二穗短柄草对光周期的代谢响应分析

doi:10.13560/j.cnki.biotech.bull.1985.2024-1099

摘要/Abstract

摘要：

目的分析不同光周期对二穗短柄草（Brachypodium distachyon,Bd21）代谢组的影响，探索Bd21适应光周期缩短的代谢机制，为研究温带主要谷物的光周期适应机制和培育广适应性新品种提供帮助。方法以长日照（16 h光照∶8 h黑暗，LD）和短日照（8 h光照∶16 h黑暗，SD）下1 d中3个时间点（ZT、ZT12、ZT24）的二穗短柄草叶片为材料，基于超高效液相色谱‒质谱联用（UPLC-MS）检测，比较LD与SD间二穗短柄草的代谢差异。结果结果检测到739种代谢物，包括135种有机酸及其衍生物、93种有机含氧化合物、92种脂质和类脂分子等。LD与SD下短柄草代谢组分离，SD条件最终使总体代谢水平提高，并上调了氨基酸类物质表达，包括天冬氨酸、异亮氨酸、组氨酸。S24 vs L24富集到19条代谢通路，包括谷氨酸、天冬氨酸和谷氨酰胺代谢，赖氨酸降解，缬氨酸、亮氨酸和异亮氨酸生物合成与降解代谢等。多条氨基酸通路通过TCA循环密切连接。结论二穗短柄草的代谢组对SD条件敏感。二穗短柄草可能通过上调天冬氨基酸下游代谢网络及支链氨基酸的合成与分解代谢以适应光周期缩短、光合不足的变化以维持代谢平衡，表明氨基酸代谢调节在二穗短柄草适应光周期缩短过程中发挥重要作用。

关键词: 二穗短柄草, 光周期, 代谢组学, 天冬氨酸, 支链氨基酸, 雌酮, UPLC-MS

Abstract:

Objective To analyze the effects of different photoperiods on the metabolome of Brachypodium distachyon (Bd21), and to investigate how photoperiod variation influences Bd21 growth and development. This study aims to provide insights into the photoperiod adaptation mechanisms of temperate grasses and assist in breeding widely adaptable new varieties. Method Leaves of B. distachyon (Bd21) were collected at three time points (ZT0, ZT12, and ZT24) under long-day (16 h light∶8 h dark, LD) and short-day (8 h light∶16 h dark, SD) conditions. Metabolic differences between LD and SD conditions in B. distachyon were compared using ultrahigh-performance liquid chromatography-mass spectrometry (UPLC-MS). Result A total of 739 metabolites were detected, including 135 organic acids and their derivatives, 93 organic oxygen-containing compounds, and 92 lipids and lipid-like molecules. Metabolomic separation between LD and SD conditions was observed, with an overall increase in metabolic levels and upregulation of amino acid-related metabolites under SD, including aspartate, isoleucine, and histidine. The S24 vs L24 comparison enriched 19 metabolic pathways, including glutamate, aspartate, and glutamine metabolism, lysine degradation, and the biosynthesis and degradation of valine, leucine, and isoleucine. Several amino acid pathways were closely linked to the TCA cycle. Conclusion The metabolome of B. distachyon is sensitive to short-day (SD) conditions. B. distachyon may adapt to the changes of shorter photoperiod and insufficient photosynthesis by upregulating the downstream metabolic network of aspartic acid and the synthesis and degradation of branched-chain amino acids, aiming to maintain metabolic balance. This indicates that amino acid metabolism regulation plays an important role in the adaptation of B. distachyon to the shortened photoperiod.

Key words: Brachypodium distachyon, photoperiod, metabolomics, aspartate, branched-chain amino acids, estrone, UPLC-MS

蒋天威, 马培杰, 李亚娇, 陈才俊, 刘晓霞, 王小利. 二穗短柄草对光周期的代谢响应分析[J]. 生物技术通报, 2025, 41(7): 237-247.

JIANG Tian-wei, MA Pei-jie, LI Ya-jiao, CHEN Cai-jun, LIU Xiao-xia, WANG Xiao-li. Metabolic Response Analysis of Brachypodium distachyon to Photoperiods[J]. Biotechnology Bulletin, 2025, 41(7): 237-247.

图/表 7

图1 代谢物分类饼图及OPLS-DA得分图、总积分统计图A：739种代谢物分类饼图；B：5个样本差异的OPLS-DA得分图；C：长短日照样本间差异的OPLS-DA得分图；D：总积分和统计图。图B、C横坐标代表第一主成分得分（PC1），用于区分不同样本组，距离越远差异越大；纵坐标代表第一正交主成分得分（OC1），用于揭示组内的相关性，重复间距离越小，重复性越好。图B、D中LS0为ZT0时的样本，L12、L24为长日照处理12 h、24 h的样本，S12、S24为短日照处理12 h、24 h的样本。图C中LS0为对照，LD表示L12、L24合并为一组，SD表示S12、S24合并为一组。图D中方差分析使用Tukey多重比较方法，字母间的差异表示差异显著（P<0.05）。下同

Fig. 1 Pie chart of metabolite classification, OPLS-DA score plot, and total score statisticsA: Pie chart showing the classification of 739 metabolites. B: OPLS-DA score plot of the five sample groups showing differences. C: OPLS-DA score plot revealing differences between long-day and short-day samples. D: Statistical plot of the total sum of integrals. In Fig. B and C, the X-axis refers to the first principal component score (PC1), which distinguishes different sample groups. The greater the distance, the larger the difference between the groups. The Y-axis refers to the first orthogonal component score (OC1), which reveals the internal correlations of the groups. The smaller the distance between replicates, the higher the reproducibility. In Fig. B and D, LS0 refers to the samples at ZT0, L12 and L24 refer to the samples subjected to long-day treatment for 12 and 24 h, respectively, while S12 and S24 refers to the samples subjected to short-day treatment for 12 and 24 h, respectively. In Fig. C, LS0 is the control group, LD refers to the combined L12 and L24 groups, and SD refers to the combined S12 and S24 groups. In Fig. D, variance analysis was performed using the Tukey multiple comparisons method. Significant differences are indicated by letters, where different letters denote a statistically significant difference (P<0.05). The same below

图2 五类代谢物聚类热图A：有机酸及其衍生物；B：有机含氧化合物；C：脂质和类脂分子；D：苯丙类和聚酮类；E：苯并杂环化合物。每个图均为行标准化、行聚类；红蓝分别代表上调及下调，小写字母a、b、c表示代谢物在长短日照间出现明显表达差异的区域

Fig. 2 Clustering heatmap of five classes of metabolitesA: Organic acids and their derivatives. B: Organic oxygen-containing compounds. C: Lipids and lipophilic molecules. D: Phenylpropanoids and polyketides. E: Benzoheterocyclic compounds. Each plot is row-normalized and row-clustered, with red and blue representing upregulation and downregulation, respectively. Lowercase letters a, b, and c indicate regions where metabolites show significant expression differences between long-day and short-day conditions

图3 各比较组差异代谢物火山图横坐标代表该组对比各物质的倍数变化（取以2为底的对数），纵坐标表示t检验的P值（取以10为底的对数）。点表示代谢物，红色、蓝色、灰色分别代表显著上调、显著下调、差异不显著，右上角点和数值表示不同颜色标注的代谢物数量

Fig. 3 Volcano plots of differential metabolites for each comparison groupThe x-axis refers to the fold change of each substance in the group comparison, taken as the logarithm base 2. The y-axis indicates the P-value from the t-test, taken as the logarithm base 10. Each dot refers to a metabolite, with red, blue, and gray colors corresponding to significantly up-regulated, significantly down-regulated, and non-significantly changed metabolites, respectively. The dots and numbers in the upper right corner indicate the number of metabolites labeled in different colors

图4 各比较组中差异倍数最大的前10种DAM从浅色到深色的颜色梯度表示|log2FC|值增加，橙色表示上调，蓝色表示下调。标签显示|log2FC|值

Fig. 4 Top 10 DAMs with the highest fold changes in each comparison groupThe color gradient from light to dark indicates increasing |log2FC| values, with orange for up-regulation and blue for down-regulation. Labels show the |log2FC| values

表1 ZT12和ZT24二穗短柄草在SD和LD间差异显著的前10种DAM

Table 1 Top 10 DAM with significant differences between SD and LD conditions at ZT12 and ZT24 in Brachypodium distachyon

组合 Group	代谢物名称 Metabolite	VIP	Log₂ 倍变比 Log₂（FC）	P值 P-value	上调或下调 Up or down
S12 vs L12	腐胺 Putrescine	2.08	11.86	1.28E-02	DOWN
	D-（-）-扁桃酸 D-（-）-mandelic acid	2.30	11.82	2.65E-09	DOWN
	D-葡萄糖-6-磷酸 D-glucose-6-phosphate	2.23	11.50	1.54E-03	DOWN
	半乳糖醇 Galactitol	1.98	11.37	2.62E-02	DOWN
	D-半乳糖 D-galactose	2.12	11.22	9.51E-03	UP
	3，4-二羟基苯乙二醇3，4-dihydroxyphenylglycol	2.30	11.11	1.37E-06	UP
	α-丙氨酸 Afalanine	2.30	11.10	2.10E-05	DOWN
	次黄嘌呤核苷 Inosine	1.94	10.83	3.65E-02	UP
	小白菊内酯 Parthenolide	2.12	5.31	9.01E-03	DOWN
	尿黑酸 Homogentisic acid	1.93	2.83	3.56E-02	DOWN
S24 vs L24	异亮氨酸 Isoleucine	2.12	12.11	7.21E-13	UP
	不枯芽菌素 A Brefeldin A	2.11	12.10	5.61E-05	DOWN
	1-β-D-阿拉伯呋喃糖基尿嘧啶 1-beta-D-arabinofuranosyluracil	2.10	11.82	1.79E-04	UP
	维生素 D₂ Vitamin D₂	2.11	11.66	2.24E-05	DOWN
	京尼平苷 Geniposide	2.11	11.47	4.54E-05	UP
	丙戊酰胺 Valpromide	2.11	11.36	2.37E-05	UP
	雌酮 Estrone	2.12	11.35	3.09E-08	DOWN
	β-甘油磷酸 Beta-Glycerophosphoric acid	2.01	11.13	3.74E-03	UP
	天冬氨酸 Aspartate	1.80	11.02	3.14E-02	UP
	（1'R，3R，5R，8'S）-二氢相酸-O-β-D-葡萄糖苷（1'R，3R，5R，8'S）-dihydrophaseic acid-O-D-glucoside	1.89	10.99	1.71E-02	DOWN

图5 KEGG代谢通路富集气泡图及氨基酸类物质热图A、B：分别为S24 vs L24、L12 vs S12组的KEGG通路富集的气泡图；C：所有氨基酸类代谢物定量聚类热图；D：氨基酸类差异代谢物定量聚类热图。气泡图气泡越大，表示该通路中富集的差异代谢物越多。图A中：1：氨基酸-tRNA生物合成；2：组氨酸代谢；3：β-丙氨酸代谢；4：赖氨酸降解；5：丙氨酸、天冬氨酸和谷氨酸代谢；6：磷酸酯和磷酰化代谢；7：缬氨酸、亮氨酸和异亮氨酸生物合成；8：α-亚麻酸代谢；9：精氨酸生物合成；10：烟酸和烟酰胺代谢；11：泛酸和辅酶A生物合成；12：柠檬酸循环（TCA循环）；13：乙二酸和二羧酸代谢；14：甘油磷脂代谢；15：不饱和脂肪酸生物合成；16：缬氨酸、亮氨酸和异亮氨酸降解；17：色氨酸代谢；18：嘌呤代谢；19：类固醇激素生物合成。图B中：1：半乳糖代谢；2：酪氨酸代谢；3：泛醌和其他萜类醌生物合成；4：谷胱甘肽代谢；5：氨基糖和核苷糖代谢；6：精氨酸和脯氨酸代谢；7：嘌呤代谢。图C、D热图均为行标准化及聚类，红色表示上调，蓝色表示下调

Fig. 5 Enrichment bubble plot of KEGG pathways and heatmap of amino acid-related metabolitesA and B: KEGG pathway enrichment bubble plots for S24 vs L24 and L12 vs S12 groups, respectively. C: Quantitative clustering heatmap of all amino acid metabolites. D: Quantitative clustering heatmap of differential amino acid metabolites. In the bubble plots, larger bubbles indicate a higher number of differentially enriched metabolites in that pathway. In Fig. A, 1: aminoacyl-tRNA biosynthesis; 2: histidine metabolism; 3: β-alanine metabolism; 4: lysine degradation; 5: alanine, aspartate, and glutamate metabolism; 6: phosphoester and phosphorylation metabolism; 7: biosynthesis of valine, leucine, and isoleucine; 8: α-linolenic acid metabolism; 9: arginine biosynthesis; 10: nicotinate and nicotinamide metabolism; 11: pantothenate and CoA biosynthesis; 12: citric acid cycle (TCA cycle); 13: oxalate and dicarboxylate metabolism; 14: glycerophospholipid metabolism; 15: unsaturated fatty acid biosynthesis; 16: degradation of valine, leucine, and isoleucine; 17: tryptophan metabolism; 18: purine metabolism; 19: steroid hormone biosynthesis. In Fig. B, 1: galactose metabolism; 2: tyrosine metabolism; 3: biosynthesis of ubiquinone and other terpenoid-quinones; 4: glutathione metabolism; 5: amino sugar and nucleotide sugar metabolism; 6: arginine and proline metabolism; 7: purine metabolism. Fig. C and D: The heatmaps are row-normalized and clustered, with red indicating upregulation and blue indicating downregulation

图6 氨基酸相关KEGG代谢通路图红框代表一条通路，蓝色文字代表在S24中显著上调的差异表达代谢物。虚线表示代谢物间接上下级关系，实线表示直接关系。为所有检测到的代谢物绘制热图，红表示上调，蓝表示下调

Fig. 6 Amino acid-related KEGG metabolic pathway diagramRed boxes indicate pathways, blue shows up-regulated metabolites in S24. Dashed lines indicate indirect superior-subordinate relationships of metabolites, and the solid lines indicate direct relationships. A heat map is drawn for all detected metabolites, red for upregulation, blue for downregulation

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