Transcriptomic Analysis of Red-Fleshed Apples Reveals the Novel Role of MdWRKY11 in Flavonoid and Anthocyanin Biosynthesis

In plants, flavonoids are important secondary metabolites that contribute to the nutritional quality of many foods. Apple is a popular and frequently consumed food because of its high flavonoid content. In this study, flavonoid composition and content were detected and compared between red- and white-fleshed apples in a BC₁ hybrid population using ultraperformance liquid chromatography–quadrupole time-of-flight mass spectrometry. Transcriptomic analysis of the red- and white-fleshed apples was then performed using RNA-seq technology. By screening differentially expressed genes encoding transcription factors, we unearthed a WRKY-family transcription factor designated MdWRKY11. Overexpression of MdWRKY11 promoted the expression of <i>F3H</i>, <i>FLS</i>, <i>DFR</i>, <i>ANS</i>, and <i>UFGT</i> and increased the accumulation of flavonoids and anthocyanin in apple calli. Our findings explored the novel role of MdWRKY11 in flavonoid biosynthesis and suggest several other genes that may also be potentially involved. This provides valuable information on flavonoid synthesis for the breeding of elite red-fleshed apples

Transcriptomic Analysis of Red-Fleshed Apples Reveals the Novel Role of MdWRKY11 in Flavonoid and Anthocyanin Biosynthesis

In plants, flavonoids are important secondary metabolites that contribute to the nutritional quality of many foods. Apple is a popular and frequently consumed food because of its high flavonoid content. In this study, flavonoid composition and content were detected and compared between red- and white-fleshed apples in a BC₁ hybrid population using ultraperformance liquid chromatography–quadrupole time-of-flight mass spectrometry. Transcriptomic analysis of the red- and white-fleshed apples was then performed using RNA-seq technology. By screening differentially expressed genes encoding transcription factors, we unearthed a WRKY-family transcription factor designated MdWRKY11. Overexpression of MdWRKY11 promoted the expression of <i>F3H</i>, <i>FLS</i>, <i>DFR</i>, <i>ANS</i>, and <i>UFGT</i> and increased the accumulation of flavonoids and anthocyanin in apple calli. Our findings explored the novel role of MdWRKY11 in flavonoid biosynthesis and suggest several other genes that may also be potentially involved. This provides valuable information on flavonoid synthesis for the breeding of elite red-fleshed apples

Comparative Transcriptomes Analysis of Red- and White-Fleshed Apples in an F₁ Population of <i>Malus sieversii f</i>. <i>niedzwetzkyana</i> Crossed with <i>M</i>. <i>domestica</i> ‘Fuji’

<div><p>Transcriptome profiles of the red- and white-fleshed apples in an F₁segregating population of <i>Malus sieversii f</i>.<i>Niedzwetzkyana</i> and <i>M</i>.<i>domestica</i> ‘Fuji’ were generated using the next-generation high-throughput RNA sequencing (RNA-Seq) technology and compared. A total of 114 differentially expressed genes (DEGs) were obtained, of which 88 were up-regulated and 26 were down-regulated in red-fleshed apples. The 88 up-regulated genes were enriched with those related to flavonoid biosynthetic process and stress responses. Further analysis identified 22 genes associated with flavonoid biosynthetic process and 68 genes that may be related to stress responses. Furthermore, the expression of 20 up-regulated candidate genes (10 related to flavonoid biosynthesis, two encoding MYB transcription factors and eight related to stress responses) and 10 down-regulated genes were validated by quantitative real-time PCR. After exploring the possible regulatory network, we speculated that flavonoid metabolism might be involved in stress responses in red-fleshed apple. Our findings provide a theoretical basis for further enriching gene resources associated with flavonoid synthesis and stress responses of fruit trees and for breeding elite apples with high flavonoid content and/or increased stress tolerances.</p></div

Phylogenetic analysis of MYB transcription factors in apple and their homologues in <i>Arabidopsis</i>.

<p>Phylogenetic analysis of MYB transcription factors in apple and their homologues in <i>Arabidopsis</i>.</p

Up-regulated genes involved in both the flavonoid biosynthetic process and stress response in red-fleshed apples.

<p>Up-regulated genes involved in both the flavonoid biosynthetic process and stress response in red-fleshed apples.</p

Hypersensitive Ethylene Signaling and <em>ZMdPG1</em> Expression Lead to Fruit Softening and Dehiscence

<div><p>‘Taishanzaoxia’ fruit rapid softening and dehiscence during ripening stage and this process is very sensitive to endogenous ethylene. In this study, we cloned five ethylene signal transcription factors (<i>ZMdEIL1, ZMdEIL2, ZMdEIL3, ZMdERF1</i> and <i>ZMdERF2</i>) and one functional gene, <i>ZMdPG1</i>, encoding polygalacturonase that could loose the cell connection which associated with fruit firmness decrease and fruit dehiscence to illustrate the reasons for this specific fruit phenotypic and physiological changes. Expression analysis showed that <i>ZMdERF1</i> and <i>ZMdEIL2</i> transcription were more abundant in ‘Taishanzaoxia’ softening fruit and dehiscent fruit and their expression was inhibited by an ethylene inhibitor 1-methylcyclopropene. Therefore, <i>ZMdERF1</i> and <i>ZMdEIL2</i> expression were responses to endogenous ethylene and associated with fruit softening and dehiscence. <i>ZMdPG1</i> expression was induced when fruit softening and dehiscence but this induction can be blocked by 1-MCP, indicating that <i>ZMdPG1</i> was essential for fruit softening and dehiscence and its expression was mediated by the endogenously occurred ethylene. <i>ZMdPG1</i> overexpression in <i>Arabidopsis</i> led to silique early dehiscence while suppressing <i>ZMdPG1</i> expression by antisense <i>ZMdPG1</i> prevented silique naturally opening. The result also suggested that <i>ZMdPG1</i> related with the connection between cells that contributed to fruit softening and dehiscence. <i>ZMdERF1</i> was more closely related with ethylene signaling but it was not directly regulated the <i>ZMdPG1</i>, which might be regulated by the synergic pattern of ethylene transcription factors because of both the ZMdERF1 and ZMdERF2 could interact with ZMdEIL2.</p> </div

The comparison of ethylene production and fruit firmness in ‘Taishanzaoxia’ and ‘Liaofu’ cultivar.

<p>(A) Changes of fruit firmness and ethylene production in different apple cultivars during fruit development. (B) Effect of different levers 1-MCP on ethylene production in ‘Taishanzaoxia’. (C) Effect of different levers 1-MCP on fruit firmness in ‘Taishanzaoxia’. TS represent ‘Taishanzaoxia’ cultivar. LF represents ‘Liaofu’ cultivar.</p

Model of the regulatory network involved in flavonoid metabolism and stress responses.

<p>Model of the regulatory network involved in flavonoid metabolism and stress responses.</p

GO terms enriched in differentially expressed genes between red- and white-fleshed apples.

<p>(A) GO terms enriched in up-regulated genes in red-fleshed apple. (B) GO terms enriched in down-regulated genes in red-fleshed apple.</p

Red- and white-fleshed apples at the ripe stage used for RNA-Seq.

<p>(A)Red-fleshed apples in the F₁ population. Scale bar = 1cm. (B) White-fleshed apples in the F₁ population. Scale bar = 1cm. (C) Flavonoid content in red- and white-fleshed apples. (D) Anthocyanin content in red- and white-fleshed apples.</p