi fruit has been delayed. Our results indicated that PpePL1 and PpePL15 play an important role in peach softening by depolymerizing pectin and degrading cell wall.</p

Additional file 4: of Transcriptomic analysis reveals the regulatory module of apple (Malus × domestica) floral transition in response to 6-BA

Figure S2 Comparison of expression profiles of representative genes as measured by RNA-seq and qRT-PCR. Columns represent expression determined by qRT-PCR (left y-axis). (A) Relative expression was calculated using the ACTIN housekeeping gene. (B) Relative expression was calculated using the HISTONE housekeeping gene. Values represent the means Âą SE of 3 biological replicates.Lines represent expression by RNA-seq in relative to FPKM values at first sample time point (right y-axis). Circles represent 6-BA treatment group, triangle represent control group. Correlations between qRT-PCR and RNA-seq expressions were calculated and their associated P-values are indicated. (PDF 272 kb

Additional file 11: of Transcriptomic analysis reveals the regulatory module of apple (Malus × domestica) floral transition in response to 6-BA

Table S9. Primers used for qRT-PCR assays in this study. (PDF 155 kb

Additional file 3: of Transcriptomic analysis reveals the regulatory module of apple (Malus × domestica) floral transition in response to 6-BA

Table S2 DEGs obtained by comparasion with B30 and C30, B50 and C50, and B70 and C70. (XLSX 1270 kb

Image_1_Genome-Wide Identification of the MdKNOX Gene Family and Characterization of Its Transcriptional Regulation in Malus domestica.tif

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.</p

Transcriptome Analysis Reveals New Insights into <i>MdBAK1</i>-Mediated Plant Growth in Malus domestica

BAK1 effects on plant stress responses have been well documented, but little is known regarding its effects on plant growth. In this study, we functionally characterized MdBAK1. Overexpressing MdBAK1 in Arabidopsis thaliana and apple trees promoted growth. Longitudinal stem cells were longer in transgenic plants than in wild-type plants. The size and number of cells and the area of the transverse stem were greater in the transgenic lines than in the wild-type plants. Moreover, transgenic A. thaliana and apple plants were more sensitive to an exogenous brassinosteroid. A transcriptome analysis of wild-type and transgenic apple revealed that MdBAK1 overexpression activated the brassinosteroid and ethylene signals, xylem production, and stress responses. Trend and Venn analyses indicated that carbohydrate, energy, and hormone metabolic activities were greater in transgenic plants during different periods. Moreover, a weighted gene coexpression network analysis proved that carbohydrate, hormone, and xylem metabolism as well as cell growth may be critical for MdBAK1-mediated apple tree growth and development. Compared with the corresponding levels in wild-type plants, the endogenous brassinosteroid, cytokinin, starch, sucrose, trehalose, glucose, fructose, and total sugar contents were considerably different in transgenic plants. Our results imply that MdBAK1 helps to regulate the growth of apple tree through the above-mentioned pathways. These findings provide new information regarding the effects of MdBAK1 onplant growth and development