Characterization of BoaCRTISO Reveals Its Role in Carotenoid Biosynthesis in Chinese Kale

Abstract

Carotenoids are organic pigments that play an important role in both plant coloration and human health; they are a critical subject in molecular breeding due to growing demand for natural molecules in both food and medicine. In this study, we focus upon characterizing BoaCRTISO, the carotenoid isomerase gene before the branch of the carotenoid biosynthetic pathway, which is expressed in all organs and developmental stages of Chinese kale, and BoaCRTISO, which is located in the chloroplast. The expression of BoaCRTISO is induced by strong light, red and blue combined light, and gibberellic acid treatment, but it is suppressed by darkness and abscisic acid treatment. We obtained BoaCRTISO-silenced plants via virus-induced gene silencing technology, and the silence efficiencies ranged from 52 to 77%. The expressions of most carotenoid and chlorophyll biosynthetic genes in BoaCRTISO-silenced plants were downregulated, and the contents of carotenoids and chlorophyll were reduced. Meanwhile, BoaCRTISO-silenced plants exhibited phenotypes of yellowing leaves and inhibited growth. This functional characterization of BoaCRTISO provides insight for the biosynthesis and regulation of carotenoid in Chinese kale.

Keywords: VIGS; BoaCRTISO; Chinese kale; carotenoid biosynthesis; color; gene expression.

FIGURE 1

Sequence characteristics of BoaCRTISO. (A) Alignment of the protein sequence of BoaCRTISO with selected homologs. The alignment was performed using DNAMAN software. The amino acids with 100% identity are shown with a black background, those with ≥75% identity are shown in red, and those with ≥50% identity are shown in blue. The species and their accession numbers in GenBank [Brassica napus (Bn): BnCRTISO (CDY09954.1), Brassica rapa (Br): BrCRTISO (AGZ62519.1), Arabidopsis thaliana (At): AtCRTISO (NP_172167.2), Capsella rubella (Cr): CrCRTISO (XP_006307051.1), and Eutrema salsugineum (Es): EsCRTISO (XP_006417877.1)] are listed here. (B) Phylogenetic analysis of BoaCRTISO and selected CRTISO from other plant species. The phylogenetic tree was generated using the neighbor-joining method by MEGA 6.0 software. The bar indicates an evolutionary distance of 0.2%. The species [B. napus (Bn), B. rapa (Br), A. thaliana (At), C. rubella (Cr), E. salsugineum (Es), Prunus persica (Pp), Lycium barbarum (Lb), Lycium ruthenicum (Lr), Prunus avium (Pa), Manihot esculenta (Me), Ziziphus jujube (Zj), Solanum lycopersicum (Sl), Theobroma cacao (Tc), Solanum pennellii (Sp), Solanum tuberosum (St), Capsicum baccatum (Cb), Capsicum annuum (Ca), and Capsicum chinense (Cc)] are listed here. (C) Subcellular localization of the BoaCRTISO-GFP fusion protein in Chinese kale protoplasts. Free green fluorescent protein served as a control. Bars = 30 μm.

FIGURE 2

Expression levels of BoaCRTISO in different developmental stages: (A) organs, (B) flower organs in flower buds stage (C), and opening flowers stage (D) of Chinese kale. The BoaCRTISO expression of germinating seeds was set as 1. The samples were a mixture from three individual plants. Data are expressed as mean ± standard deviation. The same letter in the same histogram indicates that there is no significant difference between the values tested by least significant difference (LSD) (p < 0.05).

FIGURE 3

Response of BoaCRTISO to different exogenous treatments. (A) Cis-acting elements respond to light and phytohormone in the promoter regions of BoaCRTISO. The + and − in brackets represent sense strand and antisense strand, respectively. (B) Expression levels of BoaCRTISO after treatments with darkness, weak light, and strong light. (C) Expression levels of BoaCRTISO after treatments with red light, blue light, and combined red and blue lights. (D) Expression level of BoaCRTISO after abscisic acid (ABA) treatment. (E) Expression level of BoaCRTISO after GA₃ treatment. (F) Expression level of BoaCRTISO after methyl jasmonate (MeJA) treatment.

FIGURE 4

BoaCRTISO silencing affects the color and growth of Chinese kale. (A) Expressions of BoaCRTISO in control, pTY, and pTY-BoaCRTISO plants. Data are expressed as mean ± standard deviation. The same letter indicates that there is no significant difference between the values tested by least significant difference (LSD) (p < 0.05). (B) Front view of control, pTY, and pTY-BoaCRTISO plants. Bar = 10 cm. (C) Top view of control, pTY, and pTY-BoaCRTISO plants. The red arrow points to the sampled leaves. Bar = 10 cm. (D) Plant heights of control, pTY, and pTY-BoaCRTISO plants at 1 week after the last infiltration. (E) The color parameters of control, pTY, and pTY-BoaCRTISO plants at 1 week after the last infiltration. Data are expressed as a mean ± SD. The same letter in the same column means no significant differences among values (p < 0.05) according to a least significant difference (LSD) test.

FIGURE 5

Carotenoid composition and contents in control, pTY, and pTY-BoaCRTISO plants. Samples of leaves were taken from control, pTY, and pTY-BoaCRTISO plants at 1 week after the last infiltration. Data are expressed as mean ± standard deviation. The same letter in the same histogram indicates that there is no significant difference between the values tested by least significant difference (LSD) (p < 0.05).

FIGURE 6

Heat map of carotenoid biosynthetic gene expressions in control, pTY, and pTY-BoaCRTISO plants. Samples of leaves were taken from control, pTY, and pTY-BoaCRTISO plants at 1 week after the last infiltration. GGPP, geranylgeranyl diphosphate; PSY, phytoene synthase; PDS, phytoene desaturase; ZDS, ζ-carotene desaturase; Z-ISO, ζ-carotene isomerase; CRTISO, carotenoid isomerase; LCYe, lycopene ε-cyclase; LCYb, lycopene β-cyclase; ε-OHase, ε-carotene hydroxylase; β-OHase, β-carotene hydroxylase; VDE, violaxanthin de-epoxidase; ZEP, zeaxanthin epoxidase; NXS, neoxanthin synthase.