Youmei Li

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.

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.

Photosynthetic response of tetraploid and hexaploid wheat to water stress

Photosynthetic characteristics of ear and flag leaves of wheat species, tetraploid Triticum dicoccoides Kom and hexaploid Bima1, were studied in plants grown under well-watered (WW) and water-stressed (WS) conditions. Compared to ears, flag leaves exhibited higher photosynthetic rate (PN) at the filling stage, but more severe decrease under WS. PN in the tetraploid wheat ear remained higher than that in the hexaploid wheat during the grain-filling stage. Water stress decreased PN in both the organs; this decline was caused by a reduction in Rubisco activity, not by drought-induced stomatal limitation. Tetraploid wheat ears exhibited higher relative water content and water-use efficiency than that of hexaploid wheat, under WS. The change in phosphoenolpyruvate carboxylase activity and carbon isotope composition indicated the absence of C4 metabolism in the ears of both species under both conditions. The improved performance of the tetraploid wheat ears under WS was associated with better water relations.

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.

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.