UV-A诱导大豆芽苗菜下胚轴中花青苷积累的分子机理

doi:10.3864/j.issn.0578-1752.2015.12.014

摘要/Abstract

摘要： 【目的】研究长波紫外光（UV-A）、白光（W）和蓝光（B）对大豆芽苗菜下胚轴中花青苷含量、花青苷合成相关酶活性、花青苷合成途径相关基因及光受体基因表达量的影响,以探明UV-A诱导大豆芽苗菜下胚轴中花青苷生物合成的分子机理，为光质调控技术应用于大豆芽苗菜工业化生产提供理论依据。【方法】以大豆‘东农690’为试材，以黑暗培养为对照，连续的UV-A、白光（W）和蓝光（B）光照培养作为试验处理，在处理0 h、12 h、24 h和36 h后各采样一次，分别测定花青苷含量，苯丙氨酸解氨酶（PAL）、查尔酮异构酶（CHI）以及类黄酮半乳糖苷转移酶（UFGT）活性，相关基因（PAL、CHS、CHI、DFR、ANS、UFGT、MYB75、CRY1、CRY2、UVR8）表达量。花青苷含量采用分光光度法测定，苯丙氨酸解氨酶（PAL）及查尔酮异构酶（CHI）活性采用分光光度法测定，类黄酮半乳糖苷转移酶（UFGT）活性采用超高效液相色谱法（UPLC）测定。材料总RNA采用Trizol试剂法提取，基因表达量采用qRT-PCR测定。【结果】黑暗培养下的大豆芽苗菜子叶为黄色，而白光（W）、蓝光（B）和UV-A培养下的大豆芽苗菜子叶为绿色。与黑暗培养及其他光质处理相比，UV-A显著提高大豆芽苗菜下胚轴中花青苷含量；随着处理时间的延长，花青苷积累逐渐增加。0 h处理下，大豆芽苗菜下胚轴中花青苷含量较低，约2 U·g^-1FW。经36 h的UV-A处理，大豆芽苗菜下胚轴中花青苷含量达到最大值（43 U·g^-1FW），显著高于黑暗及其他光照处理。0 h处理下，PAL和CHI酶活性较高。与黑暗培养及其他光质处理相比，24 h及36 h UV-A处理显著提高了PAL酶活性；12 h及24 h UV-A处理显著提高了UFGT酶活性。0 h处理下，不同处理间的花青苷合成相关基因表达均无差异。与黑暗培养及其他光质处理相比， UV-A处理36 h显著上调了MYB75、CRY1、CRY2及UVR8的表达，分别上调约12倍、30倍、6倍和2倍；UV-A处理12 h显著上调了花青苷合成相关结构基因（PAL、CHS、CHI、DFR、ANS、UFGT）的表达，分别上调约5倍、58倍、10倍、6倍、44倍和47倍。【结论】UV-A通过提高PAL、UFGT酶活性及上调花青苷合成和光受体相关基因的表达，诱导了大豆芽苗菜下胚轴中花青苷的积累。

关键词: 大豆芽苗菜, 花青苷, UV-A, 分子机制

Abstract: 【Objective】The objective of the experiment was to investigate the effect of UV-A, as well as white (W) and blue (B) lights on content of anthocyanin, activities of related enzymes, transcript levels of anthocyanin biosynthesis-related genes and light receptor genes. This study would find out the molecular mechanism about UV-A induces anthocyanin accumulation in the hypocotyls of soybean sprouts and provide a theoretical foundation for the application of light quality control technology in the industrialized production of soybean sprouts.【Method】The seedlings of soybean variety ‘Dongnong 690’ were treated with continuous UV-A, white light (W) and blue light (B). Samples were collected at 0, 12, 24 and 36 h after treatment, respectively. The anthocyanin content, the activities of PAL, CHI, and UFGT and the expression of related genes (PAL, CHS, CHI, DFR, ANS, UFGT, MYB75, CRY1, CRY2 and UVR8) were detected. The difference of anthocyanin content, related enzymes and genes expression under different light qualities in the hypocotyls of soybean sprouts was analyzed. The anthocyanin content was determined by spectrophotometric method, phenylalanine ammonia lyase (PAL) and chalcone isomerase (CHI) activities were measured by spectrophotometry, flavonoid galactosyl transferase (UFGT) activity by ultra performance liquid chromatography (UPLC) determination. Total RNA was extracted using the Trizol reagent. Gene expression was determined by qRT-PCR.【Result】Soybean sprouts grown in the dark showed yellow cotyledons while sprouts grown under white (W), blue (B) and UV-A lights existed green cotyledons. Compared with the dark and other light quality cultures, UV-A treatment significantly improved the content of anthocyanin in the hypocotyls of soybean sprouts. As the illumination time goes on, anthocyanin accumulation was increased gradually. The anthocyanin content in the hypocotyls of soybean sprouts was low at 0 h, about 2 U·g^-1 FW. The content of anthocyanin reached a maximum of 43 U·g^-1 FW at 36 h under UV-A, which was significantly higher than those in the other treatments. The activities of PAL and CHI were higher at 0 h. Compared to that in the dark and other light treatments, continuous UV-A light treatment for 24 h and 36 h significantly increased the activity of phenylalanine ammonia-lyase (PAL). Continuous UV-A light treatment for 12 h and 24 h significantly increased the activity of and flavonoid glycosyltransferase (UFGT). There was no difference in the expression of anthocyanin related genes among different treatments at 0 h. Continuous UV-A treatment for 36 h significantly up-regulated the expression of regulator gene MYB75 and light receptor genes (CRY1, CRY2 and UVR8) to about 12-, 30-, 6- and 2-fold, in the hypocotyls of soybean sprouts compared with those in the other treatments, respectively. Moreover, continuous UV-A treatment for 12 h significantly up-regulated the expression of anthocyanin biosynthesis-related structural genes (PAL, CHS, DFR, ANS, UFGT) to about 5, 58, 10, 6, 44 and 47 times, respectively.【Conclusion】UV-A induced anthocyanin accumulation by improving the activities of PAL and UFGT and up regulating the expression of related genes in the hypocotyls and light receptor of soybean sprouts.

Key words: soybean sprouts, anthocyanin, UV-A, molecular mechanism

戚楠楠，张晓燕，苏娜娜，邬奇，耿殿祥，齐学会，魏圣军，崔瑾. UV-A诱导大豆芽苗菜下胚轴中花青苷积累的分子机理[J]. 中国农业科学, 2015, 48(12): 2408-2416.

QI Nan-nan, ZHANG Xiao-yan, SU Na-na, WU Qi, GENG Dian-xiang, QI Xue-hui, WEI Sheng-jun, CUI Jin. The Molecular Mechanism of UV-A Induced Anthocyanin Accumulation in the Hypocotyls of Soybean Sprouts[J]. Scientia Agricultura Sinica, 2015, 48(12): 2408-2416.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2015.12.014

https://www.chinaagrisci.com/CN/Y2015/V48/I12/2408

参考文献

[1]    Mostafa M M, Rahma E H, RADY A H. Chemical and nutritional changes in soybean during germination. Food Chemistry
[2]    Kim E H, Kim S L, Kim S H, Chung I M. Comparison of isoflavones and anthocyanins in soybean [Glycine max (L.) Merrill] seeds of different planting dates. Journal of Agricultural and Food Chemistry, 2012, 60(41): 10196-10202.

[3]    Kim S L, Lee J E, Kwon Y U, Kim W H, Jung G H, Kim D W, Lee C K, Lee Y Y, Kim M J, Kim Y H, Hwang T Y, Chung I M. Introduction and nutritional evaluation of germinated soy germ. Food Chemistry, 2013, 136: 491-500.

[4]    Zhou B, Wang Y, Zhan Y G, Li Y H, Kawabata S. Chalcone synthase family genes have redundant roles in anthocyanin biosynthesis and response to blue/UV-A light in turnip. American Journal of Botany, 2013, 100(12): 2458-2467.

[5]    Tsurunaga Y, Takahashi T, Katsube T, Kudo A, Kuramitsu O, Ishiwata M, Matsumoto S. Effects of UV-B irradiation on the levels of anthocyanin, rutin and radical scavenging activity of buckwheat sprouts. Food Chemistry, 2013, 141(1): 552-556.

[6]    Emiliani J, Grotewold E, Ferreyra M L F, Casati P. Flavonols protect Arabidopsis plants against UV-B deleterious effects. Molecular Plant, 2013, 6(4): 1376-1379.

[7]    Koide T, Kamei H, Hashimoto Y. Antitumor effect of hydrolyzed anthocyanin from grape rinds and red rice. Cancer Biother Radiopharm, 1996, 11(4): 273-277.

[8]    Wang H, Nair M G, Strasburg G M. Antioxidant and anti- inflammatory activities of anthocyanins and their ahlycon, cyaniding, from tart cherries. Journal of Natural Products, 1999, 62(2): 294-296.

[9]    He J, Giusti M M. Anthocyanins: natural colorants with health- promoting properties. Annual Review of Food Science and Technology, 2010, 1: 163-187.

[10]   Feng F, Li M, Ma F W, Cheng L L. Phenylpropanoid metabolites and expression of key genes involved in anthocyanin biosynthesis in the shaded peel of apple fruit in response to sun exposure. Plant Physiology and Biochemistry, 2013, 69: 54-61.

[11]   Tan J F, Tu L L, Deng F L, Hu H Y, Nie Y C, Zhang X L. A genetic and metabolic analysis revealed that cotton fiber cell development was retarded by flavonoid naringenin. Plant Physiology, 2013, 162(1): 86-95.

[12]   Dubos C, Stracke R, Grotewold E, Weisshaar B, Martin C, Lepiniec L. MYB transcription factors in Arabidopsis. Trends in Plant Science, 2012, 15(10): 573-581.

[13]   Shin D H, Choi M, Kim K, Bang G, Cho M, Choi S B, Choi G, Park Y I. HY5 regulates anthocyanin biosynthesis by inducing the transcriptional activation of the MYB75/PAP1 transcription factor in Arabidopsis. FEBS Letters, 2013, 587 : 1543-1547.

[14]   Rizzini L, Favory J J, Cloix C, Faggionato D, O’Hara A, Kaiserli E, Baumeister R, Schäfer E, Nagy F, Jenkins G I. Perception of UV-B by the Arabidopsis UVR8 protein. Science, 2011, 332: 103-106.

[15]   Li Y Y, Mao K, Zhao C, Zhang R F, Zhao X Y, Zhang H L, Shu H R, Hao Y J. Molecular cloning of cryptochrome 1 from apple and its functional characterization in Arabidopsis. Plant Physiology and Biochemistry, 2013, 67: 169-177.

[16]   Li Y Y, Mao K, Zhao C, Zhao X Y, Zhang R F, Zhang H L, Shu H R, Hao Y J. Molecular cloning and functional analysis of a blue light receptor gene MdCRY2 from apple (Malus domestica). Plant Cell Report, 2013, 32: 555-566.

[17]   Kadomura-Ishikawa Y, Miyawaki K, Noji S, Takahashi A. Phototropin 2 is involved in blue light-induced anthocyanin accumulation in Fragaria xananassa fruits. Journal of Plant Research, 2013, 126(6): 847-857.

[18]   Morales L O, Brosché M, Vainonen J, Jenkins G I., Wargent J J, Sipari N, Strid Å, Lindfors A V, Tegelberg R, Aphalo P J. Multiple roles for UV RESISTANCE LOCUS8 in regulating gene expression and metabolite accumulation in Arabidopsis under solar ultraviolet radiation. Plant Physiology,2013, 161: 744-759.

[19] Huang W J, Zhang S L, Xiao C C, Zhang Q J, Qin G H, Wu J. Relationship between anthocyanin biosynthesis and related enzyme activities in Pyrus communis L. cv. ‘Early Red Comice’ and its green bud mutant. Xibei Zhiwu Xuebao, 2011, 31(7): 1428-1433.

[20]   Pirie A, Mullins M G. Changes in anthocyan in and phenolics content of grapevine leaf and fruit tissues treated with sucrose, nitrate abscisicacid. Plant Physiology, 1976,58: 468-472.

[21]   Lister C E, Lancaster J E, Walker J R L. Developmental changes in enzymes of flavonoid biosynthesis in the skins of red and green apple cultivars. Journal Science of Food Agriculture, 1996, 71: 313-320.

[22]   刘金, 魏景立, 刘美艳, 宋杨, 冯守千, 王传增, 陈学森. 早熟苹果花青苷积累与其相关酶活性及乙烯生成之间的关系. 园艺学报, 2012, 39(7): 1235-1242.

Liu J, Wei J L, Liu M Y, Song Y, Feng S Q, Wang C Z, Chen X S. The relationships between the enzyme activity of anthocyanin biosynthesis, ethylene release and anthocyanin accumulation in fruits of precocious apple cultivars. Acta Horticulturae Sinica, 2012, 39(7): 1235-1242. (in Chinese)

[23]   Kondo S, Hiraoka K, Kobayashi S, Honda C, Terahara N. Changes in the expression of anthocyanin biosynthetic genes during apple development. Journal of Amercia Society Horticultural Science, 2002, 127: 971-976.

[24]   Zhang Z Z, Li X X, Chu Y N, Zhang M X, Wen Y Q, Duan C Q, Pan Q H. Three types of ultraviolet irradiation differentially promote expression of shikimate pathway genes and production of anthocyanins in grape berries. Plant Physiology and Biochemistry, 2012, 57: 74-83.

[25]   Holton T A, Cornish E C. Genetics and biochemistry of anthocyanin biosynthesis. Plant Cell, 1995, 7: 1071-1083.

[26]   Pombo M A, Martínez G A, Civello P M. Cloning of FaPAL6 gene from strawberry fruit and characterization of its expression and enzymatic activity in two cultivars with different anthocyanin accumulation. Plant Science, 2011, 181(2): 111-118.

[27]   Burda S, Oleszek W, Lee C Y. Phenolic compounds and their changes in apples during maturation and cold storage. Journal of Agricultural of Food Chemistry, 1990, 38: 945-948.

[28]   Huang X Y, Cai W X, Xu B J. Kinetic changes of nutrients and antioxidant capacities of germinated soybean (Glycine max L.) and mung bean (Vigna radiata L.) with germination time. Food Chemistry, 2014, 143: 268-276.

[29]   Chen R Y, Liu H C, Huang Q, Sun G W, Huang D F. Changes of chlorophyll and anthocyanin content and related enzyme activities of flower stalk in Chinese kale under different light intensities. Acta Horticulture, 2008, 769: 103-111.

[30]   Stafford H A (Ed). Flavonoid Metabolism. Boca Raton: CRC Press, 1990: 101-132.

[31]   Zvi M M, Shklarman E, Masci T, Kalev H, Debener T, Shafir S, Ovadis M, Vainstein A. PAP1 transcription factor enhances production of phenylpropanoid and terpenoid scent compounds in rose flowers. New Phytologist, 2012, 195: 335-345.

[32]   Cominelli E, Gusmaroli G, Allegra D, Galbiatia M, Wade H K, Jenkins G I, Tonelli C. Expression analysis of anthocyanin regulatory genes in response to different light qualities in Arabidopsis thaliana. Journal of Plant Physiology, 2008, 165: 886-894.

[33]   张剑亮, 潘大仁, 周以飞, 王占成, 华树妹, 侯黎丽, 随粉粉. 观赏向日葵花青素苷合成途径同源基因的克隆与表达. 园艺学报, 2009, 36(1): 73-80.

Zhang J L, Pan D R, Zhou Y F, Wang Z C, Hua S M, Hou L L, Sui F F. Cloning and expression of genes involved in anthocyanins synthesis in ornamental sunflower. Acta Horticulturae Sinica, 2009, 36(1): 73-80. (in Chinese)

[34]   刘晓芬, 李方, 殷学仁, 徐昌杰, 陈昆松. 花青苷生物合成转录调控研究进展. 园艺学报, 2013, 40(11): 2295-2306.

Liu X F, Li F, Yin X R, Xu C J, Chen K S. Recent advances in the transcriptional regulation of anthocyanin biosynthesis. Acta Horticulturae Sinica, 2013, 40(11): 2295-2306. (in Chinese)

[35]   Maier A, Schrader A, Kokkelink L, Falke C, Welter B, Iniesto E, Rubio V, Uhrig J F, Hülskamp M, Hoecker U. Light and the E3 ubiquitin ligase COP1/SPA control the protein stability of the MYB transcription factors PAP1 and PAP2 involved in anthocyanin accumulation in Arabidopsis. The Plant Journal, 2013, 74: 638-651.
, 1987, 23(4): 257-275.