The bZIP transcription factor MdHY5 regulates anthocyanin accumulation and nitrate assimilation in apple

Abstract

The basic leucine zipper (bZIP) transcription factor HY5 plays a multifaceted role in plant growth and development. Here the apple MdHY5 gene was cloned based on its homology with Arabidopsis HY5. Expression analysis demonstrated that MdHY5 transcription was induced by light and abscisic acid treatments. Electrophoretic mobility shift assays and transient expression assays subsequently showed that MdHY5 positively regulated both its own transcription and that of MdMYB10 by binding to E-box and G-box motifs, respectively. Furthermore, we obtained transgenic apple calli that overexpressed the MdHY5 gene, and apple calli coloration assays showed that MdHY5 promoted anthocyanin accumulation by regulating expression of the MdMYB10 gene and downstream anthocyanin biosynthesis genes. In addition, the transcript levels of a series of nitrate reductase genes and nitrate uptake genes in both wild-type and transgenic apple calli were detected. In association with increased nitrate reductase activities and nitrate contents, the results indicated that MdHY5 might be an important regulator in nutrient assimilation. Taken together, these results indicate that MdHY5 plays a vital role in anthocyanin accumulation and nitrate assimilation in apple.

Figure 1

Sequence alignment and phylogenetic analysis of MdHY5. (a) Protein alignment of MdHY5 and its homologs in Arabidopsis. The zinc-finger domain is boxed. (b) Phylogenetic analysis of MdHY5 and 35 other plants HY5 protein sequences obtained from the NCBI database. MdHY5 is denoted by the red asterisk, and the scale bar indicates the branch length. RsHY5: Raphanus sativus, XP_018445811.1; AtHY5: Arabidopsis thaliana, AT5G11260.1; BrHY5: Brassica rapa, XP_009121971.1; ThHY5: Tarenaya hassleriana, XP_010541629.1; PeHY5: Populus euphratica, XP_011039711.1; MdHY5: Malus domestica, MDP0000586302; PbHY5: Pyrus bretschneideri, XP_009355719.1; FvHY5: Fragaria vesca, XP_004291469.1; ZjHY5: Ziziphus jujuba, XP_015885857.1; PmHY5: Prunus mume, XP_008219477.1; FaHY5: Fragaria ananassa, AKG58815.1; PpHY5: Prunus persica, ONI34365.1; CcHY5: Citrus clementina, XP_006450470.1; BpHY5: Betula platyphylla, AHY20043.1; EgHY5: Eucalyptus grandis, XP_010048982.1; JcHY5: Jatropha curcas, XP_012076602.1; CaHY5: Camptotheca acuminate, APD29065.1; PtHY5: Populus trichocarpa, XP_002308656.1; CmHY5: Cucumis melo, NP_001284656.1; TcHY5: Theobroma cacao, XP_007013841.2; RcHY5: Ricinus communis, XP_002515537.1; VvHY5: Vitis vinifera, XP_010648648.1; MnHY5: Morus notabilis, XP_010110356.1; SiHY5: Sesamum indicum, XP_011081579.1; NnHY5: Nelumbo nucifera, XP_010250037.1; NcHY5: Noccaea caerulescens, JAU18721.1; EsHY5: Eutrema salsugineum, XP_006399627.1; StHY5: Solanum tuberosum, XP_006361723.1; MtHY5: Medicago truncatula, XP_013459310.1; SlHY5: Solanum lycopersicum, NP_001234820.1; PdHY5: Phoenix dactylifera, XP_008785002.1; AcHY5: Ananas comosus, XP_020097860.1; NaHY5: Nicotiana attenuate, XP_019265660.1; DcHY5: Daucus carota, XP_017229054.1; GrHY5: Gentiana rigescens, AIC64080.1; CsHY5: Camelina sativa, XP_010419684.1.

Figure 2

Effects of light and ABA on the transcript level of MdHY5. (a) Apomictic crabapple (Malus hupehensis) seedlings grown in darkness for 3 days at room temperature were treated with continuous white light for 1, 3, 6, 9 and 12 h. The transcript levels of MdHY5 were determined using RT-PCR (above) and RT-qPCR (below); the value for dark-treated plants was set to 1. (b) Apomictic crabapple (M. hupehensis) seedlings were treated with 50 μM ABA at the indicated time intervals. The expression levels of MdHY5 were monitored by RT-qPCR (above) and RT-PCR (below); the value for untreated plants was set to 1.

Figure 3

MdHY5 directly and positively regulates its own expression. (a) Schematic diagram showing both the predicted E-box and mutated E-box sites in black boxes. (b) EMSA results showing that the MdHY5-GST fusion protein directly binds to its own promoter at the E-box in vitro. Biotin-labeled probes were incubated with MdHY5-GST protein, and the free and bound DNAs (arrows) were separated in an acrylamide gel. Unlabeled probes served as competitors. The 5′-CAATTG-3′ motif in mutated probes (Mut) was replaced with 5′-ACTTGT-3′. (c) Transient expression assays showing that MdHY5 promotes its own expression. A: MdHY5_pro:Luc; B: MdHY5_pro:Luc-35S_pro:MdHY5; C: MdHY5_pro(Mut):Luc-35S_pro:MdHY5. Representative images of Nicotiana benthamiana leaves 72 h after infiltration are shown. (d) Quantitative analysis of luminescence intensity in c. The value for MdHY5_pro:Luc was set to 1.

Figure 4

MdHY5 promotes the expression of MdMYB10 by directly interacting with its promoter. (a) Schematic diagram of the MdMYB10 promoter showing the potential MdHY5-binding sites. The predicated two G-box (G-box-1 and G-box-2) and mutated G-box sites and sequences are indicated with black boxes. (b) EMSA results showing that MdHY5-GST fusion protein directly binds to the MdMYB10 promoter at G-box-2 in vitro. Biotin-labeled probes were incubated with MdHY5-GST protein, and the free and bound DNAs (arrows) were separated in an acrylamide gel. Unlabeled probes served as competitors. The 5′-CACGTG-3′ motif in mutated probes (Mut) was replaced with 5′-CGTGTG-3′. (c) Transient expression assays showing that MdHY5 promotes the expression of MdMYB10. A: MdMYB10_pro:Luc; B: MdMYB10_pro(Mut):Luc-35S_pro:MdHY5; C: MdMYB10_pro:Luc-35S_pro:MdHY5. Representative images of Nicotiana benthamiana leaves 72 h after infiltration are shown. (d) Quantitative analysis of luminescence intensity in c. The value for a MdMYB10_pro:Luc was set to 1.

Figure 5

MdHY5 increases anthocyanin accumulation in transgenic apple calli. (a, b) Anthocyanin contents in transgenic calli (MdHY5-L1 and MdHY5-L2) and wild-type (WT) control grown on medium under low-temperature and high-light conditions. (c) Relative expression levels of MdMYB10, MdDFR, MdUF3GT, MdF3H, MdCHI and MdCHS in transgenic calli and WT control. The value for WT was set to 1.

Figure 6

MdHY5 influences the expression of nitrogen signaling-related genes. RT-qPCR analysis of genes related to nitrogen signaling in WT and MdHY5 transgenic calli (MdHY5-L1 and MdHY5-L2) under low-temperature and high-light conditions. The value for WT was set to 1.

Figure 7

MdHY5 influences NR activity and nitrate acquisition. (a) NR activities and (b) nitrate contents of WT and MdHY5 transgenic calli (MdHY5-L1 and MdHY5-L2) under low-temperature and high-light conditions are shown.

Figure 8

Model of MdHY5-mediated anthocyanin accumulation and nitrate assimilation. The bold arrows indicate the pathway verified in apple in the present work. The arrows indicate pathways that have been previously reported in apple or Arabidopsis. MdCOP1: apple ubiquitin E3 ligase CONSTITUTIVE PHOTOMORPHOGENIC 1; Ubi: ubiquitination. (1) Li et al. and Maier et al.; (2) Ang et al.; (3) Takos et al. and Ban et al.