The transcriptional regulatory module CsHB5-CsbZIP44 positively regulates abscisic acid-mediated carotenoid biosynthesis in citrus (Citrus spp.)

Abstract

Carotenoids contribute to fruit coloration and are valuable sources of provitamin A in the human diet. Abscisic acid (ABA) plays an essential role in fruit coloration during citrus fruit ripening, but little is known about the underlying mechanisms. Here, we identified a novel bZIP transcription activator called CsbZIP44, which serves as a central regulator of ABA-mediated citrus carotenoid biosynthesis. CsbZIP44 directly binds to the promoters of four carotenoid metabolism-related genes (CsDXR, CsGGPPs, CsBCH1 and CsNCED2) and activates their expression. Furthermore, our research indicates that CsHB5, a positive regulator of ABA and carotenoid-driven processes, activates the expression of CsbZIP44 by binding to its promoter. Additionally, CsHB5 interacts with CsbZIP44 to form a transcriptional regulatory module CsHB5-CsbZIP44, which is responsive to ABA induction and promotes carotenoid accumulation in citrus. Interestingly, we also discover a positive feedback regulation loop between the ABA signal and carotenoid biosynthesis mediated by the CsHB5-CsbZIP44 transcriptional regulatory module. Our findings show that CsHB5-CsbZIP44 precisely modulates ABA signal-mediated carotenoid metabolism, providing an effective strategy for quality improvement of citrus fruit and other crops.

Keywords: CsHB5; CsbZIP44; abscisic acid (ABA); carotenoid; citrus; transcriptional regulatory module.

Figure 1

Abscisic acid (ABA) is closely related to peel coloration and carotenoid biosynthesis. (a–c) Changes in fruit coloration, carotenoid and ABA contents during ‘Valencia’ orange fruit (Citrus sinensis Osbeck.) ripening. (a) Fruit coloration. Bars = 3 cm. Contents of total carotenoid (μg/g DW) (b) and ABA (μg/g FW) (c). (d) Correlation analysis of ABA and carotenoid content during ‘Valencia’ orange fruit ripening. (e–g) ABA treatment promotes peel coloration and carotenoid accumulation. (e) Phenotype of ‘Valencia’ orange fruit under various treatments. Bars = 3 cm. The same set of citrus fruits used in the control and ABA‐treated groups on day 0 of treatment. (f) Effect of ABA treatment on citrus colour index (CCI). Positive values for red‐yellow, negative values for blue‐green and 0 for an intermediate mixture of red, yellow and blue‐green. (g) Total carotenoid content (μg/g DW). Data represent means ± SD of three biological replicates. Asterisks indicate statistically significant differences determined by Student's t‐test (*, 0.01 < P < 0.05; **, P < 0.01; n.s., no significant difference).

Figure 2

ABA‐activated CsbZIP44 is highly associated with citrus fruit coloration. Expression of CsbZIP44 under ABA treatment in citrus fruit (a) and calli (b). (c) Spatial and temporal expression analyses of CsbZIP44. Data represent means ± SD of three biological replicates. Asterisks indicate statistically significant differences determined by Student's t‐test (*, 0.01 < P < 0.05; **, P < 0.01; n.s., no significant difference).

Figure 3

CsbZIP44 is essential for ABA‐induced carotenoid biosynthesis in citrus. (a–g) Transient expression of CsbZIP44 in ‘Valencia’ orange fruit. (a) Phenotypes. Empty vector PK7 and RNAi as control. PK7‐CsbZIP44 and RNAi‐CsbZIP44 indicate overexpressing and interfering CsbZIP44 respectively. Bars = 3 cm. Transcript levels of CsbZIP44 (c), CsDXR (d), CsGGPPs (e), CsBCH1 (f) and CsNCED2 (g). (b) Total carotenoid content (μg/g DW). (h–k) Stable transformation of CsbZIP44 in citrus calli. (h) Phenotypes. PH7‐CsbZIP44 and RNAi‐CsbZIP44 indicate overexpressing and interfering CsbZIP44 respectively. Empty vector PH7 and RNAi as control. Expression levels of CsbZIP44 (i) and CsDXR, CsGGPPs, CsBCH1 and CsNCED2 (k). (j) Total carotenoid content (μg/g DW). Data represent means ± SD of three biological replicates. Asterisks indicate statistically significant differences determined by Student's t‐test (*, 0.01 < P < 0.05; **, P < 0.01; n.s., no significant difference).

Figure 4

CsbZIP44 directly activates the transcription of carotenogenic genes. (a) Yeast one‐hybrid (Y1H) assay identified interactions of CsbZIP44 with target genes promoters. Empty PGADT7 + pAbAi‐proCBGs and PGADT7‐Rec‐p53 + p53‐AbAi as the negative (N. Control) and positive controls (P. Control) respectively. Aureobasidin A (AbA) is a yeast cell growth inhibitor. CBGs, carotenoid biosynthesis genes. SD/−Leu/AbA^x, SD/−Leu medium supplemented with 200 ng ml⁻¹ as the basal concentration of proCsDXR and proCsGGPPs. SD/−Leu/AbA^x, SD/−Leu medium supplemented with 150 ng ml⁻¹ as the basal concentration of proCsBCH1 and proCsNCED2. (b–e) Chromatin Immunoprecipitation (ChIP)‐PCR assay showed the interaction of CsbZIP44 with several regions in the promoters of CsDXR, CsGGPPs, CsBCH1 and CsNCED2 respectively. The grey lines represent the putative binding motif of bZIP family proteins in these promoters. Cross‐linked chromatin samples were extracted from GFP‐CsbZIP44 fruit calli and precipitated with an anti‐GFP antibody. The eluted DNA fragment was used to amplify by quantitative (q)‐PCR. (f) ChIP‐PCR assay showed that ABA treatment increases the binding of CsbZIP44 to the promoters of CsDXR, CsGGPPs, CsBCH1, CsNCED2. proCsDXR‐P3, proCsGGPPs‐P1, proCsBCH1‐P2 and proCsNCED2‐P3 refers to the promoter region of CsDXR, CsGGPPs, CsBCH1 and CsNCED2 in (b) to (e) respectively. Cross‐linked chromatin samples were extracted from GFP‐CsbZIP44 fruit calli treated with or without ABA (250 μm) and precipitated with an anti‐GFP antibody. Eluted DNA was used to amplify the sequences by q‐PCR. (g) Schematic representation of reporter and effector constructs used in dual‐luciferase assay. (h) Dual‐luciferase assay indicated that ABA treatment enhances the activation by CsbZIP44 of the CBG promoters. CBG, carotenoid biosynthesis gene. ABA treatment (100 μm) in the dual‐luciferase assay was conducted 3 h before determination. Data represent means ± SD of three biological replicates. Asterisks indicate statistically significant differences determined by Student's t‐test (*, 0.01 < P < 0.05; **, P < 0.01; n.s., no significant difference).

Figure 5

CsHB5 interacts with CsbZIP44 and transcriptionally activates CsbZIP44. (a) Yeast two‐hybrid (Y2H) assay revealing an interaction between CsbZIP44 and CsHB5. Yeast grown in SD/−Trp/−Leu medium and SD/−Trp/−Leu/−His/−Ade medium is shown. The interaction is indicated by yeast growth and X‐α‐Gal staining. (b) The interactions between CsbZIP44 and CsHB5 were analysed using a pull‐down assay. Fusion proteins GST‐CsHB5 and MBP‐CsbZIP44 were used in the pull‐down analysis. GST‐ and MBP‐antibodies were used for immunoblot analyses. The band detected by the GST antibody in the pull‐down protein sample indicates the interaction between CsbZIP44 and CsHB5. (c) The interaction between CsbZIP44 and CsHB5 was confirmed with a co‐immunoprecipitation (Co‐IP) assay. The fused constructs GFP‐tagged CsbZIP44, and flag‐tagged CsHB5 were co‐overexpressed in Nicotiana benthamiana leaves. GFP antibody beads were used for immunoprecipitation. GFP‐ and flag‐antibodies were used for immunoblot analyses. The band detected by the GFP antibody in the IP samples indicates an interaction between CsbZIP44 and CsHB5. (d) A luciferase complementation imaging assay shows that CsbZIP44 interacts with CsHB5, and this interaction is enhanced by ABA treatment. Agrobacterium tumefaciens strain GV3101 harbouring different constructs was infiltrated into different wild tobacco leaf regions. Luciferase activities were imaged in these regions 3 days after infiltration. cps, signal counts per second. (e) Schematic representation of reporter and effector constructs used in dual‐luciferase assay. (f) Dual‐luciferase assay showing that the interaction between CsbZIP44 and CsHB5 significantly increases the activation effect on the promoter activity of target genes, which is strengthened by ABA treatment. (g) Y1H assay showing interactions of CsHB5 with CsbZIP44 promoter. Empty PGADT7 + pAbAi‐proCsbZIP44 and PGADT7‐Rec‐p53 + p53‐AbAi as the negative (N. Control) and positive controls (P. Control) respectively. Aureobasidin A (AbA) is a yeast cell growth inhibitor. SD/−Leu/AbA²⁵⁰, SD/−Leu medium supplemented with 250 ng ml⁻¹ as the basal concentration of proCsbZIP44. (h) ChIP‐PCR assay indicating the interaction of CsHB5 with several regions in the CsbZIP44 promoter in vivo. The grey lines represent putative binding motif of HD‐ZIP family proteins in the CsbZIP44 promoter. Cross‐linked chromatin samples were extracted from GFP‐CsHB5 fruit calli treated with or without ABA (250 μm) and precipitated with an anti‐GFP antibody. Eluted DNA was used to amplify the sequences by q‐PCR. (i) Electrophoretic mobility shift assay (EMSA) assay confirming that CsHB5 directly binds the binding element of HD‐ZIP TFs in the CsbZIP44 promoter in vitro. Purified MBP‐tagged CsHB5 protein was used in EMSA assay, and purified MBP protein was used as a negative control. Black arrows indicate the position of biotin‐labelled promoter fragment (hot probe) containing the putative binding motifs of HD‐ZIP family proteins. Red arrows indicate the positions of protein‐DNA complexes or free probes. Red letters represent the binding motifs, and blue letters indicate their corresponding mutant motifs. ‘+’ and ‘–’ the presence and absence of the indicated probe or protein respectively. Increasing amounts (50 folds) of the unlabelled DNA fragments (cold probe) were added as competitors. (j) Schematic representation of reporter and effector constructs used in dual‐luciferase assay. (k) Dual‐luciferase assay showing that CsHB5 enhances the activation of the CsbZIP44 promoter, and this activation is induced by ABA treatment. ABA treatment (100 μm) in the luciferase complementation imaging assay and the dual‐luciferase assay was conducted for 3 h before determination. Data represent means ± SD of three biological replicates. Asterisks indicate statistically significant differences determined by Student's t‐test (*, 0.01 < P < 0.05; **, P < 0.01; n.s., no significant difference).

Figure 6

The transcriptional regulatory module CsbZIP44‐CsHB5 positively regulates carotenoid biosynthesis in citrus. (a–h) Transient expression of CsbZIP44 and CsHB5 in ‘Valencia’ orange fruit. (a) Phenotypes. Empty vector PK7 as control. PK7‐CsbZIP44 and RNAi‐CsbZIP44 indicates overexpressing CsbZIP44 and CsHB5 respectively. Bars = 3 cm. Transcript levels of CsbZIP44 (b), CsHB5 (c), CsDXR (e), CsGGPPs (f), CsBCH1 (g) and CsNCED2 (h). (d) Total carotenoid content (μg/g DW). (i–p) Stable transformation of CsbZIP44 and CsHB5 in citrus calli. (i) Phenotypes. PH7 indicates the transformation of PH7 empty vector in citrus calli as control. PH7‐CsbZIP44 indicates overexpressing CsbZIP44. PH7‐CsbZIP44 + PK7‐CsHB5 indicates co‐overexpressing CsbZIP44 and CsHB5. The expression levels of CsbZIP44 (j) CsHB5 (k), CsDXR (m), CsGGPPs (n), CsBCH1 (o) and CsNCED2 (p). (l) Total carotenoid content (μg/g DW). Data represent means ± SD of three biological replicates. Asterisks indicate statistically significant differences determined by Student's t‐test (*, 0.01 < P < 0.05; **, P < 0.01; n.s., no significant difference).

Figure 7

Model for the positive feedback regulatory loop between ABA and carotenoid metabolisms mediated by the transcriptional regulatory module CsbZIP44‐CsHB5 in citrus. ABA induces CsHB5 and activates CsbZIP44 expression through binding the AATNATT cis‐elements in the CsbZIP44 promoter, and then CsbZIP44 promotes carotenoid accumulation by directly enhancing the transcriptional levels of carotenogenic genes (including CsDXR, CsGGPPs, CsBCH1 and CsNCED2) in citrus fruit. Moreover, CsbZIP44 interacts with CsHB5 to further positively regulate carotenoid biosynthesis by directly activating the expressions of CsBCH1 and CsNCED2. In turn, ABA as ripening signal significantly induces the regulation and interaction of CsbZIP44 and CsHB5 in feedback regulation, thereby promoting carotenoid biosynthesis during citrus fruit ripening. ‘+’ represents promotion. CsDXR, 1‐deoxy‐D‐xylulose 5‐phosphate; CsGGPPs, geranylgeranyl diphosphates; CsBCH1, β‐carotene hydroxylase 1; CsNCED2, 9‐cisepoxycarotenoid dioxygenase 2.