Carotenoid components and their biosynthesis in a bud mutant of Shiranui mandarin (Citrus reticulata Blanco) with citrine flavedo

Carotenoid components and their biosynthesis in a bud mutant of Shiranui mandarin ( Citrus reticulata Blanco) with citrine flavedo “不知火”黄色果皮芽变品种中类胡罗卜素组分及生物合成 1 equal first-author 2 equal 2 3 4 1 2 2 corresp Zhihui WANG, wangzhihui318@126.com Zhihui WANG, wangzhihui318@126.com wangzhihui318@126.com https://orcid.org/0000-0003-2234-144X 四川农业大学果蔬研究所， 中国成都， 611130 四川农业大学果蔬研究所， 中国成都， 611130 Institute of Pomology and Olericulture, Sichuan Agricultural University, Chengdu 611130, China Institute of Pomology and Olericulture, Sichuan Agricultural University, Chengdu 611130, China 四川农业大学园艺学院， 中国成都， 611130 四川农业大学园艺学院， 中国成都， 611130 College of Horticulture, Sichuan Agricultural University, Chengdu 611130, China College of Horticulture, Sichuan Agricultural University, Chengdu 611130, China 四川省园艺作物技术推广总站， 中国成都， 610041 四川省园艺作物技术推广总站， 中国成都， 610041 Sichuan Horticulture Techniques Promotion Station, Chengdu 610041, China Sichuan Horticulture Techniques Promotion Station, Chengdu 610041, China 西南大学柑桔研究所， 中国重庆， 400712 西南大学柑桔研究所， 中国重庆， 400712 Citrus Research Institute, Southwest University, Chongqing 400712, China Citrus Research Institute, Southwest University, Chongqing 400712, China 宽皮柑橘“柠檬不知火”是“不知火”的自然芽变品种，其果皮呈类似柠檬果皮的亮黄色，明显区别于“不知火”果皮的橘红色。本研究通过分析上述两种柑橘果皮中类胡罗卜素的含量和组分，及与其合成代谢相关基因的表达量，解析两种柑橘果皮颜色的差异机制。测定结果显示，“不知火”中总类胡罗卜素含量显著高于芽变品种“柠檬不知火”，推测这是造成两种果皮颜色差异的主要原因。芽变品种中呈现橙色色素的组分β-隐黄质占比低于“不知火”；而与绿-黄色调有关的叶黄素和新黄质这两种类胡罗卜素在芽变品种中的占比高于“不知火”。“不知火”中 PSY 基因的高表达可能为其类胡罗卜素合成提供了充足的底物，从而促使其果皮中较高水平的类胡罗卜素合成； HYb 基因在所有基因中表达量最高，推测与其参与催化类胡罗卜素代谢途径中多个反应有关；而 ZEP 基因的低表达量可能与类胡罗卜素代谢途径下游产物的反馈调节有关。 柠檬不知火 果皮色泽 高效液相色谱（HPLC） 类胡罗卜素组分 基因表达 Carotenoids are secondary metabolite responsible for colored pigments in plants and microbes ( Li et al., 2022 Li et al., 2022 ). They are a class of C 40 tetraterpenoids consisting of eight isoprenoid units, and can be classified into carotenes and xanthophylls on the basis of their functional groups ( Saini et al., 2015 Saini et al., 2015 ). Carotenes can be linear (phytoene, phytofluene, and ζ‍-carotene) or branched (β‍-carotene and α‍-carotene). Xanthophylls comprise β,β‍-xanthophylls (β‍-cryptoxanthin, zeaxanthin, violaxanthins, and neoxanthin) and β,ε‍-xanthophylls (α-cryptoxanthin, α-carotene, and lutein). Citrus fruits are complex sources of carotenoids, which are the principal pigments responsible for the typical orange color of most types ( Chen, 2020 Chen, 2020 ). The difference in total carotenoid content and the diversity of carotenoid isomer proportion also accounts for other colors of citrus fruits, such as yellow, red, and pink ( Chen, 2020 Chen, 2020 ). Carotenoid biosynthesis in higher plants is initiated by the linkage of two C 20 precursors ( Hirschberg, 2001 Hirschberg, 2001 ). More than nine catalases participate in this pathway, including phytoene synthase (PSY), phytoene desaturase (PDS), ζ-carotene desaturase (ZDS), those responsible for β,β‍-carotenoids such as lycopene β‍-cyclase (LCYb), β‍-carotene hydroxylase (HYb), zeaxanthin epoxidase (ZEP), and neoxanthin synthase (NSY), and those responsible for β,ε-‍carotenoids such as LCYb, lycopene ε-cyclase (LCYe), and ε/β‍-carotene hydroxylase (HYe and HYb). The accumulation of carotenoid components is usually coincident with the difference in catalase gene transcript levels in the biosynthetic pathway. Occasionally, there is a feedback regulatory mechanism resulting in inconsistency between the transcription of carotenoid genes and the accumulation of carotenoid components ( Feng et al., 2021 Feng et al., 2021 ). Citrine Shiranui (also named “Jinlegan”) is a spontaneous bud mutation of Shiranui mandarin ( Citrus reticulata Blanco) found in Southwest China. Its most striking feature is the brighter colored (citrine/yellow) flavedo of mature fruit, whereas the normal flavedo of Shiranui is a rich orange color ( Fig. 1 Fig. 1 a). In this research, the differences in carotenoid biochemistry and biosynthesis of Citrine Shiranui and Shiranui were analyzed to illuminate the phenotype of this citrus mutation. We analyzed the total carotenoid content, carotenoid component proportions (using high-performance liquid chromatography (HPLC) analysis), and transcript levels of carotenoid biosynthesis genes (using quantitative real-time polymerase chain reaction (qRT-PCR)) in the two cultivars at six stages of fruit ripening, including breaker stage (Stage I), coloring stage (Stage II), full coloring stage (Stage III), one month after full coloring (Stage IV), two months after full coloring (Stage V), and at the full ripening stage (Stage VI). The detailed methods are included in the supplementary. The total carotenoid contents were 4.23‍‒‍7.33 times higher in Shiranui than in Citrine Shiranui at all stages of fruit ripening ( Fig. 1 Fig. 1 b), which was considered to be the primary cause of the difference in flavedo color. The same mechanism generally occurs in flesh or flavedo pigmentation mutants of citrus. In the red-pigmented sweet orange mutant Cara Cara ( Citrus. sinensis Osbeck), the total carotenoid is increasingly accumulated compared with the yellowish-‍orange color of the parent Navel orange flavedo ( Alquezar et al., 2008 Alquezar et al., 2008 ). The sweet orange Pinalate is a mutant of Navelate and produces distinctive yellow fruit instead of the typical bright orange fruit of Navelate. In full-colored Pinalate fruit, the total carotenoid content is significantly lower than that in Navelate fruit ( Rodrigo et al., 2003 Rodrigo et al., 2003 ). The yellow flavedo of lemon ( Citrus. limon (L.) Osbeck) has a low total carotenoid content compared with the orange flavedo of Satsuma mandarin ( Citrus. unshiu Marc.) and Valencia sweet orange ( Kato et al., 2004 Kato et al., 2004 ). All of twelve prevalent components of the carotenoid biosynthesis pathway were identified in the flavedo of Shiranui and Citrine Shiranui ( Fig. 2 Fig. 2 ). Five carotenoids, namely 9- cis- violaxanthin, trans- violaxanthin, β‍-cryptoxanthin, phytoene, and α‍-‍cryptoxanthin, were increased in proportion with the fruit coloring and ripening processes in both cultivars ( Fig. 3 Fig. 3 ). Conversely, β‍-carotene, neoxanthin, α-carotene, and lutein were the major components at the breaker stage (Stage I) in both cultivars, but they were decreased in proportion with the coloring and ripening of the fruit ( Fig. 3 Fig. 3 ). Notably, the proportions of lutein and neoxanthin decreased sharply in Shiranui (both with P < 0.001), but to a lower degree in Citrine Shiranui. Violaxanthins (including 9- cis -violaxanthin and trans -violaxanthin) are the carotenoids mainly accumulated in the flavedo of mature fruit in various citrus with an orange color, especially in mandarin ( Kato et al., 2004 Kato et al., 2004 ; Agócs et al., 2007 Agócs et al., 2007 ). In the present research, violaxanthins were the principal conventional carotenoids in both Shiranui and Citrine Shiranui ( Fig. 3 Fig. 3 ) from the full coloring stage (Stage III) to the full ripening stage (Stage VI). In Shiranui, β‍-cryptoxanthin was another predominant carotenoid after violaxanthins (including 9- cis -violaxanthin and trans -violaxanthin), whereas it was less accumulated in Citrine Shiranui ( Fig. 3 Fig. 3 ). β‍-Cryptoxanthin is mainly accumulated in the flavedo of mature fruit in various mandarin citruses with an orange color, but is present at a low concentration in green‍-‍yellow organs, such as immature fruits and leaves ( Sumiasih et al., 2017 Sumiasih et al., 2017 ). Consequently, the lower accumulation of β‍-cryptoxanthin in the Citrine Shiranui flavedo was a possible reason for its off-orange color. A more notable difference in the two citrus cultivars was observed in the accumulation pattern of lutein and neoxanthin. These carotenoids were present in the flavedo at relatively high proportions at the beginning of the color transition (break stage/Stage I) and decreased as the fruit ripened in the both two citrus cultivars ( Fig. 3 Fig. 3 ). However, the transition in Citrine Shiranui seemed incomplete, resulting in the retention of greater quantities of lutein and neoxanthin compared to Shiranui ( Fig. 3 Fig. 3 ). In particular, lutein in Citrine Shiranui was the second most abundant carotenoid after violaxanthins during the period after color transition. Generally, lutein and neoxanthin are the preponderant carotenoid components in immature fruits and leaves ( Rodrigo et al., 2003 Rodrigo et al., 2003 ), and they are derivatively strongly associated with their green-yellow color. Therefore, the retention of lutein and neoxanthin contributed to the yellow flavedo of Citrine Shiranui. The analysis of the carotenoid components of so-called “colorless” citrus cultivars with a bright yellow flavedo indicated the excessive accumulation of colorless compounds (phytoene, phytofluene, or ζ-carotene), such as in lemon ( Chen, 2020 Chen, 2020 ), yellow-pigmented sweet orange Pinalate ( Rodrigo et al., 2003 Rodrigo et al., 2003 ), and white grapefruit ( Citrus paradisi Macf.) ( Alquezar et al., 2013 Alquezar et al., 2013 ). In the present research on Citrine Shiranui, some differences from the results of previous research were noted. Less β-cryptoxanthin (responsible for an orange color), coupled with higher proportions of lutein and neoxanthin (associated with a green-yellow color), was a considerable factor underlying these deviations. The expression of the above nine carotenoid biosynthesis-related genes was investigated. All of them showed an initial increase and subsequent decrease in transcript level during fruit ripening in both Shiranui and Citrine Shiranui ( Fig. 4 Fig. 4 ). Comparing the two cultivars, all genes were generally more highly expressed in Shiranui than in Citrine Shiranui, especially during the initial three stages ( Fig. 4 Fig. 4 ), which might explain the greater accumulation of total carotenoids and most carotenoid components in the Shiranui fruit flavedo. Especially, higher transcript levels of PSY may provide abundant substrate for the subsequent steps and sustain a higher total carotenoid content in the flavedo of Shiranui, since PSY catalyzes a rate-limiting step in carotenoid biosynthesis ( Rodríguez-Villalón et al., 2009 Rodríguez-Villalón et al., 2009 ). In each seperated cultivar, the relative expression levels of all nine carotenoid genes during all stages were calculated ( Fig. 5 Fig. 5 ). The relative transcript level of HYb was higher than that of other carotenoid biosynthetic genes in Shiranui and Citrine Shiranui ( Fig. 5 Fig. 5 ). This HYb is the isogene of β-carotene hydroxylase 2 (BCH2/At5g52570) of Arabidopsis , which mainly catalyzes the hydroxylation of β‍‍-carotene and β‍‍-cryptoxanthin, and partially participates in α‍‍-carotene hydroxylation ( Ruiz-Sola and Rodríguez-Concepción, 2012 Ruiz-Sola and Rodríguez-Concepción, 2012 ). The high transcript level of HYb here might accord with its multiple roles. A similar phenomenon was observed by several scholars, such as CHYB2 in watermelon ( Citrullus lanatus ) ( Yuan et al., 2021 Yuan et al., 2021 ) and Ag10G02588.1 in celery ( Apium graveolens ) ( Song et al., 2022 Song et al., 2022 ). Moreover, since the differential expression of HYb / BCH genes provides flexibility to regulate the accumulation of downstream xanthophylls or upstream carotenes ( Ruiz-Sola and Rodríguez-Concepción, 2012 Ruiz-Sola and Rodríguez-Concepción, 2012 ), the accumulation of violaxanthins in the present study might be predicted from the high transcript level of HYb . The ZEP transcript level was lower than the upstream HYb and downstream NSY for both cultivars ( Fig. 5 Fig. 5 ), although the product of ZEP protain (violaxanthins) was massively accumulated. Regulations of carotenoid accumulation at multiple levels, such as feedback regulation by end products, were frequently detected ( Stanley and Yuan, 2019 Stanley and Yuan, 2019 ). The finding of the present research might implicate a potential negative feedback regulation in ZEP transcription, i.e., the accumulation of violaxanthins or downstream carotenoids/products might lead to the downregulation of ZEP . Further study is needed to confirm this notion. The analyses of this paper provide new insights into the carotenoid biochemistry and biosynthesis of the mutant citrus with brighter color flavedo. Lower β-cryptoxanthin (a carotenoid associated with an orange color) combined with higher proportions of lutein and neoxanthin (associated with a green-yellow color) were found as additional factors contributing to the brighter color flavedo of Citrine Shiranui. The significantly higher transcript levels of PSY in the early stages of fruit ripening in the flavedo of Shiranui might provide abundant substrate to sustain its higher total carotenoid content. The HYb gene was more highly expressed than the other genes in both cultivars, which is in accord with its multiple hydroxylation functions. Meanwhile, the low transcript levels of ZEP probably imply potential negative feedback regulation through violaxanthins or downstream carotenoids/metabolic products. Materials and methods 1 Detailed methods are provided in the electronic supplementary materials of this paper. References Agócs A , Nagy V , Szabó Z , et al. , 2007 . Comparative study on the carotenoid composition of the peel and the pulp of different citrus species . Innov Food Sci Emerg Technol , 8 ( 3 ): 390 - 394 . https://doi.org/10.1016/j.ifset.2007.03.012 https://doi.org/10.1016/j.ifset.2007.03.012 Comparative study on the carotenoid composition of the peel and the pulp of different citrus species Alquezar B , Rodrigo MJ , Zacarías L , 2008 . Regulation of caro tenoid biosynthesis during fruit maturation in the red-fleshed orange mutant Cara Cara. Phytochemistry , 69 ( 10 ): 1997 - 2007 . https://doi.org/10.1016/j.phytochem.2008.04.020 https://doi.org/10.1016/j.phytochem.2008.04.020 Regulation of caro Alquezar B , Rodrigo MJ , Lado J , et al. , 2013 . A comparative physiological and transcriptional study of carotenoid biosynthesis in white and red grapefruit ( Citrus paradisi Macf .). Tree Genet Genomes , 9 ( 5 ): 1257 - 1269 . https://doi.org/10.1007/s11295-013-0635-7 https://doi.org/10.1007/s11295-013-0635-7 A comparative physiological and transcriptional study of carotenoid biosynthesis in white and red grapefruit (Citrus paradisi Macf Chen CX , 2020 . Pigments in citrus fruit: mutants, compounds, genes, and beyond . In: Gentile A, la Malfa S, Deng ZN (Eds.), The Citrus Genome . Springer , Cham , p. 195 - 209 . https://doi.org/10.1007/978-3-030-15308-3 https://doi.org/10.1007/978-3-030-15308-3 Pigments in citrus fruit: mutants, compounds, genes, and beyond Feng GZ , Wu JX , Xu YH , et al. , 2021 . High-spatiotemporal-resolution transcriptomes provide insights into fruit devel opment and ripening in Citrus sinensis. Plant Biotechnol J , 19 ( 7 ): 1337 - 1353 . https://doi.org/10.1111/pbi.13549 https://doi.org/10.1111/pbi.13549 High-spatiotemporal-resolution transcriptomes provide insights into fruit devel Hirschberg J , 2001 . Carotenoid biosynthesis in flowering plants . Curr Opin Plant Biol , 4 ( 3 ): 210 - 218 . https://doi.org/10.1016/S1369-5266(00)00163-1 https://doi.org/10.1016/S1369-5266(00)00163-1 Carotenoid biosynthesis in flowering plants Kato M , Ikoma Y , Matsumoto H , et al. , 2004 . Accumulation of carotenoids and expression of carotenoid biosynthetic genes during maturation in citrus fruit . Plant Physiol , 134 ( 2 ): 824 - 837 . https://doi.org/10.1104/pp.103.031104 https://doi.org/10.1104/pp.103.031104 Accumulation of carotenoids and expression of carotenoid biosynthetic genes during maturation in citrus fruit Li MY , Li J , Tan HH , et al. , 2022 . Comparative metabolomics provides novel insights into the basis of petiole color differences in celery ( Apium graveolens L .). J Zhejiang Univ-Sci B (Biomed & Biotechnol) , 23 ( 4 ): 300 - 314 . https://doi.org/10.1631/jzus.B2100806 https://doi.org/10.1631/jzus.B2100806 Comparative metabolomics provides novel insights into the basis of petiole color differences in celery (Apium graveolens L Rodrigo MJ , Marcos JF , Alférez F , et al. , 2003 . Characterization of Pinalate, a novel Citrus sinensis mutant with a fruit-specific alteration that results in yellow pigmentation and decreased ABA content . J Exp Bot , 54 ( 383 ): 727 - 738 . https://doi.org/10.1093/jxb/erg083 https://doi.org/10.1093/jxb/erg083 Characterization of Pinalate, a novel Citrus sinensis mutant with a fruit-specific alteration that results in yellow pigmentation and decreased ABA content Rodríguez-Villalón A , Gas E , Rodríguez-Concepción M , 2009 . Phytoene synthase activity controls the biosynthesis of carotenoids and the supply of their metabolic precursors in dark-grown Arabidopsis seedlings . Plant J , 60 ( 3 ): 424 - 435 . https://doi.org/10.1111/j.1365-313X.2009.03966.x https://doi.org/10.1111/j.1365-313X.2009.03966.x Phytoene synthase activity controls the biosynthesis of carotenoids and the supply of their metabolic precursors in dark-grown Arabidopsis seedlings Ruiz-Sola MÁ , Rodríguez-Concepción M , 2012 . Carotenoid biosynthesis in Arabidopsis: a colorful pathway . Arabidopsis Book , 2012 ( 10 ): e0158 . https://doi.org/10.1199/tab.0158 https://doi.org/10.1199/tab.0158 Carotenoid biosynthesis in Arabidopsis: a colorful pathway Saini RK , Nile SH , Park SW , 2015 . Carotenoids from fruits and vegetables: chemistry, analysis, occurrence, bioavailability and biological activities . Food Res Int , 76 (Part 3 ): 735 - 750 . https://doi.org/10.1016/j.foodres.2015.07.047 https://doi.org/10.1016/j.foodres.2015.07.047 Carotenoids from fruits and vegetables: chemistry, analysis, occurrence, bioavailability and biological activities Song XM , Li N , Zhang YC , et al. , 2022 . Transcriptomics and genomics analysis uncover the differentially expressed chlorophyll and carotenoid-related genes in celery . Int J Mol Sci , 23 ( 16 ): 8986 . https://doi.org/10.3390/ijms23168986 https://doi.org/10.3390/ijms23168986 Transcriptomics and genomics analysis uncover the differentially expressed chlorophyll and carotenoid-related genes in celery Stanley L , Yuan YW , 2019 . Transcriptional regulation of caro tenoid biosynthesis in plants: so many regulators, so little consensus. Front Plant Sci , 10 : 1017 . https://doi.org/10.3389/fpls.2019.01017 https://doi.org/10.3389/fpls.2019.01017 Transcriptional regulation of caro Sumiasih IH , Poerwanto R , Efendi D , et al. , 2017 . β‍- β‍- Cryptoxanthin and zeaxanthin pigments accumulation to induce orange color on citrus fruits . IOP Conf Ser Mater Sci Eng , 299 : 012074 . https://doi.org/10.1088/1757-899X/299/1/012074 https://doi.org/10.1088/1757-899X/299/1/012074 Cryptoxanthin and zeaxanthin pigments accumulation to induce orange color on citrus fruits Yuan PL , Umer MJ , He N , et al. , 2021 . Transcriptome regulation of carotenoids in five flesh-colored watermelons ( Citrullus lanatus ) . BMC Plant Biol , 21 : 203 . https://doi.org/10.1186/s12870-021-02965-z https://doi.org/10.1186/s12870-021-02965-z Transcriptome regulation of carotenoids in five flesh-colored watermelons (Citrullus lanatus) Xun WANG, Jinqiu HUANG, and Zongyan YIN performed the experimental research and data analysis. Ke XU contributed to the study design. Dong JIANG provided the information about the plant materials. Xun WANG, Lijin LIN, and Xiaoai ZHANG contributed to data analysis, writing and editing of the manuscript. Zhihui WANG financially supported this work. All authors have read and approved the final manuscript, and therefore, have full access to all the data in the study and take responsibility for the integrity and security of the data. This work was supported by the National Key Research and Development Program of China (No. 2021YFD1600802-02). We also thank Robert McKenzie, PhD, from Liwen Bianji (Edanz) ( https://www.liwenbianji.cn https://www.liwenbianji.cn ) for editing the English text of a draft of this manuscript. 10.1631/jzus.B2200431 1673-1581（2023）01-0094-07 王迅,黄金秋,尹宗艳等.“不知火”黄色果皮芽变品种中类胡罗卜素组分及生物合成[J].浙江大学学报（英文版）（B辑：生物医学和生物技术）,2023,24(01):94-100. 王迅,黄金秋,尹宗艳等.“不知火”黄色果皮芽变品种中类胡罗卜素组分及生物合成[J].浙江大学学报（英文版）（B辑：生物医学和生物技术）,2023,24(01):94-100. This work was supported by the National Key Research and Development Program of China (No. 2021YFD1600802-02). 2022-08-30 2022-10-26 2023-01-15 2023-02-06 2022-12-19 Copyright © Zhejiang University and Springer-Verlag GmbH Germany, part of Springer Nature 2022 2023 浙江大学出版社 Xun WANG, Jinqiu HUANG, Zongyan YIN, Ke XU, Dong JIANG, Lijin LIN, Xiaoai ZHANG, and Zhihui WANG declare that they have no conflict of interest. This article does not contain any studies with human or animal subjects performed by any of the authors. Compliance with ethics guidelines Supplementary information 1 Materials and methods 浙江大学学报（英文版）（B辑：生物医学和生物技术） 1 24