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. 2018 Jun 1:5:27.
doi: 10.1038/s41438-018-0032-3. eCollection 2018.

Tomato SlAN11 regulates flavonoid biosynthesis and seed dormancy by interaction with bHLH proteins but not with MYB proteins

Yongfeng Gao  1 , Jikai Liu  1 , Yongfu Chen  1 , Hai Tang  1 , Yang Wang  1 , Yongmei He  2 , Yongbin Ou  1 , Xiaochun Sun  3 , Songhu Wang  4 , Yinan Yao  1
Affiliations

Tomato SlAN11 regulates flavonoid biosynthesis and seed dormancy by interaction with bHLH proteins but not with MYB proteins

Yongfeng Gao et al. Hortic Res. .

VSports手机版 - Abstract

The flavonoid compounds are important secondary metabolites with versatile human nutritive benefits and fulfill a multitude of functions during plant growth and development VSports手机版. The abundance of different flavonoid compounds are finely tuned with species-specific pattern by a ternary MBW complex, which consists of a MYB, a bHLH, and a WD40 protein, but the essential role of SlAN11, which is a WD40 protein, is not fully understood in tomato until now. In this study, a tomato WD40 protein named as SlAN11 was characterized as an effective transcription regulator to promote plant anthocyanin and seed proanthocyanidin (PA) contents, with late flavonoid biosynthetic genes activated in 35S::SlAN11 transgenic lines, while the dihydroflavonol flow to the accumulation of flavonols or their glycosylated derivatives was reduced by repressing the expression of SlFLS in this SlAN11-overexpressed lines. The above changes were reversed in 35S::SlAN11-RNAi transgenic lines except remained levels of flavonol compounds and SlFLS expression. Interestingly, our data revealed that SlAN11 gene could affect seed dormancy by regulating the expressions of abscisic acid (ABA) signaling-related genes SlABI3 and SlABI5, and the sensitivity to ABA treatment in seed germination is conversely changed by SlAN11-overexpressed or -downregulated lines. Yeast two-hybrid assays demonstrated that SlAN11 interacted with bHLH but not with MYB proteins in the ternary MBW complex, whereas bHLH interacted with MYB in tomato. Our results indicated that low level of anthocyanins in tomato fruits, with low expression of bHLH (SlTT8) and MYB (SlANT1 and SlAN2) genes, remain unchanged upon modification of SlAN11 gene alone in the transgenic lines. These results suggest that the tomato WD40 protein SlAN11, coordinating with bHLH and MYB proteins, plays a crucial role in the fine adjustment of the flavonoid biosynthesis and seed dormancy in tomato. .

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1. Simplified flavonoid biosynthetic pathway
. First, chalcone synthase (CHS) catalyzes the condensation of one molecule of 4-coumaroyl-CoA with three molecules of malonyl-CoA. The later steps in this pathway are catalyzed by a series of enzymes, leading to the production of three main types of final products: flavonols (including quercetin, kaempferol, and myricetin); anthocyanins; and proanthocyanidins (PAs). CHS chalcone synthase, CHI chalcone isomerase, F3H flavanone 3-hydroxylase, F3’H flavonoid 3’-hydroxylase, DFR dihydroflavanol 4-reductase, FLS flavonol synthase, LDOX leucoanthocyanidin dioxygenase, BAN anthocyanidin reductase, TT19 glutathione-S-transferase, TT12 multidrug and toxic efflux transporter, AHA10 H+ ATPase
Fig. 2
Fig. 2. The phylogentic tree of AN11/WD40 protein sequences
. The name of the proteins, their source, and GenBank accession numbers refer to figure legend of the Supplementary Fig.S1
Fig. 3
Fig. 3. The expression pattern analysis of SlAN11 in different tissues.
a The expression levels of SlAN11 in different tissues. Total RNAs were extracted from roots, stem, leaves, flowers, green fruit pericarps (GF), BF (breaker phase fruit), red fruit pericarps (RF), and mature seeds. The SlAN11 mRNA levels were quantified by quantitative RT-PCR and are indicated as relative expression levels compared with the internal control SlUBI3 mRNA. Results represent mean values ± SD from three biological replicates. b–i Histochemical analysis of GUS staining of SlAN11Pro::GUS transgenic plants. The expression of SlAN11Pro::GUS transgene was determined by the GUS staining of roots (b), transverse sections of roots (c), stems (d), transverse sections of stems (e), leaves (f), flowers (g), flower buds (h), and fruits (i)
Fig. 4
Fig. 4. Subcellular localization of SlAN11 protein
. The PCR-amplified GFP fragment, encoding green fluorescent protein, was cloned into XbaI and SalI sites of pTEX to generate pTEX-GFP. The coding sequences of SlAN11 genes were PCR-amplified from tomato leaf cDNA and cloned in the pTEX-GFP vector to generate SlAN11-GFP construct. Subcellular localization assay was carried out via transformation of tomato protoplasts. After DAPI staining the protoplasts was examined using confocal microscope to simultaneously capture DAPI and GFP signals. pTEX-GFP construct was included as control. Left to right: green, GFP fluorescence; red, chlorophyll autofluorescence; blue, nucleus stained with DAPI; merged, combined fluorescence from GFP, chlorophyll, and DAPI. Bars, 5 μm
Fig. 5
Fig. 5. Phenotypes of wild-type and SlAN11 transgenic plants.
a, b Phenotypes of anthocyanin accumulation in leaves (a) and stems (b) from field-grown plants of wild-type Ailsa Craig (WT), 35S::SlAN11 (SlAN11-OX), and SlAN11-RNAi (SlAN11-Ri) transgenic lines. c Microscopic observation of transverse sections of stem from field-grown plants of wild-type, 35S::SlAN11, and SlAN11-RNAi transgenic lines. d, e Quantitative analysis of anthocyanins contents of leaves (d) and stems (e) from wild-type, 35S::SlAN11, and SlAN11-RNAi transgenic lines. The plants were grown in greenhouse and harvested 30 days after germination. Results represent mean values ± SD from three biological replicates. Asterisks indicate statistically significant differences (**P < 0.01; t-test)
Fig. 6
Fig. 6. Expression level analysis of genes involved anthocyanin and flavonol biosynthetic pathway in wild-type and SlAN11 transgenic plants
. Total RNA were extracted from seedlings of WT, 35S::SlAN11 (lines OX-3, OX-4, and OX-5), and SlAN11-RNAi (lines Ri-7, Ri-8, and Ri-11) transgenic plants. Quantitative real-time PCR analysis revealed the relative mRNA levels of these genes, including SlAN11 (a), SlCHS (b), SlCHI (c), SlF3’H (d), SlDFR (e), and SlFLS1 (f). Results represent mean values ± SD from three biological replicates. Asterisks indicate statistically significant differences (**P < 0.01, ***P < 0.001; t-test)
Fig. 7
Fig. 7. Kaempferol and quercetin (flavonols) accumulation in wild-type and SlAN11 transgenic seedlings
. Seven-day-old wild-type (WT), 35S::SlAN11 (SlAN11-OX), and SlAN11-RNAi (SlAN11-Ri) transgenic seedlings grown on 1/2 MS medium were used for DPBA staining for the visualization of kaempferol and quercetin accumulation (bar 100 μm). The roots shown were typical of three separate experiments performed at 23 °C (n = 10 seedlings per experiment)
Fig. 8
Fig. 8. Phenotypes of wild-type and SlAN11 transgenic seeds.
a Phenotypes of proanthocyanidin accumulation in seeds from plants of wild-type (WT), 35S::SlAN11 (SlAN11-OX), and SlAN11-RNAi (SlAN11-Ri) transgenic lines. b Germination phenotype of wild-type (WT), 35S::SlAN11 (OX), and SlAN11-RNAi (Ri) transgenic seeds in the different concentrations of ABA. c Quantitative analysis of anthocyanin contents of seeds from wild-type, 35S::SlAN11, and SlAN11-RNAi transgenic lines. Results represent mean values ± SD from three biological replicates. Asterisks indicate statistically significant differences (**P < 0.01, ***P < 0.001; t-test). d Germination ratio of WT and transgenic T2 seeds at the different concentrations of ABA. The percentage of seeds showing root emergence was scored 8 days post stratification. Standard error bars represent three independent experiments. e Germination ratio of WT and transgenic T2 seeds in 1/2 MS plates with 2.5 μM ABA. Germinated seeds (radicle protruding) were counted daily for 12 days. Germination ratio refers to the number of germinated seeds as a proportion of the total number of seeds. Standard deviation bars represent three independent experiments
Fig. 9
Fig. 9. Expression levels analysis of genes regulating seed development and dormancy in wild-type and SlAN11 transgenic seeds
. Total RNAs were extracted from these mature seeds of WT, 35S::SlAN11 (lines OX-3, OX-4, and OX-5), and SlAN11-RNAi (lines Ri-7, Ri-8, and Ri-11) T2 homozygous transgenic plants. Quantitative real-time PCR analysis revealed the relative mRNA levels of SlAN11 (a), SlBAN (b), SlTTG2 (c), and ABA signal transduction genes SlABI3 (d) and SlABI5 (e). Results represent mean values ± SD from three biological replicates. Asterisks indicate statistically significant differences (**P < 0.01, t-test)
Fig. 10
Fig. 10. The interaction of SlAN11 with the bHLH TFs but not with the MYB TFs in yeast.
a Transcriptional activity of SlAN11, SlANT1, SlAN2, SlGL3, and SlTT8 determined by the oNPG assay in yeast. Yeast strain EGY48 containing the pSH18-34 reporter plasmid was transformed pEG202-SlAN11, pEG202-SlANT1, pEG202-SlAN2, pEG202-SlGL3, and pEG202-SlTT8, respectively. Yeast colony grown on X-Gal plates were showed on the left panel with blue color indicating activation of LacZα marker gene by SlANT1 or SlAN2. b SlAN11 has interaction with SlGL3 and SlTT8 but not with SlANT1 and SlAN2. c bHLH TFs SlGL3 and SlTT8 interact with the MYB TFs SlANT1 and SlAN2, and can also homodimerize or heterodimerize with SlGL3 or SlTT8. The interaction was determined by the oNPG assay. Results represent mean values ± SD from three independent a-galactosidase assays
Fig. 11
Fig. 11. SlAN11 could not enhance anthocyanins a ccumulation in tomato fruits.
a Expression level analysis of SlAN11 in wild-type and SlAN11 transgenic fruits. Total RNAs were extracted from these red fruit pericarps of WT, 35S::SlAN11 (lines OX-3, OX-4, and OX-5), and SlAN11-RNAi (lines Ri-7, Ri-8, and Ri-11) T2 homozygous transgenic plants. b Quantitative analysis of anthocyanin contents of red fruit pericarps from wild-type, 35S::SlAN11, and SlAN11-RNAi transgenic lines. c The expression pattern analysis of SlAN11, SlANT1, SlAN2, SlTT8, and SlGL3 in different tissues. Total RNAs were extracted from roots, stem, leaves, flowers, green fruit pericarps (GF), BF (breaker phase fruit), red fruit pericarps (RF), and mature seeds. Relevant mRNA levels were quantified by quantitative RT-PCR and are indicated as relative expression levels compared with the internal control SlUBI3 mRNA. The vertical axis is log scale. Results represent mean values ± SD from three biological replicates. Asterisks indicate statistically significant differences ***P < 0.001; t-test)
Fig. 12
Fig. 12. The model for SlAN11-dependent regulation of anthocyanin/PA accumulation and seed dormancy.
Solid lines indicate interactions between members of a complex. Solid arrows indicate positive regulations. Dashed arrows indicate a multi-step differentiation pathway. Colored lines and arrows indicate specific regulator combinations and the pathway controlled
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"VSports app下载" References

    1. Li S. Transcriptional control of flavonoid biosynthesis: fine-tuning of the MYB-bHLH-WD40 (MBW) complex. Plant Signal. Behav. 2014;9:e27522. doi: 10.4161/psb.27522. - DOI (V体育ios版) - PMC - PubMed
    1. Koes R, Verweij W, Quattrocchio F. Flavonoids: a colorful model for the regulation and evolution of biochemical pathways. Trends Plant. Sci. 2005;10:236–242. doi: 10.1016/j.tplants.2005.03.002. - "V体育官网入口" DOI - PubMed
    1. Ben-Simhon Z, et al. A pomegranate (punica granatum l.) WD40-repeat gene is a functional homologue of Arabidopsis TTG1 and is involved in the regulation of anthocyanin biosynthesis during pomegranate fruit development. Planta. 2011;234:865–881. doi: 10.1007/s00425-011-1438-4. - "V体育官网" DOI - PubMed
    1. Nesi N, et al. The TT8 gene encodes a basic helix-loop-helix domain protein required for expression of DFR and BAN genes in Arabidopsis siliques. Plant Cell. 2000;12:1863–1878. doi: 10.1105/tpc.12.10.1863. - DOI - PMC - PubMed
    1. Gonzalez A, Zhao M, Leavitt JM, Lloyd AM. Regulation of the anthocyanin biosynthetic pathway by the TTG1/bHLH/Myb transcriptional complex in Arabidopsis seedlings. Plant J. 2008;53:814–827. doi: 10.1111/j.1365-313X.2007.03373.x. - DOI - PubMed