Regulation Analysis Of Auxin-Related Transcription Factor Family Genes On Watermelon Fruit Expansion

Watermelon, being cultivated globally, is an important horticultural crop with significant economic value. Fruit expansion is one of the unique traits for Cucurbitaceae crops. Unveiling the expansion mechanism of watermelon fruit can provide valuable information not only for the studies on fruit development biology, but also for the genetic improvement on watermelon fruit traits. Plant hormones including auxin play crucial roles in watermelon fruit expansion. Auxin related transcription factors are the important regulatory genes that perceive auxin signal and regulate the expression of downstream genes. However, the questions that whether the auxin related transcription factor family genes are involved in the regulation of watermelon fruit expansion, which auxin related transcription factor family genes are involved, and how to regulate watermelon fruit expansion, are still unknown. To address these problems, the members of auxin related transcription factor families, including ARF, Aux/IAA, and GH3, were identified on watermelon genome. The relationships between expression patterns of these genes and the traits of fruit size, IAA content, and number and size of cells were analyzed during watermelon fruit development, with the aims of elucidating the regulation mechanisms of auxin related transcription factor family genes on watermelon fruit expansion at transcriptional level. The major results are as the following.1. Suitable reference genes systematically validated for relative gene expression quantification in q RT-PCR analysis of watermelon. To address the problem of lack of validated reference genes in watermelon in gene expression analysis by q RT-PCR, a total of 15 reference genes used in plant species were selected as the candidates, and their transcription abundances were measured in different organs of watermelon inbred line 97103 under normal growth conditions, or suffering abiotic and biotic stresses by q RT-PCR analysis. The algorithms of ge Norm and Norm Finder were used to evaluate expression stability for the candidate reference genes. Cl YLS8 and Cl PP2 A were ranked as the best reference gene combination under all conditions. The catalase family genes were identified on watermelon genome and their expression levels under low temperature stress were normalized by using the identified reference genes. The consistence of catalase gene expression profiles normalized by different top ranked genes further supported the reliability of the identified reference genes. To screen the most stable reference genes in watermelon fruit, a total of 12 commonly used reference genes were selected as the candidates, and their expression stability was evaluated and compared among 48 samples obtained from the fertilized fruits and parthenocarpic fruits of inbred line 97103 and F1 hybrid 8424. Ge Norm analysis showed that Cl TUA5, Cl CAC and Cl ACT were the best reference gene combination in watermelon fruit. Meanwhile, Norm Finder identified Cl CAC and Cl SAND as the best reference gene combination. A good correlation between lycopene contents and relative expression levels of Cl PSY1 normalized by the top ranked reference genes was observed during watermelon fruit development, which further validated the suitability of the identified reference genes.2. Dynamic analyses of the fruit traits during watermelon fruit development. Several fruit traits including fruit weight, fruit diameter, fruit longitude diameter, cell number, single cell area, cell ploidy, and IAA content, were measured during fruit development of 97103. The results showed that cell divisions and 2C ploidy cells were predominant before 7 DAP, and no significant changes on fruit weight were observed. The highest IAA content(68.89ng/g.fw) appeared on 12 h after pollination. During the mid-term of fruit development(7~20 DAP), cell enlargements were dominant. Cell number and single cell area increased greatly. The growth rate of fruit weight reached the highest level at 13 DAP. Meanwhile, the highest IAA content at this stage appeared at 15 DAP. The ratio of cells with 4C ploidy was higher than that of 2C. At the late stage of fruit development(20~34 DAP), no significant changes on cell enlargement and division were observed. IAA contents at this stage reached the highest level on 26 DAP. The ratio of cells with larger 4C ploidy was higher than that of 2C and 4C.3. ARF family genes identification and their expression analyses during watermelon development. A total of 17 ARF family genes were identified on watermelon genome by using ARF family HMM model. These family genes scattered on the 10 of all 11 watermelon chromosomes. Most of these genes were expressed in the vegetative organs including roots, leaves, stems, and tendrils of watermelon. However, only 13 and 11 genes were expressed in the female and male flowers, respectively. During watermelon fruit development, expression of 9 ARF genes was upregulated and showed positive correlation with fruit expansion. Expression of 6 ARF genes was upregulated during the early stage of fruit development, exhibiting positive correlation with cell division. No correlations were observed between the expression patterns and watermelon development for 5 ARF genes. Co-expression analyses were conducted for micro RNAs and their targeted ARF genes. During the late stage of fruit development, expression of mi RNA160 was upregulated, while expression of its targeted genes, namely Cl ARF6 and Cl ARF12, was down-regulated. Similarly, with the increasing expression of mi R167 a and mi R167 c, expression of their targeted gene Cl ARF2 decreased. These results demonstrated that both mi RNA and ARF participated in the regulation of watermelon fruit development on transcription level.4. Aux/IAA family genes identification and their expression analyses during watermelon development. A total of 38 Aux/IAA family genes were identified on watermelon genome, which scattered on the 10 watermelon chromosomes except for the No. 11 chromosome. There were 21, 24, 26, 14, 11, and 28 Aux/IAA genes that were expressed in roots, stems, leaves, female flowers, male flowers, and tendrils of watermelon, respectively. Expression of 4 genes was upregulated during the early stage of fruit development and showed positive correlation with cell division. Meanwhile, there were 7 genes exhibiting positive correlation with fruit expansion with their upregulating expression at this stage.5. GH3 family genes identification and their expression analyses during watermelon development. A total of 7 GH3 family genes were identified on watermelon genome, which scattered on the No.1, No. 3, No. 5, and No. 10 watermelon chromosome. There were 6, 4, 5, 4, 3, and 4 GH3 genes that were expressed in roots, stems, leaves, female flowers, male flowers, and tendrils of watermelon, respectively. Expression of Cl GH3-7 was upregulated during the early stage of fruit development. Cl GH3-2 exhibited upregulated expression during the mid-term and late stage of fruit developments.