Citrus PH4-Noemi regulatory complex is involved in proanthocyanidin biosynthesis via a positive feedback loop

Abstract

Proanthocyanidins (PAs; or condensed tannins) are a major class of flavonoids that contribute to citrus fruit quality. However, the molecular mechanism responsible for PA biosynthesis and accumulation in citrus remains unclear. Here, we identify a PH4-Noemi regulatory complex that regulates proanthocyanidin biosynthesis in citrus. Overexpression of PH4 or Noemi in citrus calli activated the expression of PA biosynthetic genes and significantly increased the PA content. Interestingly, Noemi was also shown to be up-regulated in CsPH4-overexpressing lines compared with wild-type calli. Simultaneously, CsPH4 partially complemented the PA-deficient phenotype of the Arabidopsis tt2 mutant and promoted PA accumulation in the wild-type. Further analysis revealed that CsPH4 interacted with Noemi, and together these proteins synergistically activated the expression of PA biosynthetic genes by directly binding to the MYB-recognizing elements (MRE) of the promoters of these genes. Moreover, CsPH4 could directly bind to the promoter of Noemi and up-regulate the expression of this gene. These findings explain how the CsPH4-Noemi regulatory complex contributes to the activation of PA biosynthetic genes via a positive feedback loop and provide new insights into the molecular mechanisms underlying PA biosynthesis, which can be effectively employed for metabolic engineering to improve citrus fruit quality.

Keywords: Citrus; positive feedback; proanthocyanidin biosynthesis; regulatory complex; transcriptional regulation.

Fig. 1.

Characterization of PA levels in ‘Anliu’, ‘Hong Anliu’, and ‘Succari’ sweet oranges. (A) Mature seeds without testae from three sweet orange varieties. Scale bar: 1 cm. (B) The contents and extracts after DMACA staining of soluble PAs in seeds. (C) The contents and extracts after DMACA staining of insoluble PAs in seeds. (D) The contents and extracts after DMACA staining of soluble PAs in pulps. (E) The contents and extracts after DMACA staining of insoluble PAs in pulps. (1) Blank control, (2) ‘Succari’, (3) ‘Hong Anliu’, (4) ‘Anliu’. DW, dry weight. Error bars represent the mean ±SD of three biological replicates. (This figure is available in colour at JXB online.)

Fig. 2.

Transcriptomic differences among the three sweet orange varieties. (A) Fruits from the three citrus varieties 90 DAF. Scale bar: 1 cm. (B) Cluster heat map based on the expression of phenylpropanoid-related genes in the pulps of the three citrus varieties. AL_F, ‘Anliu’ pulps; HAL_F, ‘Hong Anliu’ pulps; S_F, ‘Succari’ pulps. (C) Cluster heat map based on the expression of phenylpropanoid-related genes in the seeds of the three citrus varieties. AL_S, ‘Anliu’ seeds; HAL_S, ‘Hong Anliu’ seeds; S_S, ‘Succari’ seeds. (D) Relative expression patterns of PA biosynthetic genes (CsDFR, CsANS, CsANR, CsLAR), CsPH4, and Noemi in the pulps of the three citrus varieties. (E) Relative expression patterns of PA biosynthetic genes (CsDFR, CsANS, CsANR, CsLAR), CsPH4, and Noemi in the seeds of the three citrus varieties. The gene expression data for ‘Succari’ were normalized to 1. Error bars represent the mean ±SD of three biological replicates. (This figure is available in colour at JXB online.)

Fig. 3.

Phylogenetic analysis of CsPH4 and Noemi. (A) Phylogenetic analysis of predicted peptide sequences of CsPH4 and related genes from other plants. (B) Phylogenetic analysis of predicted peptide sequences of Noemi and related genes from other plants. Scale bar represents 0.05 substitutions per site and numbers next to the nodes are bootstrap values from 1000 replicates. Phylogenetic trees were constructed using the neighbour-joining method of MEGA v.5.1 software. Putative regulatory functions of most of the proteins in the control of flavonoid biosynthesis are indicated. (This figure is available in colour at JXB online.)

Fig. 4.

Functional characterization of CsPH4 and Noemi overexpression in citrus calli. (A) Phenotypes of transgenic citrus calli. RM, wild-type citrus callus; OE-CsPH4, CsPH4-overexpressing callus; OE-Noemi, Noemi-overexpressing callus. (B) Semi-quantitative RT-PCR and western blotting analysis of CsPH4 and Noemi transcript and protein levels. An anti-HA antibody was used for immunoblotting. Actin, Actin gene (internal control). (C) DMACA staining of transgenic citrus calli. (D) Quantification of soluble and insoluble PA levels in transgenic citrus calli. DW, dry weight. (E) Relative expression of flavonoid biosynthetic genes in CsPH4-overexpressing calli. (F) Relative expression of flavonoid biosynthetic genes in Noemi-overexpressing calli. After several rounds of subculture, stable transgenic callus lines were established on selectable media. Calli grown for 15 d were collected for each assay. The gene expression data in ‘RM’ were normalized to 1. Error bars represent the mean ±SD of three biological replicates. Asterisks indicate significant differences using Student’s t-test: *P<0.05; **P<0.01. (This figure is available in colour at JXB online.)

Fig. 5.

Functional characterization of CsPH4 overexpression in Arabidopsis. (A) Unstained and DMACA-stained seeds from tt2 mutants, tt2/OE-CsPH4 transformants, wild-type (Col-0), and Col-0/OE-CsPH4 transformants. Three independent transgenic lines were obtained and showed similar results. (B, C) Quantification of soluble and insoluble PAs in seed from tt2 mutants, tt2/OE-CsPH4 transformants, wild-type (Col-0), and Col-0/OE-CsPH4 transformants. DW, dry weight. Error bars represent the mean ±SD of three biological replicates. Asterisks indicate significant differences using Student’s t-test: **P<0.01; n.s., no significant difference. (This figure is available in colour at JXB online.)

Fig. 6.

CsPH4 interacts with Noemi. (A) Yeast two-hybrid assay revealing an interaction between CsPH4^ΔC2 and Noemi. The full-length coding sequences of Noemi and the truncated coding sequence of CsPH4^ΔC2 were cloned into PGADT7 (AD–Noemi) and PGBKT7 (BD–CsPH4^ΔC2), respectively. The interaction is indicated by yeast growth and X-α-Gal staining. Yeast grown in SD/−Trp/−Leu medium and SD/−Trp/−Leu/−His/−Ade medium is indicated. (B) BiFC assay of the interaction between CsPH4 and Noemi in epidermal cells of N. benthamiana. CsPH4–nYFP and Noemi–cYFP were used for the interaction assay, while nYFP plus Noemi–cYFP and CsPH4–nYFP plus cYFP were used as the controls. Yellow indicates a positive interaction signal. Scale bar: 10 μm. The experiment was repeated independently three times with similar results obtained each time. (C) Pull-down assays showing the interaction of CsPH4 and Noemi. The recombinant GST–CsPH4 or GST was incubated with 6His–Noemi. Blots were first probed with anti-His antibody and then with anti-GST antibody. (This figure is available in colour at JXB online.)

Fig. 7.

The CsPH4–Noemi complex activates the promoters of PA biosynthetic genes and Noemi by binding to these promoters. (A) Schematic diagrams of vectors used for the dual-luciferase assay. The reporter vector contained the promoter of CsDFR, CsANS, CsANR, CsLAR, and Noemi fused to LUC. (B) Transient promoter activity assays were carried out using LUC reporter gene under the control of the promoters of CsDFR, CsANS, CsANR, CsLAR, or Noemi, along with effectors (CsPH4+Noemi) and the empty vector as an internal control. Error bars represent the mean ±SD of eight biological replicates. Different letters indicate a significant difference using Duncan’s test: P<0.01. (C) qRT-PCR analysis of flavonoid biosynthetic genes (NtCHS, NtCHI, NtF3H, NtDFR, NtANS, NtANR, NtLAR, and NtUFGT) in tobacco leaves overexpressing CsPH4, Noemi, and CsPH4 plus Noemi and leaves infiltrated with the empty vector control. All transient overexpression experiments were conducted three times. The gene expression data in ‘Control’ were normalized to 1. Error bars represent the mean ±SD of three biological replicates. (D) EMSAs showing the binding of CsPH4 to the MREs of the CsDFR, CsANR, CsLAR, and Noemi promoters. For competition, 10- and 20-fold excess of non-labelled probes or mutant unlabelled probes were used. ‘+’ and ‘−’ indicate the presence and absence, respectively, of the indicated probe or protein.

Fig. 8.

A proposed model of the mechanism by which the CsPH4–Noemi regulatory complex regulates PA biosynthesis. Top: under low-expression conditions, neither CsPH4 nor Noemi can be translated to the corresponding protein and form the complex, resulting in the inability to induce PA biosynthetic genes and Noemi expression; thus, PAs could not be effectively accumulated. Bottom: under high-expression conditions, CsPH4 interacts with the Noemi protein to form a regulatory complex, which is mediated by CsPH4 recognition by the consensus MRE site and induction of the expression of PA biosynthetic genes and Noemi. Simultaneously, the regulatory complex provides further positive feedback, which regulates the expression of Noemi, thereby enhancing the accumulation of PAs in citrus. The green parts represent the CsPH4 protein, and the purple parts represent the Noemi protein. (This figure is available in colour at JXB online.)