A kiwifruit (Actinidia deliciosa) R2R3-MYB transcription factor modulates chlorophyll and carotenoid accumulation

Abstract

MYB transcription factors (TFs) regulate diverse plant developmental processes and understanding their roles in controlling pigment accumulation in fruit is important for developing new cultivars. In this study, we characterised kiwifruit TFMYB7, which was found to activate the promoter of the kiwifruit lycopene beta-cyclase (AdLCY-β) gene that plays a key role in the carotenoid biosynthetic pathway. To determine the role of MYB7, we analysed gene expression and metabolite profiles in Actinidia fruit which show different pigment profiles. The impact of MYB7 on metabolic biosynthetic pathways was then evaluated by overexpression in Nicotiana benthamiana followed by metabolite and gene expression analysis of the transformants. MYB7 was expressed in fruit that accumulated carotenoid and Chl pigments with high transcript levels associated with both pigments. Constitutive over-expression of MYB7, through transient or stable transformation of N. benthamiana, altered Chl and carotenoid pigment levels. MYB7 overexpression was associated with transcriptional activation of certain key genes involved in carotenoid biosynthesis, Chl biosynthesis, and other processes such as chloroplast and thylakoid membrane organization. Our results suggest that MYB7 plays a role in modulating carotenoid and Chl pigment accumulation in tissues through transcriptional activation of metabolic pathway genes.

Keywords: carotenoid; chlorophyll; kiwifruit; overexpression; transcription factor; transcriptomics.

Figure 1

A schematic of the carotenoid biosynthetic pathway in plants. Enzymes involved in the various conversion steps are indicated with arrows. GGPPS, geranylgeranyl pyrophosphatase; PSY, phytoene synthase; PDS, phytoene desaturase; ZISO, zeta‐carotene isomerase; ZDS, zeta‐carotene desaturase; CRTISO, carotenoid isomerase; LCY‐β, lycopene beta‐cyclase; LCY‐ɛ, lycopene epsilon‐cyclase; BCH, beta‐carotene hydroxylase; CYP97A, cytochrome P450 beta‐ring carotenoid hydroxylase; CYP97C, cytochrome P450 epsilon‐ring carotenoid hydroxylase; ZEP, zeaxanthin epoxidase; VDE, violaxanthin de‐epoxidase.

Figure 2

Transient activation of AdLCY‐β promoter by kiwifruit MYB transcription factors. Ratio of luminescence (LUC) signals measured from Nicotiana benthamiana leaves coinfiltrated with Agrobacterium constructs of MYB genes and a vector with firefly luciferase under the control of LCY‐β promoter and Renilla luciferase (REN) under the control of CaMV 35S promoter. Luminescence signals were normalised to the basal promoter activity. Bars represent means ± SE of four biological replicates.

Figure 3

Phylogenetic tree of MYB transcription factors showing the relationship between kiwifruit MYB sequences in this study, MYBs implicated in carotenoid regulation (ElRCP1 and CrMYB68) and sequences retrieved from Arabidopsis databases. Protein sequences were aligned using ClustalW and evolutionary relationships were determined using maximum‐likelihood analysis with 1000 bootstrap replicates. The kiwifruit sequences are indicated by triangles, ElRCP1 and CrMYB68 by circles. GenBank accession numbers are provided in Supporting Information Table S1.

Figure 4

Ad MYB7 binds and enhances the activity of the AdLCY‐β promoter. (a) EMSA analysis using a ³²P‐labelled 200 bp promoter fragment showed that MYB7 recombinant protein binds to the LCY‐β promoter fragment. The migration of the labelled probe only is shown in lane 1. Complexes formed between labelled probe and MYB7 protein (lane 2), with competition from 50‐fold and 350‐fold increase of unlabelled probe, are shown in lanes 3 and 4, and with nonspecific competitor DNA (NS) in lane 5. (b) Constructs of AdLCY‐β promoter deletion fragments (showing MYB binding sites predicted by Plant PAN 2.0) infiltrated with Ad MYB7 construct as described above. Data are means ± SE of four biological replicates.

Figure 5

(a) Ripe fruit of Actinidia arguta (green) and Actinidia macrosperma (orange). (b) Total Chl (left) and total carotenoid concentration (right) in fruit of A. arguta (top panel) and A. macrosperma (bottom panel) during fruit development. Error bars indicate ± SE from three biological replicates.

Figure 6

Gene expression profile of MYB7 and lycopene beta‐cyclase (LCY‐β) in kiwifruit genotypes during fruit development. Relative gene expression of MYB7 and LCY‐β in fruit of Actinidia arguta (a, c) and Actinidia macrosperma (b, d). Data were analysed using target : reference ratios (measured with Lightcycler 480 software) using actin as reference gene and presented as means ± SE from three biological replicates.

Figure 7

Nicotiana benthamiana plants expressing AdMYB7 show changes in Chl and carotenoid profiles. MYB7 transgenic plants displayed darker green leaves in tissue culture (a) and in soil (b). (c) Transcript levels of MYB7 in the transgenic N. benthamiana plants measured by RT‐qPCR (arranged in decreasing order). Bars represent means and SE from four biological replicates. (d, e) Graphs showing concentrations of Chl pigments, pheophytin (Pheo) a, Chla, Pheo b, Chlb (d) and carotenoid pigments (e) in transgenic N. benthamiana plants measured by HPLC from leaf tissue. Bars represent means and standard errors from three biological replicates. (f) HPLC pigment profiles showing pigments detected in 50 mg of leaves of control (top), T5‐9 (middle) and T5‐3 (bottom) transgenic plants. Labelled peaks: 1, violaxanthin; 2, lutein; 3, zeaxanthin; 4, Chlb; 5, Chla; 6, pheophytin a; 7, pheophytin b; 8, beta‐carotene.

Figure 8

Relative expression of carotenoid (a) and Chl (b) biosynthetic pathway genes in transgenic MYB7 Nicotiana benthamiana plants. Values are means ± SE from three biological replicates. PSY, phytoene synthase; PDS, phytoene desaturase; ZDS, zeta‐carotene desaturase; CRTISO, carotene isomerase; LCY‐β, lycopene beta‐cyclase; LCY‐ɛ, lycopene epsilon‐cyclase; BCH, beta‐carotene hydroxylase; NbCYP97C, cytochrome P450 epsilon‐ring carotenoid hydroxylase; ALAD, delta‐aminolevulinic acid dehydratase; PGBD, porphobilinogen deaminase; PROTOX, protoporphyrinogen oxidase; MGCH, magnesium chelatase; MPECYC, magnesium protoporphyrin ester cyclase; POR, protochlorophyllide oxidoreductase.

Figure 9

A GO plot of differentially expressed genes (P < 0.05) of AdMYB7‐treated leaves in the biological process category at T1 (a) and T2 (b). The outer wheel shows a scatter plot of log fold change (FC) for each gene under the gene ontology (GO) terms. Red dots indicate upregulated genes and blue dots show downregulated genes; the inner wheel shows the z‐score, a measure of up‐ or downregulation, of each identified process.