欢迎访问作物学报，今天是
作物学报

作物学报 ›› 2025, Vol. 51 ›› Issue (4): 1050-1060.doi: 10.3724/SP.J.1006.2025.44136

• 耕作栽培·生理生化 • 上一篇    下一篇

4种马铃薯内源激素提取方法优化及其在块茎解除休眠过程中的含量分析

赵喜娟1(), 张帆2, 刘圣宣2, 覃骏2, 陈惠兰2, 林原1, 罗红兵1, 刘易3, 宋波涛2, 胡新喜1,*(), 王恩爽2,*()   

  1. 1湖南农业大学 / 湖南省蔬菜生物学重点实验室 / 岳麓山实验室, 湖南长沙 410128
    2华中农业大学园艺林学学院 / 农业农村部马铃薯生物学与生物技术重点实验室 / 果蔬园艺作物种质创新与利用全国重点实验室, 湖北武汉 430070
    3新疆农业科学院综合试验场, 新疆乌鲁木齐 830013
  • 收稿日期:2024-08-25 接受日期:2024-12-12 出版日期:2025-04-12 网络出版日期:2025-01-02
  • 通讯作者: 胡新喜, E-mail: hu_xinxi@126.com; 王恩爽, E-mail: wangenshuang@126.com
  • 作者简介:赵喜娟, E-mail: 704986451@qq.com第一联系人：

    **同等贡献

  • 基金资助:
    新疆维吾尔自治区智力援疆创新拓展人才计划“小组团”援疆团队—“马铃薯生物技术与早熟育种团队”项目和国家重点研发计划项目(2023YFD1201505);新疆维吾尔自治区智力援疆创新拓展人才计划“小组团”援疆团队—“马铃薯生物技术与早熟育种团队”项目和国家重点研发计划项目(2022YFD1100201)

Optimization of extraction methods for four endogenous hormones in potatoes and analysis of their content during the process of releasing dormancy in tubers

ZHAO Xi-Juan1(), ZHANG Fan2, LIU Sheng-Xuan2, QIN Jun2, CHEN Hui-Lan2, LIN Yuan1, LUO Hong-Bing1, LIU Yi3, SONG Bo-Tao2, HU Xin-Xi1,*(), WANG En-Shuang2,*()   

  1. 1Hunan Agricultural University / Key Laboratory for Vegetable Biology of Hunan Province / Yuelu Mountain Laboratory, Changsha 410128, Hunan, China
    2College of Horticulture & Forestry Sciences, Huazhong Agricultural University / Key Laboratory of Potato Biology and Biotechnology, Ministry of Agriculture and Rural Affairs / National Key Laboratory of Germplasm Innovation and Utilization of Horticulture Crops, Wuhan 430070, Hubei, China
    3Comprehensive Test Site of Xinjiang Academy of Agricultural Sciences, Urumqi 830013, Xinjiang, China
  • Received:2024-08-25 Accepted:2024-12-12 Published:2025-04-12 Published online:2025-01-02
  • Contact: E-mail: hu_xinxi@126.com; E-mail: wangenshuang@126.com
  • About author:First author contact:

    **Contributed equally to this work

  • Supported by:
    Xinjiang Uygur Autonomous Region Intelligence Assistance Xinjiang Innovation and Expansion Talent Plan “Group Team” Assistance Xinjiang Team—“Potato Biotechnology and Early Maturing Breeding Team” Project and the National Key Research and Development Program of China(2023YFD1201505);Xinjiang Uygur Autonomous Region Intelligence Assistance Xinjiang Innovation and Expansion Talent Plan “Group Team” Assistance Xinjiang Team—“Potato Biotechnology and Early Maturing Breeding Team” Project and the National Key Research and Development Program of China(2022YFD1100201)

RichHTML

13

PDF

143

摘要：

植物激素是生长发育的重要调节物质, 但马铃薯内源激素还没有统一、高效的检测方法, 难以满足深入研究的需求。本研究利用超高效液相色谱技术, 设计单因素试验和正交试验, 优化了SA、IAA、ABA和JA这4种植物激素的提取方法, 其重复性和稳定性的相对标准偏差均小于10%, 提取效率显著高于其他提取方法。利用该方法, 对生长30 d的马铃薯植株的根、茎、叶、匍匐茎和顶芽的激素含量进行测定发现, 顶芽和叶片中SA含量最高, 顶芽和匍匐茎中IAA含量最高, 叶片中ABA含量最高, 根和匍匐茎中JA含量偏高。对马铃薯栽培种华薯1号和中薯5号的块茎分别在4℃、22℃和28℃条件下储存, 并检测了其休眠打破过程中激素含量。 结果表明,在整个休眠期, IAA含量整体呈上升趋势, SA含量呈先上升后下降而后快速上升的趋势, ABA含量整体呈下降趋势。本研究优化的提取方法提取效率高、灵敏度高、稳定性和重复性好, 适用于马铃薯4种内源激素的检测分析, 研究数据为马铃薯不同组织和块茎休眠过程中激素合成及作用机制提供理论参考。

关键词: 马铃薯, 激素, 提取方法, 超高效液相色谱, 休眠

Abstract:

Plant hormones are essential regulators of growth and development. However, the lack of a unified and efficient method for detecting endogenous hormones in potato has hindered in-depth research. In this study, ultra-high performance liquid chromatography (UPLC) was combined with single-factor and orthogonal experimental designs to optimize the extraction methods for four phytohormones: salicylic acid (SA), indole-3-acetic acid (IAA), abscisic acid (ABA), and jasmonic acid (JA). The optimized method demonstrated excellent repeatability and stability, with relative standard deviations (RSDs) below 10%, and significantly improved extraction efficiency compared to conventional methods. Using this optimized method, the hormone contents in the roots, stems, leaves, apical buds, and stolons of potato plants at 30 days of growth were quantified. The results revealed that endogenous SA levels were highest in the apical buds and leaves, IAA was most abundant in the shoot apex and stolons, ABA was concentrated in the leaves, and JA levels were higher in the roots and stolons. Additionally, the tubers of potato cultivars Huashu 1 and Zhongshu 5 were stored at 4°C, 22℃, and 28℃, and hormone levels during dormancy breaking were analyzed. The findings showed a general upward trend in IAA levels throughout storage, a fluctuating pattern in SA levels (initial increase, subsequent decline, and rapid rise), and a consistent decrease in ABA levels. The optimized extraction method developed in this study offers high efficiency, sensitivity, stability, and repeatability, making it suitable for the detection and analysis of these four endogenous hormones in potato. This research provides valuable insights into hormone synthesis and their regulatory mechanisms during dormancy and across various tissues in potato.

Key words: potato, hormones, extraction method, ultra high-performance liquid chromatography, dormancy

表1

色谱洗脱梯度表"

序号
No.		 时间
Time (min)		 流动相A
Mobile phase A (%)		 流动相B
Mobile phase B (%)
1 0 Run
2 0 90 10
3 2.0 90 10
4 8.0 10 90
5 11.0 10 90
6 11.5 90 10
7 14.0 Stop

图1

4种激素的标准曲线 SA: 水杨酸; IAA: 吲哚-3-乙酸; ABA: 脱落酸; JA: 茉莉酸。"

表2

正交设计表"

处理编号
Treatment number		 A有机相比例
Organic phase ratio (%)		 B超声时间
Ultrasound time (min)		 C酸度
Acidification degree (%)		 D提取时间
Extraction time (h)
1 (A1B1C1D1) 80 20 0.7 9
2 (A1B2C2D2) 80 25 1.0 12
3 (A1B3C2D2) 80 30 1.0 12
4 (A2B1C2D2) 90 20 1.0 12
5 (A2B2C2D1) 90 25 1.0 9
6 (A2B3C1D2) 90 30 0.7 12
7 (A3B1C2D2) 100 20 1.0 12
8 (A3B2C1D2) 100 25 0.7 12
9 (A3B3C2D1) 100 30 1.0 9

表3

6种激素提取方法"

提取方法
Extraction method		 有机相
Organic phase		 超声时间
Ultrasound time (min)		 提取时间
Extraction time
(h)		 酸化程度
Acidification degree
1 80%甲醇水 80% Methanol water 12
2 80%异丙醇水 80% Isopropanol water 12
3 50%乙腈水 50% Acetonitrile water 12
4 100%乙腈 100% Acetonitrile 12
5 100%乙腈 100% Acetonitrile 1
6 100%乙腈 100% Acetonitrile 25 12 0.7%乙酸 0.7% Acetic acid

图2

单因素条件下E3叶片植物激素回收效果 缩写同图1。A~D依次是不同有机相比例、超声时间、酸化程度、提取时间处理的E3叶片中4种激素回收浓度。数据为3个重复的平均值±标准偏差。不同字母表示由方差分析确定的各组间的统计学差异(Tukey多重比较, P < 0.05)。"

图3

4种激素提取的正交试验效果 缩写同图1。A~D依次是不同处理下E3叶片中ABA、IAA、JA、SA的含量。横坐标中A1、A2、A3依次是有机相比例80%、90%、100%; B1、B2、B3依次是超声20、25和30 min; C1、C2依次是酸化程度0.7%、1.0%; D1、D2依次是提取时间9 h和12 h。数据为3个重复的平均值±标准偏差。不同字母表示由方差分析确定的各组间的统计学差异(Tukey多重比较, P < 0.05)。"

表4

方法A3B2C1D2提取4种激素稳定性及重复性结果"

激素
Hormone		 稳定性相对标准偏差
Stability relative standard deviation (RSD)		 重复性相对标准偏差
Repetitive relative standard deviation (RSD)
ABA 3.29 4.35
IAA 7.80 3.51
SA 4.36 4.32
JA 5.55 4.40

图4

优化方法与其他5种方法提取效果比较 缩写同图1。A~D依次是利用6种方法提取的E3叶片的IAA、ABA、SA、JA的含量。横坐标1~6依次对应表3中提取方法1-6, 6为优化方法。数据为3个重复的平均值±标准偏差。不同字母表示由方差分析确定的各组间的统计学差异(Tukey多重比较, P < 0.05)。"

图5

华1和中5植株中各部位激素含量 缩写同图1。A~D和E~H分别是华1和中5根、茎、叶、顶芽、匍匐茎中JA、IAA、SA、ABA含量, 数据为3个重复的平均值±标准偏差。不同字母表示由方差分析确定的各组间的统计学差异(Tukey多重比较, P < 0.01)。"

图6

华1块茎休眠过程中顶芽长度统计 A: 华1块茎在不同温度顶芽状态图, 比例尺长度为1.5 cm; B: 不同温度下华1块茎顶芽达到2 mm数目占总数的比例变化; C: 华1块茎顶芽达到5 mm数目占总数的比例变化。数据为3个重复的平均值±标准偏差。"

图7

中5块茎休眠过程中顶芽长度统计 A: 中5块茎在不同温度顶芽状态图, 比例尺长度为1.5 cm; B: 不同温度下中5块茎顶芽达到2 mm数目占总数的比例变化; C: 中5块茎顶芽达到5 mm数目占总数的比例变化。数据为3个重复的平均值±标准偏差。"

图8

华1和中5休眠期顶芽内源激素随时间变化检测结果 缩写同图1。A~C和D~F分别是华1和中5不同储存阶段的激素含量。数据为3个重复的平均值±标准偏差。"

[1] Yang C H, Wang D D, Zhang C, Ye M H, Kong N N, Ma H L, Chen Q. Comprehensive analysis and expression profiling of PIN AUX/LAX and ABCB auxin transporter gene families in Solanum tuberosum under phytohormone stimuli and abiotic stresses. Biology, 2021, 10: 127.
[2] Swarup R, Perry P, Hagenbeek D, Van Der Straeten D, Beemster G T S, Sandberg G, Bhalerao R, Ljung K, Bennett M J. Ethylene upregulates auxin biosynthesis in Arabidopsis seedlings to enhance inhibition of root cell elongation. Plant Cell, 2007, 19: 2186-2196.
[3] 邓文红, 赵欣蕊, 张俊琦, 郭惠红. UPLC-MS/MS测定植物组织中植物激素的含量. 北京林业大学学报, 2019, 41(8): 154-160.
Deng W H, Zhao X R, Zhang J Q, Guo H H. Determination of plant hormones in plant tissues by UPLC-MS/MS. J Beijing For Univ, 2019, 41(8): 154-160 (in Chinese with English abstract).
[4] Cui K Y, Lin Y Y, Zhou X, Li S C, Liu H, Zeng F, Zhu F, Ou-Yang G F, Zeng Z X. Comparison of sample pretreatment methods for the determination of multiple phytohormones in plant samples by liquid chromatography-electrospray ionization-tandem mass spectrometry. Microchem J, 2015, 121: 25-31.
[5] Durgbanshi A, Arbona V, Pozo O, Miersch O, Sancho J V, Gómez-Cadenas A. Simultaneous determination of multiple phytohormones in plant extracts by liquid chromatography-electrospray tandem mass spectrometry. J Agric Food Chem, 2005, 53: 8437-8442.
[6] He Y. Recent advances in application of liquid-based micro- extraction: a review. Chem Pap, 2014, 68: 995-1007.
[7] Jalili V, Barkhordari A, Ghiasvand A. A comprehensive look at solid-phase microextraction technique: a review of reviews. Microchem J, 2020, 152: 104319.
[8] Atapattu S N, Temerdashev A. Recent advances in gas chromatography injection port derivatization in analytical method development. Trac Trends Anal Chem, 2023, 168: 117334.
[9] Rawlinson C, Kamphuis L G, Gummer J P A, Singh K B, Trengove R D. A rapid method for profiling of volatile and semi-volatile phytohormones using methyl chloroformate derivatisation and GC-MS. Metabolomics, 2015, 11: 1922-1933.
pmid: 26491427
[10] Izumi Y, Okazawa A, Bamba T, Kobayashi A, Fukusaki E. Development of a method for comprehensive and quantitative analysis of plant hormones by highly sensitive nanoflow liquid chromatography-electrospray ionization-ion trap mass spectrometry. Anal Chim Acta, 2009, 648: 215-225.
doi: 10.1016/j.aca.2009.07.001 pmid: 19646587
[11] Ziegler J, Qwegwer J, Schubert M, Erickson J L, Schattat M, Bürstenbinder K, Grubb C D, Abel S. Simultaneous analysis of apolar phytohormones and 1-aminocyclopropan-1-carboxylic acid by high performance liquid chromatography/electrospray negative ion tandem mass spectrometry via 9-fluorenylmethoxycarbonyl chloride derivatization. J Chromatogr A, 2014, 1362: 102-109.
doi: 10.1016/j.chroma.2014.08.029 pmid: 25160953
[12] Basharat R, Kotra V, Loong L Y, Mathews A, Kanakal M M, Dev C B P, Nyamathulla S, Varala R, Ming L C, Rao K S, et al. A mini-review on ultra performance liquid chromatography. Orient J Chem, 2021, 37: 847-857.
[13] Kojima M, Kamada-Nobusada T, Komatsu H, Takei K, Kuroha T, Mizutani M, Ashikari M, Ueguchi-Tanaka M, Matsuoka M, Suzuki K, et al. Highly sensitive and high-throughput analysis of plant hormones using MS-probe modification and liquid chromatography-tandem mass spectrometry: an application for hormone profiling in Oryza sativa. Plant Cell Physiol, 2009, 50: 1201-1214.
[14] 查秋艳, 王雪, 王少阳, 吴永超, 刘浩, 李俊花, 邓纲. 内源激素基因和生化物质调控马铃薯块茎休眠的研究进展. 中国种业, 2023, (1): 26-32.
Zha Q Y, Wang X, Wang S Y, Wu Y C, Liu H, Li J H, Deng G. Research progress in regulation of potato tuber dormancy by endogenous hormone genes and biochemical substances. China Seed Ind, 2023, (1): 26-32 (in Chinese with English abstract).
[15] 肖关丽, 郭华春. 马铃薯内源激素研究进展. 云南农业大学学报, 2007, 22: 431-434.
Xiao G L, Guo H C. Advances on the research on potato endogenous hormones. J Yunnan Agric Univ, 2007, 22: 431-434 (in Chinese with English abstract).
[16] Wang Z W, Lyu J, Xie S Z, Zhang Y, Qiu Z N, Chen P, Cui Y T, Niu Y F, Hu S K, Jiang H Z, et al. OsSLA4 encodes a pentatricopeptide repeat protein essential for early chloroplast development and seedling growth in rice. Plant Growth Regul, 2018, 84: 249-260.
[17] Abelenda J A, Barrero-Gil J. ABA signaling branches out: emerging ABA-related signaling functions in Solanum tuberosum. J Exp Bot, 2023, 74: 6405-6408.
doi: 10.1093/jxb/erad395 pmid: 37988178
[18] Zhou F, Last R L, Pichersky E. Degradation of salicylic acid to catechol in Solanaceae by SA 1-hydroxylase. Plant Physiol, 2021, 185: 876-891.
doi: 10.1093/plphys/kiaa096 pmid: 33793924
[19] 陈玉珍, 唐广彬, 马宪新, 田贵云, 于宏心, 骆璎珞, 樊明寿, 贾立国. 马铃薯块茎发育的四大调控途径. 作物杂志, 2022, (4): 9-13.
Chen Y Z, Tang G B, Ma X X, Tian G Y, Yu H X, Luo Y L, Fan M S, Jia L G. Four major regulatory pathways of potato tuber development. Crops, 2022, (4): 9-13 (in Chinese with English abstract).
[20] Walton A, Stes E, De Smet I, Goormachtig S, Gevaert K. Plant hormone signalling through the eye of the mass spectrometer. Proteomics, 2015, 15: 1113-1126.
doi: 10.1002/pmic.201400403 pmid: 25404421
[21] Van Ittersum M K. Relation between growth conditions and dormancy of seed potatoes: 2. Effects of temperature. Potato Res, 1992, 35: 365-375.
[22] Chemat F, Vian M A, Cravotto G. Green extraction of natural products: concept and principles. Int J Mol Sci, 2012, 13: 8615-8627.
doi: 10.3390/ijms13078615 pmid: 22942724
[23] Fu J H, Sun X H, Wang J D, Chu J F, Yan C Y. Progress in quantitative analysis of plant hormones. Chin Sci Bull, 2011, 56: 355-366.
[24] Chen X H, Zhao Y G, Shen H Y, Jin M C. Application of dispersive solid-phase extraction and ultra-fast liquid chromatography-tandem quadrupole mass spectrometry in food additive residue analysis of red wine. J Chromatogr A, 2012, 1263: 34-42.
[25] Deng T, Wu D P, Duan C F, Yan X H, Du Y, Zou J, Guan Y F. Spatial profiling of gibberellins in a single leaf based on microscale matrix solid-phase dispersion and precolumn derivatization coupled with ultraperformance liquid chromatography-tandem mass spectrometry. Anal Chem, 2017, 89: 9537-9543.
doi: 10.1021/acs.analchem.7b02589 pmid: 28783368
[26] Deng W H, Zhang J Q. Determination of four endogenous phytohormones in Artemisia ordosica leaves by ultra-performance liquid chromatography-tandem mass spectrometry. Bot Res, 2015, 4: 1-7.
[27] Wang Z K, Ma R, Zhao M S, Wang F F, Zhang N, Si H J. NO and ABA interaction regulates tuber dormancy and sprouting in potato. Front Plant Sci, 2020, 11: 311.
doi: 10.3389/fpls.2020.00311 pmid: 32322258
[28] Krishna Reddy S, Finlayson S A. Phytochrome B promotes branching in Arabidopsis by suppressing auxin signaling. Plant Physiol, 2014, 164: 1542-1550.
[29] Jing S L, Begum S, Yu L, Abu Kawochar M, Wang E S, Chen Y, Zhao J, Song B T. StJAZ1-like mediated root architecture plays critical roles in drought susceptibility in potato. Environ Exp Bot, 2022, 202: 105008.
[30] Mani F, Taoufik B, Doudech N, Hanène C. Physiological mechanisms for potato dormancy release and sprouting: a review. Afr Crop Sci J, 2014, 2: 155-174.
[31] Suttle J C, Hultstrand J F. Role of endogenous abscisic acid in potato microtuber dormancy. Plant Physiol, 1994, 105: 891-896.
doi: 10.1104/pp.105.3.891 pmid: 12232251
[32] Ordaz-Ortiz J J, Foukaraki S, Terry L A. Assessing temporal flux of plant hormones in stored processing potatoes using high-definition accurate mass spectrometry. Hortic Res, 2015, 2: 15002.
[33] Sorce C, Lorenzi R, Ceccarelli N, Ranalli P. Changes in free and conjugated IAA during dormancy and sprouting of potato tubers. Funct Plant Biol, 2000, 27: 371-377.
[1] 陈国欢, 张锐, 李艳迪, 赵佳琪, 任湧涛, 张天赐, 郭华春, 李俊, 杨芳. 外源硒叶面喷施对浅紫色马铃薯块茎花青素合成的影响[J]. 作物学报, 2026, 52(6): 1876-1890.
[2] 金昱何, 王雪菲, 徐张一娃, 缪怡宁, 蒋云杰, 伊莹, 缪德麟, 朱静仪, 钟一帆, 陈铭亨, 方芳, 刘鹏. 外源激素对低温胁迫下大豆叶片叶绿素荧光参数及抗氧化酶系统的影响[J]. 作物学报, 2026, 52(6): 1817-1829.
[3] 王文辕, 燕雪嘉, 刘玉霖, 孙晓彤, 李亚楠, 唐鑫华, 石瑛. 耐弱光马铃薯品种筛选及转录因子编码基因StPIF3的克隆与功能分析[J]. 作物学报, 2026, 52(6): 1631-1645.
[4] 刘昕萌, 任昊, 张继波, 张吉旺, 赵斌, 任佰朝, 刘鹏, 王洪章. 茉莉酸甲酯(MeJA)缓解高温影响玉米雌穗分化的生理机制[J]. 作物学报, 2026, 52(5): 1561-1572.
[5] 蔡兆琴, 何观咏, 何文, 阮丽霞, 梁振华, 李永珍, 李恒锐, 陈会鲜. 木薯分枝发育过程的动态转录组分析与关键基因发掘[J]. 作物学报, 2026, 52(5): 1430-1441.
[6] 宋松泉, 唐翠芳, 梁裕荣, 程红焱, 王伟青. sRNA的生物合成和作用机制及其对种子发育和休眠与萌发调控的研究进展[J]. 作物学报, 2026, 52(4): 959-981.
[7] 田立涛, 丁宁, 王树林, 齐恩芳, 张荣, 王蕊蕊, 马利雯, 李建武, 杨江伟. 马铃薯Argonaute基因家族鉴定及晚疫病诱导表达分析[J]. 作物学报, 2026, 52(4): 1116-1126.
[8] 张宇, 刘芳, 蔡诚诚, 杨小华, 吉阿么石扎, 杨元军, 王西瑶. 溴乙烷与赤霉素协同处理破除马铃薯块茎休眠的机理初探[J]. 作物学报, 2026, 52(3): 825-838.
[9] 汪玲, 胡好, 宋家凤, 程洁蓝, 陈颖, 郑婷婷, 吕钊彦, 朱晓彪, 侯华兰. 马铃薯UDP-糖基转移酶基因StUGT52的克隆及功能验证[J]. 作物学报, 2026, 52(3): 665-676.
[10] 马亮, 马璐, 张舒钰, 章慧敏, 王仁明, 宋旭东, 张振良, 冒宇翔, 陆虎华, 陈国清, 郝德荣, 周广飞. 玉米苞叶数目转录组分析及候选基因鉴定[J]. 作物学报, 2026, 52(3): 790-801.
[11] 徐强, 谢奎忠, 胡新元, 岳云, 董博, 罗爱花. 连作对马铃薯根际土壤线虫群落结构与功能的影响[J]. 作物学报, 2026, 52(2): 527-538.
[12] 杨飚, 杜帅康, 张继旺, 石瑛, 张丽莉. 马铃薯III类POD基因家族的全基因组鉴定及其表达谱分析[J]. 作物学报, 2026, 52(2): 405-420.
[13] 姬炫彤, 卞春松, 金黎平, 李森, 秦军红, 李广存. 不同耐旱型马铃薯根际微生物对干旱的响应[J]. 作物学报, 2026, 52(1): 165-177.
[14] 田甲春, 葛霞, 李守强, 李梅, 田世龙, 张亚倩, 程建新, 李玉梅. 低O2高CO2贮藏环境延缓马铃薯块茎衰老的作用机制[J]. 作物学报, 2026, 52(1): 262-278.
[15] 王雅致, 杨飚, 季香林, 石瑛, 张丽莉. 二倍体马铃薯抗旱资源鉴定及抗旱基因初步筛选[J]. 作物学报, 2026, 52(1): 72-84.
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!
京ICP备15008789号-2