Zongying Zhang

Genome re-sequencing reveals the history of apple and supports a two-stage model for fruit enlargement

Human selection has reshaped crop genomes. Here we report an apple genome variation map generated through genome sequencing of 117 diverse accessions. A comprehensive model of apple speciation and domestication along the Silk Road is proposed based on evidence from diverse genomic analyses. Cultivated apples likely originate from Malus sieversii in Kazakhstan, followed by intensive introgressions from M. sylvestris. M. sieversii in Xinjiang of China turns out to be an “ancient” isolated ecotype not directly contributing to apple domestication. We have identified selective sweeps underlying quantitative trait loci/genes of important fruit quality traits including fruit texture and flavor, and provide evidences supporting a model of apple fruit size evolution comprising two major events with one occurring prior to domestication and the other during domestication. This study outlines the genetic basis of apple domestication and evolution, and provides valuable information for facilitating marker-assisted breeding and apple improvement.Apple is one of the most important fruit crops. Here, the authors perform deep genome resequencing of 117 diverse accessions and reveal comprehensive models of apple origin, speciation, domestication, and fruit size evolution as well as candidate genes associated with important agronomic traits.

Hypersensitive ethylene signaling and ZMdPG1 expression lead to fruit softening and dehiscence.

‘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 (ZMdEIL1, ZMdEIL2, ZMdEIL3, ZMdERF1 and ZMdERF2) and one functional gene, ZMdPG1, 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 ZMdERF1 and ZMdEIL2 transcription were more abundant in ‘Taishanzaoxia’ softening fruit and dehiscent fruit and their expression was inhibited by an ethylene inhibitor 1-methylcyclopropene. Therefore, ZMdERF1 and ZMdEIL2 expression were responses to endogenous ethylene and associated with fruit softening and dehiscence. ZMdPG1 expression was induced when fruit softening and dehiscence but this induction can be blocked by 1-MCP, indicating that ZMdPG1 was essential for fruit softening and dehiscence and its expression was mediated by the endogenously occurred ethylene. ZMdPG1 overexpression in Arabidopsis led to silique early dehiscence while suppressing ZMdPG1 expression by antisense ZMdPG1 prevented silique naturally opening. The result also suggested that ZMdPG1 related with the connection between cells that contributed to fruit softening and dehiscence. ZMdERF1 was more closely related with ethylene signaling but it was not directly regulated the ZMdPG1, which might be regulated by the synergic pattern of ethylene transcription factors because of both the ZMdERF1 and ZMdERF2 could interact with ZMdEIL2.

The molecular mechanism underlying anthocyanin metabolism in apple using the MdMYB16 and MdbHLH33 genes

Key messageMdMYB16 forms homodimers and directly inhibits anthocyanin synthesis via its C-terminal EAR repressor. It weakened the inhibitory effect of MdMYB16 on anthocyanin synthesis when overexpressing MdbHLH33 in callus overexpressing MdMYB16. MdMYB16 could interact with MdbHLH33.AbstractAnthocyanins are strong antioxidants that play a key role in the prevention of cardiovascular disease, cancer, and diabetes. The germplasm of Malus sieversii f. neidzwetzkyana is important for the study of anthocyanin metabolism. To date, only limited studies have examined the negative regulatory mechanisms underlying anthocyanin synthesis in apple. Here, we analyzed the relationship between anthocyanin levels and MdMYB16 expression in mature Red Crisp 1–5 apple (M. domestica) fruit, generated an evolutionary tree, and identified an EAR suppression sequence and a bHLH binding motif of the MdMYB16 protein using protein sequence analyses. Overexpression of MdMYB16 or MdMYB16 without bHLH binding sequence (LBSMdMYB16) in red-fleshed callus inhibited MdUFGT and MdANS expression and anthocyanin synthesis. However, overexpression of MdMYB16 without the EAR sequence (LESMdMYB16) in red-fleshed callus had no inhibitory effect on anthocyanin. The yeast one-hybrid assay showed that MdMYB16 and LESMdMYB16 interacted the promoters of MdANS and MdUFGT, respectively. Yeast two-hybrid, pull-down, and bimolecular fluorescence complementation assays showed that MdMYB16 formed homodimers and interacted with MdbHLH33, however, the LBSMdMYB16 could not interact with MdbHLH33. We overexpressed MdbHLH33 in callus overexpressing MdMYB16 and found that it weakened the inhibitory effect of MdMYB16 on anthocyanin synthesis. Together, these results suggested that MdMYB16 and MdbHLH33 may be important part of the regulatory network controlling the anthocyanin biosynthetic pathway.

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

Transcriptome profiles of the red- and white-fleshed apples in an F1 segregating population of Malus sieversii f.Niedzwetzkyana and M.domestica ‘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.

Identification of Differentially Expressed Genes Associated with Apple Fruit Ripening and Softening by Suppression Subtractive Hybridization

Apple is one of the most economically important horticultural fruit crops worldwide. It is critical to gain insights into fruit ripening and softening to improve apple fruit quality and extend shelf life. In this study, forward and reverse suppression subtractive hybridization libraries were generated from ‘Taishanzaoxia’ apple fruits sampled around the ethylene climacteric to isolate ripening- and softening-related genes. A set of 648 unigenes were derived from sequence alignment and cluster assembly of 918 expressed sequence tags. According to gene ontology functional classification, 390 out of 443 unigenes (88%) were assigned to the biological process category, 356 unigenes (80%) were classified in the molecular function category, and 381 unigenes (86%) were allocated to the cellular component category. A total of 26 unigenes differentially expressed during fruit development period were analyzed by quantitative RT-PCR. These genes were involved in cell wall modification, anthocyanin biosynthesis, aroma production, stress response, metabolism, transcription, or were non-annotated. Some genes associated with cell wall modification, anthocyanin biosynthesis and aroma production were up-regulated and significantly correlated with ethylene production, suggesting that fruit texture, coloration and aroma may be regulated by ethylene in ‘Taishanzaoxia’. Some of the identified unigenes associated with fruit ripening and softening have not been characterized in public databases. The results contribute to an improved characterization of changes in gene expression during apple fruit ripening and softening.

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 F3H, FLS, DFR, ANS, and UFGT 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.

MdMYB3 helps regulate anthocyanin accumulation in apple calli under moderately acidic conditions

Anthocyanins are the key factors controlling the coloration of plant tissues. However, the molecular mechanism underlying the effects of environmental pH on the synthesis of apple anthocyanins is unclear. In this study, we analyzed the anthocyanin contents of apple calli cultured in media at different pHs (5.5, 6.0, and 6.5). The highest anthocyanin content was observed at pH 6.0. Additionally, the moderately acidic conditions up-regulated the expression of MdMYB3 as well as specific anthocyanin biosynthesis structural genes (MdDFR and MdUFGT). Moreover, the anthocyanin content was higher in calli overexpressing MdMYB3 than in the wild-type controls at different pHs. Yeast one-hybrid assay results indicated that MdMYB3 binds to the MdDFR and MdUFGT promoters in vivo. An analysis of the MdDFR and MdUFGT promoters revealed multiple MYB-binding sites. Meanwhile, electrophoretic mobility shift assays confirmed that MdMYB3 binds to the MdDFR and MdUFGT promoters in vitro. Furthermore, GUS promoter activity assays suggested that the MdDFR and MdUFGT promoter activities are enhanced by acidic conditions, and the binding of MdMYB3 may further enhance activity. These results implied that an acid-induced apple MYB transcription factor (MdMYB3) promotes anthocyanin accumulation by up-regulating the expression of MdDFR and MdUFGT under moderately acidic conditions.

Molecular characterization and expression analysis of the critical floral gene MdAGL24-like in red-fleshed apple

The transition from vegetative to reproductive growth is the most dramatic phase change in plants. To better understand the molecular regulation of floral transition and flower development in red-fleshed apple (Malus sieversii f. niedzwetzkyana), we isolated and characterized a floral MADS-box gene, MdAGL24-like, which shares sequence similarity with AGAMOUS-LIKE 24 (AGL24) from other species. Spatial expression analysis showed that MdAGL24-like dynamically expressed in flowers, followed by roots and fruits. Subcellular localization analysis indicated that, like other transcript factors, MdAGL24-like was localized in the nucleus. Protein interaction analysis showed that MdAGL24-like could interact with MdSOC1 and MdAP1 in vivo and in vitro. MdAGL24-like and MdSOC1 could increase each others expression by binding the CArG motifs in their promoters. Unlike MdSOC1, MdAGL24-like might indirectly promote the expression of MdLFY by upregulating the expression of MdSOC1. Ectopic expression of MdAGL24-like in wild-type Arabidopsis induced early flowering like the phenotypes induced by other AGL24 genes. Similar to AGL24 in Arabidopsis, MdAGL24-like could rescue the late-flowering phenotype of the agl24 mutant to some extent. These results help clarify the molecular mechanism underlying flowering and provide a means of shortening the juvenile period in red-fleshed apples and other fruit trees.

The ethylene response factor MdERF1B regulates anthocyanin and proanthocyanidin biosynthesis in apple

AbstractKey message The regulator MdERF1B in the apple (Malus × domestica) ethylene pathway mainly acts on MdMYB9 and MdMYB11 to regulate anthocyanin and proanthocyanidin accumulation.Abstract Dietary anthocyanins and proanthocyanidins (PAs) have health benefits for humans, and are associated with decreased risks of coronary heart disease and cancer. Ethylene can enhance reddening of apple (Malus × domestica), but the regulatory mechanism is poorly understood. In this study, an ethylene response factor (ERF), MdERF1B, was identified and functionally characterized. ‘Orin’ calli overexpressing MdERF1B were generated and then analyzed by quantitative reverse transcription-PCR. Compared with the control calli, the MdERF1B-overexpressing calli showed increased expression levels of MdACO1, MdERF1, and MdERF3 in the ethylene pathway and MdCHS, MdCHI, MdF3H, MdDFR, MdANS, MdLAR, MdANR, MdMYB9 and MdMYB11 in the flavonoid pathway. As a result, the levels of anthocyanins and PAs were significantly increased in the MdERF1B-overexpressing calli. MdERF1B interacted with MdMYB9, MdMYB1, and MdMYB11 proteins in yeast two-hybrid, pull-down, and bimolecular fluorescence complementation assays. Furthermore, in yeast one-hybrid and electrophoretic mobility shift assays, MdERF1B also bound to the promoters of MdMYB9, MdMYB1, and MdMYB11. In a luciferase reporter assay, MdERF1B mainly activated proMdMYB9 and proMdMYB11, promoting their expression levels. This was in agreement with MdERF1B’s overexpression in calli, which barely affected MdMYB1 expression. Taken together, our findings provide an insight into the regulatory mechanisms in the ethylene pathway that increase anthocyanin and PA accumulation in apple.

Methyl jasmonate enhances apple’ cold tolerance through the JAZ–MYC2 pathway

Improving the cold resistance of plants is important, because their growth and development are negatively affected by cold stress. In this study, exogenous applications of methyl jasmonate could enhance the cold resistance of ‘Orin’ apple (Malus × domestica) calli by increasing the expression levels of the cold-signal response genes MdCIbHLH1, MdCBF1, MdCBF2 and MdCBF3. In addition, yeast two-hybrid and pull-down assays demonstrated that MdCIbHLH1 interacts with MdJAZ1/4 and MdMYC2 in vitro and in vivo. Protein degradation experiments demonstrated that the stability of the MdJAZ1/4 proteins were affected by the application of exogenous methyl jasmonate, which was followed by their degradation by the 26S proteasome. MdJAZ1/4 act as repressors, binding MdMYC2 in the jasmonate-signaling pathway. The overexpression of MdMYC2 in ‘Orin’ calli increased the expression levels of MdCIbHLH1, MdCBF1, MdCBF2 and MdCBF3, resulting in an increased freeze tolerance. Furthermore, the overexpression of MdJAZ1 or MdJAZ4 in transgenic red-fleshed apple calli weakened the promotive effect of MdMYC2 on cold tolerance. Yeast one-hybrid and chromatin immunoprecipitation-PCR analyses showed that MdMYC2 could bind the G-box element found in the MdCBF1 promoter. Thus, jasmonate may function as a critical upstream signal in the ICE–CBF/DREB1 pathway to positively regulate apple freeze tolerance.