G C A T T A C G G C A T genes Article Characterization of the Role of the Neoxanthin Synthase Gene BoaNXS in Carotenoid Biosynthesis in Chinese Kale Yue Jian 1,2,†, Chenlu Zhang 1,†, Yating Wang 1,†, Zhiqing Li 1, Jing Chen 1, Wenting Zhou 1, Wenli Huang 1, Min Jiang 1, Hao Zheng 1, Mengyao Li 1 , Huiying Miao 2, Fen Zhang 1, Huanxiu Li 1, Qiaomei Wang 2,* and Bo Sun 1,* 1 College of Horticulture, Sichuan Agricultural University, Chengdu 611130, China; [email protected] (Y.J.); [email protected] (C.Z.); [email protected] (Y.W.); [email protected] (Z.L.); [email protected] (J.C.); [email protected] (W.Z.); [email protected] (W.H.); [email protected] (M.J.); [email protected] (H.Z.); [email protected] (M.L.); [email protected] (F.Z.); [email protected] (H.L.) 2 Department of Horticulture, Zhejiang University, Hangzhou 310058, China; [email protected] * Correspondence: [email protected] (Q.W.); [email protected] (B.S.); Tel.: +86-571-85909333 (Q.W.); +86-28-86291840 (B.S.) † These authors contributed equally to this work. Abstract: Chinese kale (Brassica oleracea var. alboglabra) is rich in carotenoids, and neoxanthin is one of the most important carotenoids in Chinese kale. In this study, the function of the neoxanthin synthase gene (BoaNXS) in Chinese kale was investigated. BoaNXS, which had a 699-bp coding sequence, was cloned from the white flower cultivar of Chinese kale and was expressed in all developmental stages and organs of Chinese kale; its expression was highest in young seeds. The subcellular local- ization indicated that BoaNXS was localized in the chloroplast. BoaNXS-overexpressed plants were Citation: Jian, Y.; Zhang, C.; Wang, obtained via Agrobacterium-mediated transient overexpression methodology, and the gene overex- Y.; Li, Z.; Chen, J.; Zhou, W.; Huang, BoaNXS W.; Jiang, M.; Zheng, H.; Li, M.; et al. pression efficiencies ranged from 2.10- to 4.24-fold. The color in the leaves of -overexpressed Characterization of the Role of the plants changed from green to yellow-green; the content of total and individual carotenoids, such as Neoxanthin Synthase Gene BoaNXS neoxanthin, violaxanthin, and lutein, was significantly increased, and the expression levels of most in Carotenoid Biosynthesis in Chinese carotenoid biosynthetic genes were notably increased. These findings indicated that BoaNXS is of Kale. Genes 2021, 12, 1122. vital importance in carotenoid biosynthesis in Chinese kale and could be used as a candidate gene https://doi.org/10.3390/genes12081122 for enriching the carotenoid accumulation and color of Chinese kale and other Brassica vegetables. Academic Editors: Tomas Laursen Keywords: Chinese kale; BoaNXS; carotenoid biosynthesis; subcellular localization; gene tran- and Bruna Marques dos Santos sient overexpression Received: 7 June 2021 Accepted: 23 July 2021 Published: 24 July 2021 1. Introduction Publisher’s Note: MDPI stays neutral Carotenoids are a major group of pigments that are distributed abundantly in a with regard to jurisdictional claims in variety of plants [1]. They typically contain 40 carbons in their polyene backbones with published maps and institutional affil- conjugated double bonds and rings at the ends [2]. They play significant roles in plant iations. development, including light harvesting, photoprotection against excess light, and the synthesis of plant hormones [3]. Most animals obtain necessary carotenoids through their diet [4]. The accumulation of carotenoids enhances both the sensory and nutritional quality of fruits, flowers, and vegetables; carotenoids are also beneficial to human health [5]. Furthermore, they are essential precursors to several phytohormones, such as abscisic acid Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. and strigolactones [6]. In humans, carotenoids can help prevent several major diseases, This article is an open access article including certain cancers and eye diseases [7]. The carotenoid biosynthesis pathway distributed under the terms and in higher plants consists of the condensation of small molecular substances into C40 conditions of the Creative Commons compounds and a series of reactions, including dehydrogenation, isomerization, cyclization, Attribution (CC BY) license (https:// hydroxylation, and epoxidation, to produce different carotenoids [8]. creativecommons.org/licenses/by/ Neoxanthin, which is present in light-harvesting complexes, is a precursor of ab- 4.0/). scisic acid and a xanthophyll that is the last product of carotenoid biosynthesis in green Genes 2021, 12, 1122. https://doi.org/10.3390/genes12081122 https://www.mdpi.com/journal/genes Genes 2021, 12, 1122 2 of 13 plants [9,10]. The neoxanthin synthase gene (NXS) encodes a 56-kDa plastid-targeted protein that, when expressed in Escherichia coli, catalyzes the conversion of violaxanthin to neoxanthin [9]. In plants, one of the possible pathways that is thought to underlie this conversion is as follows: violaxanthin ! neoxanthin ! abscisic acid [11]. Previous studies have identified NXS from Arabidopsis thaliana [12], tomato (Solanum lycopersicum)[9], and potato (Solanum tuberosum)[13]. However, little research has been conducted on NXS compared with other carotenoid biosynthetic genes. Chinese kale (Brassica oleracea var. alboglabra) is a vegetable within the Cruciferae family native to South China, where it is distributed widely [14]. The tender leaves and bolting stems are the most commonly used edible parts because of their high content of nutrients, such as glucosinolates, vitamin C, and carotenoids [15,16]. In our previous study, four carotenoids (lutein, neoxanthin, violaxanthin, and β-carotene) were observed in Chinese kale, among which the content of neoxanthin is second only to lutein [16]. However, the synthesis of the xanthophyll neoxanthin in Chinese kale remains unclear. In this study, the full-length coding sequence (CDS) of BoaNXS was cloned. Sequence analysis and subcellular location were performed. The role of BoaNXS in carotenoid biosynthesis was characterized by phenotypic analysis, gene expression, pigment composition, and content analysis in BoaNXS overexpression plants. 2. Materials and Methods 2.1. Plant Materials The cultivar ‘Sijicutiao’ of white flower Chinese kale was used in this study. The plants were grown in trays containing a mixture of peat, perlite, and vermiculite (3:1:1) in an artificial climate chamber with a light intensity of 160 µmol m−2 s−1, a temperature of 25/20 ◦C (day/night), a 12/12 h (day/night) light cycle, and relative humidity maintained at approximately 70%. Fertilizer and water were applied as needed. Chinese kale materials were sampled at different developmental stages (germinating seeds, cotyledons, fifth to sixth true leaves, and mature leaves), various organs were sampled at the maturity stage (roots, bolting stems, leaves, petioles, inflorescences, seed pods, and young seeds), and floral organs were sampled at the flower bud stage and the opening flower stage (sepals, petals, stamens, and pistils) [17]. The samples were then quickly frozen in liquid nitrogen and were stored in a refrigerator at −80 ◦C for subsequent studies. 2.2. Molecular Cloning and Sequence Analysis Specific primers for the BoaNXS gene were designed according to the sequences of NXS of homologous species such as cabbage and Chinese cabbage obtained from Brassica database (BRAD) (http://brassicadb.org accessed on 1 May 2019) (Supplementary Table S1). The primers were synthesized by Sangon Biotech Co. Ltd. (Sangon, Shanghai, China). The cDNA of fifth to sixth true leaves was used as the template, and PCR amplification was performed using TransStart FastPfu fly DNA polymerase Taq (TransGene, Beijing, China). The method of gene cloning refers to Sun et al. [18]. The NXS amino acid sequences of other species were downloaded from NCBI (https://www.ncbi.nlm.nih.gov/ accessed on 31 July 2019) and Brassica database (BRAD) (http://brassicadb.org accessed on 31 July 2019) and then subjected to multiple sequence alignment using DNAMAN software (Lynnon Biosoft, Foster City, CA, USA). Subcellular localization was predicted by WoLF PSORT (http://www.genscript.com/wolf-psort.html accessed on 1 June 2019). 2.3. Subcellular Localization The complete CDS of BoaNXS was amplified by primers NXS-GFP-F and NXS-GFP-R (Table S1) containing BamH I and Sal I restriction sites. Then, BoaNXS and pC2300-35S-eGFP plasmid digested with BamH I and Sal I were mixed to construct the pC2300-35S-NXS-eGFP plasmid. The recombinant plasmid pC2300-35S-NXS-eGFP and empty vector pC2300- 35S-eGFP were transformed into Chinese kale mesophyll protoplasts, respectively. After Genes 2021, 12, 1122 3 of 13 being cultured in the dark at 23 ◦C for 24 h, the protoplasts were observed using a BX51 fluorescence microscope equipped with a DP70 camera (Olympus, Tokyo, Japan) [19]. 2.4. Transient Overexpression of BoaNXS Transient overexpression assay was conducted using the methods of a previous study [20]. The complete CDS of BoaNXS was amplified by primers NXS-pCAM-F and NXS-pCAM-R (Supplementary Table S1) containing BamH I and Sal I restriction sites. Then, BoaNXS and pCAMBIA1301-35S-Nos overexpression vector (kindly supplied by Associate Professor Chen Qing, Sichuan Agricultural University, China) digested with BamH I and Sal I were mixed to construct the pCAMBIA1301-BoaNXS plasmid. The recombinant plasmid pCAMBIA1301-BoaNXS and empty vector pCAMBIA3101 were transferred into Agrobacterium GV3101 by freeze-thaw