• ISSN 1008-505X
  • CN 11-3996/S

柳枝稷生长、生理和基因差异表达对供磷水平的响应

刘威帆, 赵匆, 吴娜, 刘吉利

刘威帆, 赵匆, 吴娜, 刘吉利. 柳枝稷生长、生理和基因差异表达对供磷水平的响应[J]. 植物营养与肥料学报, 2024, 30(11): 2181-2196. DOI: 10.11674/zwyf.2024169
引用本文: 刘威帆, 赵匆, 吴娜, 刘吉利. 柳枝稷生长、生理和基因差异表达对供磷水平的响应[J]. 植物营养与肥料学报, 2024, 30(11): 2181-2196. DOI: 10.11674/zwyf.2024169
LIU Wei-fan, ZHAO Cong, WU Na, LIU Ji-li. Response of growth, physiology and differential gene expression to phosphate supply level in switchgrass (Panicum virgatum L.)[J]. Journal of Plant Nutrition and Fertilizers, 2024, 30(11): 2181-2196. DOI: 10.11674/zwyf.2024169
Citation: LIU Wei-fan, ZHAO Cong, WU Na, LIU Ji-li. Response of growth, physiology and differential gene expression to phosphate supply level in switchgrass (Panicum virgatum L.)[J]. Journal of Plant Nutrition and Fertilizers, 2024, 30(11): 2181-2196. DOI: 10.11674/zwyf.2024169

柳枝稷生长、生理和基因差异表达对供磷水平的响应

基金项目: 国家自然科学基金项目(31860344);宁夏自然科学基金项目(2022AAC03062)。
详细信息
    作者简介:

    刘威帆 E-mail: 12022131346@stu.nxu.edu.cn

    通讯作者:

    刘吉利 E-mail: tim11082003@163.com

Response of growth, physiology and differential gene expression to phosphate supply level in switchgrass (Panicum virgatum L.)

  • 摘要:
    目的 

    探究柳枝稷 (Panicum virgatum L.) 在不同磷水平下的生理代谢及转录水平变化,以解析柳枝稷磷响应特征,挖掘磷高效利用基因。

    方法 

    以‘Pathfinder’品种柳枝稷为供试材料,采用水培方法(Hoagland 营养液)进行培养试验。营养液设置4个KH2PO4供应水平:20、100、200、500 μmol/L (分别记为P20、P100、P200、P500)。在生长箱中培养45天后,取样测定柳枝稷生长和根系表型指标、抗逆酶活性,并进行叶片和根系转录组测序,分析不同磷水平下柳枝稷叶片和根系的响应差异。最后,从糖酵解和苯丙素生物合成途径挑选了部分基因进行qRT-PCR分析,以证实转录组测序的准确性。

    结果 

    随着供磷水平的提高,叶片超氧化物歧化酶(SOD)、过氧化物酶(POD)和过氧化氢酶(CAT)活性呈先降低后增加的趋势;根系总长度、根系总面积和根系活力呈先增加后减少的趋势;叶片和根系中酸性磷酸酶活性和磷含量呈先增加后减少的趋势。不同供磷浓度下,叶片和根系中各磷处理共同表达差异表达基因(DEGs)分别有1091、1762个。GO富集注释显示,叶片中DEGs富集在离子跨膜转运、氧化还原酶上,根系则富集在特异DNA序列结合、主动跨膜转运和裂合酶上。两器官共同富集的DEGs主要定位于抗氧化酶、转移酶、无机分子跨膜转运蛋白上。KEGG富集分析发现,柳枝稷叶片和根系中共有9个共同代谢通路,其中氨基糖和核苷糖代谢、糖酵解/糖异生、苯丙素生物合成、蔗糖和淀粉代谢通路DEGs显著富集,具体为:糖酵解和苯丙素生物合成途径各有16个DEGs,涉及磷吸收转运基因共20个。糖酵解途径中关键酶基因 (AEPGMHKPFK),苯丙素生物合成途径中关键酶基因 (PALCCRCADC4H4CL) 和磷吸收转运调控基因 (GPT2PHT2;1TPTPPT1PPT2PPT3PiC3APC2) 的差异化表达,引起柳枝稷叶片和根系对磷营养的代谢反应差异。

    结论 

    适当提高供磷水平,可改善柳枝稷根系特征,降低叶片抗逆酶活性,提高叶片和根系中酸性磷酸酶活性,从而提高植株磷含量和干物质量。此外,糖酵解、苯丙素生物合成和磷吸收转运相关的关键基因在叶片和根系中均表现出显著差异表达。

    Abstract:
    Objectives 

    We explored the physiological metabolism and transcriptional changes of switchgrass (Panicum virgatum L.) under different phosphorus levels, aiming to understand the phosphorus response of root morphology and growth of switchgrass.

    Methods 

    A hydroponics experiment of switchgrass was carried out, based on the Hoagland nutrient solution, 4 KH2PO4 supply levels: 20, 100, 200, 500 μmol/L were setup (recorded as P20, P100, P200, P500). After 45 days of treatment, the seedlings were harvested for determination of physiological, root phenotype indexes and antistress enzyme activities, and transcriptome sequencing of leaves and roots was performed to identify the differentially expressed genes (DEGs) in leaves and roots. Some high expressed DEGs from glycolysis and phenylpropyl biosynthesis pathways were selected for qRT-PCR analysis to confirm the accuracy of transcriptome sequencing.

    Results 

    With the increase of P supply level, the activities of SOD, POD, and CAT in the leaves showed a trend of initially decreasing and then increasing; the total length, total area, and vitality of the roots increased first and then decreased; the acid phosphatase activities and phosphorus contents in both leaves and roots showed an initial increase followed by a decrease trend. There were 1091 and 1762 commonly expressed genes in the leaves and roots under all the phosphorus levels. GO enrichment annotation showed that the DEGs in leaves were mainly enriched in ion transmembrane transport and oxidoreductase, and the DEGs in roots were mainly enriched in specific DNA sequence binding, active transmembrane transport and lyase, and the DEGs in both the two organs in antioxidant enzyme, transferase and inorganic molecular transmembrane transporter. KEGG enrichment analysis showed that there were 9 common metabolic pathways in switchgrass leaves and roots, the DEGs were significantly enriched in amino sugar and nucleoside glucose metabolism, glycolysis/gluconeogenesis, phenylpropanoid biosynthesis, sucrose and starch metabolism pathways, and both glycolysis and phenylpropanoid biosynthesis pathways were enriched 16 DEGs each, and a total of 20 DEGs involved in phosphorus uptake transportion. The differential expression of AE, PGM, HK, PFK in key enzyme genes of glycolysis pathway, that of PAL, CCR, CAD, C4H, 4CL in key enzyme genes in the phenylpropanoid biosynthesis pathway, and GPT2, PHT2;1, TPT, PPT1, PPT2, PPT3, PiC3, APC2 in phosphorus uptake and absorption transporter explained the metabolic response differences of switchgrass to phosphorus nutrition.

    Conclusions 

    The suitable phosphorus supply level is conducive to good switchgrass root morphology, low anti-stress enzyme activities in leaves, and high acid phosphatase activity in leaves and roots, and high dry matter weight, as a result. The number and levels of differentially expressed genes related to glycolysis, phenylpropanoid biosynthesis and phosphorus absorption and transport in leaves and roots, which are caused by the P supply levels, could partially explain the mechanism.

  • 柳枝稷 (Panicum virgatum L.) 是一种多年生C4植物,原产于北美大草原,具有生长速度快、环境适应性强、生物质产量潜力高和投入需求相对较低的特点,被视为理想的生物能源作物和牧草作物[12]。磷是植物体内蛋白质、磷脂、脂肪酸、核苷酸和ATP等重要有机物的组成元素,对于能量代谢、酶促反应、信号转导等生命活动至关重要[3]。因此,研究柳枝稷在不同供磷水平下的生理和分子调控机制,不仅可以为能源物质生产和农业生产提供重要的资源,还有助于优化磷素的循环和利用。

    在供磷水平不足条件下,植物会表现出一系列适应性变化,这些变化涉及表型、生理生化和分子层面,最终影响植株的生长。磷缺乏会抑制叶片代谢过程,导致活性氧 (ROS) 过度积累,从而损伤DNA、蛋白质和脂质[4]。为了抵抗ROS的破坏,植物会激活抗氧化系统,包括超氧化物歧化酶 (SOD)、过氧化物酶 (POD) 和过氧化氢酶 (CAT) 等酶类[5]。根系作为吸收磷的主要器官,在供磷不足的环境下会增加根毛长度,扩大根系与土壤的接触面积,从而提高磷的吸收和利用效率[67]。除了通过分析叶片和根系的生长发育来研究植物对磷水平变化的适应性外,转录组学也是解析植物磷调控机制的有效方法。研究人员在拟南芥 (Arabidopsis thaliana)[8]、玉米 (Zea mays)[9]、水稻 (Oryza sativa)[10]和小麦 (Triticum aestivum)[11]叶片和根系中发现了多个响应磷水平变化的差异表达基因 (DEGs),这些基因参与调控糖酵解途径、离子转运、活性氧清除、信号转导、苯丙素生物合成等过程。其中,糖酵解是植物细胞能量代谢的核心途径,可确保植物在低磷环境下仍能合成必要的能量物质[12]。苯丙素生物合成途径产生的次生代谢产物,如黄酮类、花青素、单宁和木质素等,不仅提高植物的抗氧化能力,还参与病害防御和胁迫信号传递,有助于植物在不利环境条件下生存和发育[13]。同时,Yan等[14]通过对水稻 (Oryza sativa) 在磷充足和磷缺乏条件下的第一叶片 (上部) 和第四叶片 (下部) 进行转录组分析发现,OsPHO1;3基因在植物正常生长的无机磷酸盐 (Pi) 再动员过程中发挥着重要作用。

    已有大量研究探讨了植物适应磷营养水平的表型变化以及生化和分子反应机制,但这些研究的材料绝大多数为模式作物,研究结果对特定作物的适应性机制的代表性缺少验证。本研究选择高地型柳枝稷作为研究对象,探究不同供磷水平下叶片和根系的表型和生理调控效应;通过转录组测序技术,采用FPKM (Fragments Per Kilobases per Millionreads) 方法和P-adjust≤0.05且|log2FC|≥1标准,鉴定叶片和根系的差异表达基因 (DEGs) ,通过GO (gene ontology) 功能富集分析和KEGG (Kyoto Encyclopedia of Genes and Genomes) 代谢途径分析,对低磷水平下的DEGs进行功能注释,以揭示叶片和根系在分子层面上的适应性差异,为进一步探索柳枝稷耐低磷候选基因提供理论依据。

    试验于2022年4—10月在宁夏银川市宁夏大学试验研究基地大棚温室开展。以Hoagland营养液为基础,参考Ding等[15]设置4个营养液KH2PO4 水平:20 μmol/L (P20)、100 μmol/L (P100)、200 μmol/L (P200) 和500 μmol/L (P500)。供试材料为高地型柳枝稷 (Panicum virgatum L.) Pathfinder品种,由北京市农林科学院草业与环境发展研究中心提供。

    试验采用无土盆栽试验,培养基质为石英砂、蛭石和珍珠岩混合物 (体积比1∶3∶1)。柳枝稷种子经3% H2O2消毒后育苗,三叶期幼苗移栽至准备好的花盆中,每盆4株,每个处理3次重复,共12盆,于光照箱中培养45天,生长条件为光周期12 h光照/12 h黑暗,光照强度600 μmol/(m2·s),昼夜温度(25±2)℃/(20±2)℃,相对湿度60%~70%。每间隔5 天浇1.5 L含不同KH2PO4配比的Hoagland培养液,浇培养液前用2.0 L蒸馏水浸润花盆中培养基质,以保证营养充足以及磷浓度相对稳定。于第46 天上午9:30,各处理挑选3株长势均匀的柳枝稷幼苗测定生理指标,再采集3株幼苗分为叶片和根系样品,用超纯水清洗3次,用滤纸吸干植株表面水分后置于液氮中冷冻,在−80℃下保存,用于后续转录组测序。

    幼苗干物质量采用烘干称重法测定,将幼苗分为地上部和地下部置于烘箱中,70℃烘干至恒重。根系形态采用全自动根系扫描分析仪STD1600 (Epson, Japan) 进行测定,然后使用分析软件Win-RHIZO (Regent instrument,Canada) 对根系形态进行分析。根系活力采用TTC法测定[16]。叶片SOD、POD和CAT活性分别采用NBT法、愈创木酚法和TBA法测定[17]。叶片和根系酸性磷酸酶活性采用试剂盒 (Solarbio,货号BC2130) 测定。叶片和根系全磷含量采用钒钼比色法测定[18]

    委托苏州帕诺米克生物医药科技有限公司进行转录组测序。采用天根多糖多酚试剂盒(QIAGEN,Germany) 从柳枝稷叶片和根系组织中提取RNA,随后对RNA样品进行严格质控,主要利用Agilent 2100 bioanalyzer精确检测RNA完整性。文库构建采用NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina®试剂盒。库检合格后,采用illumina NovaSeq 6000 (illumina, USA) 进行双端测序,获得原始数据 (raw reads),通过质控软件fastp (version 0.19.7) 去除低质量的读数,获得高质量的序列数据 (clean reads)。使用HISAT2软件将clean reads与参考基因组 (Phytozome Panicum virgatum v4.1) 进行精确比对,获取在参考基因组上的位置信息以及测序样本特有的序列特征信息。计算每千个碱基转录每百万映射读取的fragments (FPKM值),分别使用差异基因Padj数值BH法和DESeq2软件计算P-adjust和Fold Change值,以P-adjust≤0.05且|log2FC|≥1为筛选标准,获得不同处理下DEGs。然后对 DEGs进行GO富集和 KEGG富集功能注释。

    从糖酵解和苯丙素生物合成途径挑选6个基因,采用qRT-PCR方法对叶片和根系转录组测序结果进行验证,利用Primer 3 plus设计扩增引物(表1),以柳枝稷Actin为内参基因。每个处理进行3次生物学重复,采用2–ΔΔCt方法计算差异基因相对表达量。

    表  1  qRT-PCR引物信息
    Table  1.  Information of qRT-PCR primers
    基因名称 Gene name 正向引物 Forward primer (5′→3′) 反向引物 Reverse primer (5′→3′)
    CAD GACATCCACCAGGCCAAGAA GAGCTCACTAGGTACGCGTC
    PAL ATGTCCAGAGTGCCGAACAG GAATCGGTAGAGCGGGTACG
    PGK CCCCACCATCAAGTTCCTCC CTTCGCCAGCAGAGACTCAA
    PGM TCTGCTGCTGTTGACTCCAG GCCCTGAAACTCTTACGCCT
    PDC ACTTCAACCTCACGCTGCTT GCTTCACGGCCTTGTTCAAG
    ACS ATGATCACCCCTTTGCCTGG GGCGCCCAATGAATCTTGTC
    Actin CCACGTGCTGTTTTCCCAAG AAAGAGTAGCCCCTCTCCGT
    下载: 导出CSV 
    | 显示表格

    利用Microsoft Excel 2019和SPSS 20.0进行数据处理和分析,采用单因素方差分析(ANOVA) 比较处理间差异,采用相关性分析和随机森林模型分析植株磷变化的主控因子,采用Origin 2024软件以及R 4.3.2的ggplot2、pheatmap、rfPermute、psych、reshape2、patchwork、randomForest、rfUtilities、linkET、cols4all、dplyr包等绘制图表。

    供磷水平显著影响柳枝稷叶片的抗氧化酶 (POD、SOD和CAT) 活性 (图1)。随供磷水平的提高,叶片抗氧化酶活性均呈先减后增趋势,POD、SOD和CAT活性在P200处理达到最低,较P20处理分别显著降低了54.15%、38.60%和80.42%,P100和P500处理的叶片抗氧化酶活性差异不显著。总根长、总根面积和根系活力随磷水平提高均呈先增加后降低的趋势 (图1),P200处理下的总根长、总根面积和根系活力达到峰值,分别较P20显著提高了103.80%、82.55%和160.21%。干物质量、磷含量和酸性磷酸酶活性同样在P200处理达到峰值(图1),其幼苗地上部、地下部干物质量分别较P20处理显著增加77.73%、150.46%,叶片和根系酸性磷酸酶活性分别显著增加42.77%和278.49%,叶片和根系磷含量分别显著增加23.81%和15.56%。

    图  1  不同供磷水平下柳枝稷的生理和表型指标
    注:P20、P100、P200、P500分别代表KH2PO4供应水平为20、100、200和500 μmol/L。柱上或柱下不同小写字母表示处理间差异显著 (P<0.05)。
    Figure  1.  The biological and phynotype indicator values of switchgrass as affected by KH2PO4 supply levels
    Note: P20, P100, P200 and P500 stand for KH2PO4 supply levels of 20, 100, 200 and 500 μmol/L, respectively. Different lowercase letters above or below the bars indicate significant difference among treatments (P<0.05).

    进行柳枝稷磷含量与各指标之间的相关性分析及随机森林模型分析,结果 (图2)显示,叶片磷含量与叶片磷酸酶活性、总根面积极显著正相关 (P<0.01),与叶片POD活性显著负相关 (P<0.05),地下部干物质量、地上部干物质量是显著影响因素;根系磷含量与根系磷酸酶活性极显著正相关 (P<0.01),与叶片POD活性显著负相关 (P<0.05),地下部干物质量、地上部干物质量和根系磷酸酶活性是显著影响因素。综上所述,供磷水平显著影响柳枝稷的表型特征与生理特性,低磷胁迫显著提高了柳枝稷叶片的抗氧化酶活性,且提高供磷水平有利于植株干物质积累,促进磷的吸收。

    图  2  柳枝稷叶片和根系磷含量与生化和根系表型指标的相关性及主控因子分析
    Figure  2.  Correlation of switchgrass leaf and root phosphorus content with the biological and phynotype indicators and the random forest analysis for the main driving factors
    Note: *—P<0.05, **—P<0.01.

    和P20处理比,随磷水平升高叶片的差异表达基因 (上调、下调) 总数逐渐降低,而根系呈先增加后减少趋势 (图3)。叶片中,和P20处理比,P100中上调基因3979个,下调基因4334个;P200中上调基因2651个,下调基因3393个;P500中上调基因4075个,下调基因1669个,检测到各磷处理共同表达的差异表达基因有1091个。根系中,和P20处理比,P100中上调基因1918个,下调基因2157个;P200中上调基因3418个,下调基因4022个;P500中上调基因3484个,下调基因3292个,检测到各磷处理共同表达的差异表达基因有1762个。

    图  3  叶片和根系不同磷水平相比于P20处理的差异表达基因数
    注:P100、P200、P500分别代表KH2PO4供应水平为100、200和500 μmol/L。
    Figure  3.  Number of DEGs in leaves and roots under different phosphate levels compared to P20
    Note: P100, P200 and P500 stand for KH2PO4 supply levels of 100, 200 and 500 μmol/L, respectively.

    从生物合成过程看,和P20处理比,P100处理的叶片差异表达基因(DEGs)显著富集于阳离子运输、有机磷代谢、碳水化合物衍生物代谢、小分子生物合成、核苷酸、有机酸生物合成、氧化应激、细胞脂质代谢和有机磷生物合成等过程 (图4),根系DEGs显著富集于小分子生物合成、氧化应激、有机酸生物合成、细胞脂质代谢、脂质生物合成、单羧酸代谢、单羧酸生物合成、脂肪酸代谢和脂肪酸生物合成等过程。从功能看,叶片DEGs显著富集于特异DNA序列结合、离子跨膜转运蛋白活性、无机分子跨膜转运蛋白活性、抗氧化活性、裂合酶活性、氧化还原酶活性、过氧化物酶、主动跨膜转运蛋白活性和阳离子跨膜转运蛋白活性等条目,根系DEGs显著富集于酰基转移酶活性、非氨基酰基转移酶活性、抗氧化活性、过氧化物酶活性、主动跨膜转运蛋白活性和三磷酸腺苷酶活性等条目。

    图  4  与P20处理相比P100处理下叶片和根系差异表达基因在生物代谢(BP)和分子功能(MF)调节过程的GO富集分析
    注:P20和P100分别代表KH2PO4供应水平为20和100 μmol/L。
    Figure  4.  GO analysis on the enrichment of differentially expressed genes (DEGs) in the biosynthetic process (BP) and molecular function (MF) regulation of leaf and root in P100 compared to P20
    Note: P20 and P100 stand for KH2PO4 supply levels of 20 μmol/L and 100 μmol/L, respectively.

    和P20比,P200处理的生物学过程中,叶片DEGs显著富集于氧化应激和碳水化合物代谢等过程,根系DEGs显著富集于小分子生物合成、有机酸生物合成、单羧酸代谢和氧化应激等过程 (图5)。分子功能中,叶片DEGs显著富集于调节过氧化物酶活性、氧化还原酶活性、抗氧化活性、序列特异性DNA结合、酰基转移酶活性、非氨基酰基转移酶活性、丝氨酸水解酶活性和裂合酶活性等条目,根系DEGs显著富集于调节酰基转移酶活性、非氨基酰基转移酶活性、裂合酶活性、过氧化物酶活性、氧化还原酶活性、抗氧化活性和主动跨膜转运蛋白活性等条目。

    图  5  与P20处理相比P200处理下叶片和根系差异表达基因在生物代谢(BP)和分子功能(MF)调节过程的GO富集分析
    注:P20和P200分别代表KH2PO4供应水平为20与200 μmol/L。
    Figure  5.  GO analysis on the enrichement of differentially expressed genes (DEGs) involved in the biosynthetic process (BP) and molecular function(MF) regulation under P200, compared to P20
    Note: P20 and P200 stand for KH2PO4 supply levels of 20 μmol/L and 200 μmol/L, respectively.

    和P20处理比, P500处理的生物学过程中,叶片DEGs显著富集于氧化应激等过程,根系DEGs显著富集于小分子生物合成、碳水化合物衍生物代谢、有机酸合成、有机磷合成、单羧酸代谢、辅因子代谢、细胞脂质代谢和核苷酸代谢等过程 (图6)。分子功能中,叶片DEGs显著富集于特异DNA序列结合、过氧化物酶活性、氧化还原酶活性、抗氧化活性、非氨基酰基转移酶活性和主动跨膜转运蛋白活性等条目,根系DEGs主要富集于酰基转移酶活性、非氨基酰基转移酶活性、裂合酶活性、电子传递活性、三磷酸腺苷酶活性、抗氧化活性和主动跨膜转运蛋白活性等条目。

    图  6  与P20处理相比P500处理下叶片和根系差异表达基因在生物代谢(BP)和分子功能(MF)调节过程的GO富集分析
    注:P20和P500分别代表KH2PO4供应水平为20与500 μmol/L。
    Figure  6.  GO analysis on the enrichment of differentially expressed genes (DEGs) in the biosynthetic process (BP) and molecular function (MF) regulation under P500, compared to P20
    Note: P20 and P500 stand for KH2PO4 supply levels of 20 μmol/L and 500 μmol/L, respectively.

    根据KEGG数据库进行通路富集分析,解析差异表达基因对代谢通路的影响,揭示柳枝稷缓解低磷胁迫的分子机制 (图7)。环境信息处理相关通路中,叶片和根系共有通路为植物激素信号转导和植物−病原互作通路,其中植物激素信号转导通路在叶片中显著表达。遗传信息处理相关通路中,叶片和根系中共有通路为内质网蛋白质加工通路,叶片和根系中均未达到显著水平。代谢相关通路中,叶片和根系富集到的共同通路有氨基糖和核苷糖代谢、半胱氨酸和蛋氨酸代谢、脂肪酸代谢、谷胱甘肽代谢、糖酵解/糖异生、苯丙素生物合成以及蔗糖和淀粉代谢通路,其中氨基糖和核苷糖代谢、糖酵解/糖异生、苯丙素生物合成、蔗糖和淀粉代谢通路在叶片和根系中显著表达,而脂肪酸代谢和谷胱甘肽代谢通路主要在根系中显著表达。

    图  7  叶片及根系差异表达基因KEGG富集分析
    注:P20、P100、P200、P500分别代表KH2PO4供应水平为20、100、200和500 μmol/L。EIP、GIP和MET是KEGG富集分析的生物学分类,分别代表环境信息处理、遗传信息处理和代谢。
    Figure  7.  KEGG enrichment analysis of differentially expressed genes (DEGs) in leaves and roots
    Note: P20, P100, P200 and P500 stand for KH2PO4 supply levels of 20, 100, 200 and 500 μmol/L, respectively. EIP, GIP and MET are the biological classifications analyzed for KEGG enrichment, representing environmental information processing, genetic information processing and metabolism, respectively.

    糖酵解是代谢的重要途径之一,在能量代谢和生物合成中起着举足轻重的作用。不同供磷水平下糖酵解途径的相关基因差异表达,本试验中共筛选出16个差异表达基因 (图8a)。由β-葡萄糖切入生成α-葡萄糖进入糖酵解途径的变旋酶 (AE) 基因在叶片中的表达量高于根系;由1-磷酸葡萄糖切入糖酵解途径的磷酸葡萄糖变位酶 (PGM) 基因主要在根系中表达。己糖激酶 (HK) 基因和6-磷酸果糖激酶 (PFK) 基因在叶片中的表达量高于根系,三磷酸异构酶 (TPI) 基因在叶片中表达量较高,3-磷酸甘油醛脱氢酶 (GAPD) 基因在根系中表达量高于叶片,磷酸甘油醛激酶 (PGK) 基因LOC120664222LOC120698254分别在叶片的P20处理以及根系的P500处理中表达量较高。最后,糖酵解产物丙酮酸通过丙酮酸脱羧酶 (PDC) 和乙酰辅酶A合成酶 (ACS) 进入三羧酸循环。

    图  8  叶片和根系糖酵解途径及苯丙素生物合成途径不同磷水平下的差异表达基因表达水平
    注:虚线代表多步反应,实线代表一步反应。热图是不同处理间差异表达基因的 FPKM 值,颜色越红,数值越高。FPKM—每千个碱基的转录每百万映射读取的fragments。从左往右处理依次为叶Leaf_P20、叶Leaf_P100、叶Leaf_P200、叶Leaf_P500、根Root_P20、根Root_P100、根Root_P200、根Root_P500。P20、P100、P200、P500分别代表KH2PO4供应水平为20、100、200和500 μmol/L。
    Figure  8.  Expression levels of differentially expressed genes (DEGs) in glycolysis and phenylpropyl biosynthesis pathway of leaves and roots as affected by P treatments
    Note: The dashed line represents a multi-step reaction, and the solid line represents a one-step reaction. The heatmap shows the FPKM values of the differential genes between treatments, the redder the colour, the higher the value. FPKM is fragments per kilobase of exon model per million mapped fragments. From left to right treatments are Leaf_P20、Leaf_P100, Leaf_P200, Leaf_P500, Root_P20, Root_P100, Root_P200, Root_P500. P20, P100, P200 and P500 stand for KH2PO4 supply levels of 20, 100, 200 and 500 μmol/L, respectively.

    叶片及根系的苯丙素生物合成途径共涉及到16个差异表达基因 (图8b)。催化苯丙氨酸生成对香豆酸的苯丙氨酸裂解酶 (PAL) 基因LOC120642393主要在柳枝稷叶片中表达,并在P200和P500处理中表达量较高;LOC120709328LOC120683054基因主要在根系中表达,同样在P200和P500处理中表达量较高。反式肉桂酸-4-羧化酶 (C4H) 基因和香豆酸辅酶A连接酶 (4CL) 基因在叶片和根系中表达量无明显差异。参与对香豆酸合成木质素过程的肉桂酰辅酶A还原酶 (CCR) 基因、肉桂醇脱氢酶 (CAD) 基因、Class Ⅲ型植物过氧化物酶 (CIII Prxs) 基因主要在柳枝稷叶片中表达,并主要以P20处理下的基因表达量较高。

    共有20个磷吸收、转运及调控基因在叶片和根系中差异表达 (图9)。其中,编码G6P/Pi转运蛋白的GPT2基因,编码无机Pi转运蛋白的PHT2;1基因,编码磷酸三糖/Pi转运蛋白的TPT基因,编码磷酸烯醇式丙酮酸(PEP)/Pi转运蛋白的PPT1PPT2以及PPT3基因在叶片中的表达量高于根系,而编码线粒体Pi载体蛋白的PiC3基因和编码钙依赖性线粒体ATP-镁/Pi载体蛋白的APC2基因在根系中的表达量高于叶片。

    图  9  叶片及根系磷吸收、转运及调控差异表达基因
    注:P20、P100、P200、P500分别代表KH2PO4供应水平为20、100、200和500 μmol/L。热图是不同处理间差异表达基因的 FPKM 值,颜色越红,数值越高。FPKM—每千个碱基的转录每百万映射读取的fragments。
    Figure  9.  Leaf and root phosphorus uptake, transport and regulation of DEGs
    Note: P20, P100, P200 and P500 stand for KH2PO4 supply levels of 20, 100, 200 and 500 μmol/L, respectively. The heatmap shows the FPKM values of the differentially expressed genes (DEGs) between treatments, the redder the colour, the higher the value. FPKM—Fragments per kilobase of exon model per million mapped fragments.

    为验证柳枝稷幼苗叶片及根系转录组测序结果中基因表达量的准确性,从糖酵解和苯丙素生物合成途径挑选了6个基因进行不同供磷水平处理下的qRT-PCR验证 (图10)。将qRT-PCR结果与转录组测序数据比对发现,6个差异表达基因变化趋势表现与转录组数据基本一致。

    图  10  糖酵解和苯丙素生物合成途径6个差异表达基因的qRT-PCR验证
    注:P20、P100、P200、P500分别代表KH2PO4供应水平为20、100、200和500 μmol/L。FPKM—每千个碱基的转录每百万映射读取的fragments。
    Figure  10.  qRT-PCR verification of 6 differentially expressed genes involved in glycolysis and phenylpropanoid biosynthesis pathways
    Note: P20, P100, P200 and P500 stand for KH2PO4 supply levels of 20, 100, 200 and 500 μmol/L, respectively. FPKM—Fragments per kilobase of exon model per million mapped fragments.

    供磷水平对植物的生长和生理过程有着显著影响。在供磷不足条件下,植物可以通过激活SOD、POD和CAT 等抗氧化酶,增强植物的抗氧化能力,从而减轻活性氧 (ROS)的损害[1920]。本试验发现,柳枝稷的SOD、POD和CAT活性显著受到供磷水平的影响,与Su等[21]研究结果相似。随供磷水平的提高,柳枝稷叶片抗氧化酶活性呈现先降低后增加的趋势,P200处理下的POD、SOD和CAT的活性达到最低,较P20处理分别显著降低了54.15%、38.60%和80.42%。值得注意的是,P500处理提高了抗氧化酶活性。根据前人研究推测,过高的磷水平增加了叶片中的丙二醛 (MDA) 和过氧化氢 (H2O2) 含量,刺激SOD、POD和CAT活性提高,加快自由基清除进度,进而减少胁迫对植株的损害[22]

    调整根系形态是植物适应磷水平变化的关键策略之一,根系总长度和根系表面积反映了植物根系的吸收能力[23]。Wang等[24]发现,施磷显著降低了低密度种植下的柳枝稷根平均直径,而对比根长无显著影响,导致根系较细,根系组织密度较高。在本试验中,同P20处理相比,P200处理下的总根长、总根面积和根系活力分别显著提高了103.80%、82.55%和160.21%。随着磷水平提高,柳枝稷叶片和根系的酸性磷酸酶活性、磷含量以及干物质量呈现先上升后下降的趋势,这与根系总长度、根系总面积和根系活力的变化规律相吻合。随机森林模型分析发现,植株干物质量和磷酸酶活性是影响叶片和根系磷含量的重要因素。同时发现,根系中的酸性磷酸酶活性高于叶片,而磷含量则表现出相反的趋势。这反映了柳枝稷对磷的重新分配,提高磷水平促使根系中的酸性磷酸酶分解磷并将其输送到柳枝稷地上部分,参与如光合作用、碳分配、能量代谢、信号转导和酶调节等关键代谢途径,从而影响了叶片酸性磷酸酶的活性和磷含量。然而,过高的供磷水平并没有进一步提高柳枝稷对磷素的吸收和利用。在实际生产中,柳枝稷通常比其他植物更能适应低磷土壤环境[25]。根据形态和生境偏好,柳枝稷分为低地生态型和高地生态型,高地生态型在较干燥和寒冷的地区生长良好,并且我国北方地区土壤中有效磷含量通常较低[26]。因此,探究高地生态型柳枝稷品种的表型、生理生化对供磷水平的响应,对北方地区发展可再生饲料资源和生物质能源资源具有重要意义。

    在柳枝稷适应不同磷供应水平的过程中,差异表达基因发挥了关键作用。与P20相比,P100及P200处理中,叶片和根系的差异表达基因主要呈现下调趋势,与Hou等[27]和Shao等[28]研究结果相似。而在P500处理中,叶片和根系的差异表达基因则以上调为主,这表明过量的磷素可能会导致细胞内磷酸盐水平失衡,影响细胞的能量代谢和信号传导,柳枝稷通过上调一系列防御基因来减轻磷毒性的影响。通过GO富集注释分析得知,差异表达基因主要在分子功能上显著富集。在叶片中,主要受影响的是离子跨膜转运蛋白活性、无机分子跨膜转运蛋白活性、酰基转移酶活性、抗氧化活性、氧化还原酶活性、过氧化物酶活性等,这与叶片中SOD、POD和CAT活性的变化相符合,表明柳枝稷可能通过调控抗氧化酶相关基因的表达来响应磷水平变化,与朱晨璐等[29]研究结果相似。在根系中,主要受影响的是酰基转移酶活性、非氨基酰基转移酶活性、抗氧化活性、特异DNA序列结合、主动跨膜转运蛋白活性、裂合酶活性、无机分子跨膜转运蛋白活性等。KEGG富集分析进一步揭示了柳枝稷叶片和根系中差异表达基因调控的生物代谢途径,包括植物激素信号转导、植物−病原互作、苯丙素生物合成、氨基糖和核苷糖代谢、半胱氨酸和蛋氨酸代谢、脂肪酸代谢、谷胱甘肽代谢、糖酵解和糖异生、蔗糖和淀粉代谢通路等。通过这些调控机制,柳枝稷能够在各种磷供应条件下维持正常的生长和发育。

    糖酵解代谢途径是生物体碳代谢的中心途径,连通了核酸代谢、蛋白质代谢、脂类代谢及次生代谢等多个代谢途径[3031],磷水平变化直接影响糖酵解途径,进而调控对磷的吸收和运输[3234]。本研究发现,有16个基因在柳枝稷叶片和根系中表现出差异化表达。AE基因在叶片中的表达量高于根系,而PGM基因在根系中表达量更高。这一表达模式表明,在叶片中主要通过AE催化β-葡萄糖生成α-葡萄糖;而在根系中主要通过PGM催化1-磷酸葡萄糖生成6-磷酸葡萄糖,从而参与糖酵解途径,揭示了柳枝稷不同器官磷代谢反应和调控机制的差异。己糖激酶(HK)和磷酸果糖激酶(PFK)是糖酵解中的关键酶和限制酶,磷胁迫将抑制两者基因的表达[35]。但本试验研究发现,叶片内的HK基因和PFK基因的表达水平在不同磷水平下差异不明显,后续糖酵解酶基因表达量总体高于根系。根据前人研究推测,供磷不足将降低HK和PFK代谢底物 (ATP) 的含量,而焦磷酸二酯 (PPi) 可以作为一种ATP的替代能量供体,参与UDP-葡萄糖合成6-磷酸果糖的过程[36]。因此,为了适应磷水平变化,叶片提高了UDP-葡萄糖焦磷酸化酶基因 (UGP)和磷酸葡萄糖异构酶基因 (GPI) 的表达。在PPi作用下,UGP和GPI将UDP-葡萄糖转化为6-磷酸葡萄糖,从而维持柳枝稷叶片中糖酵解代谢途径的进行。

    在植物体内,苯丙氨酸 (或酪氨酸) 经过脱氨基、羟基化、甲基化、氧化还原反应和聚合生成H木质素、G木质素和S木质素等木质素单体[37],苯丙氨酸解氨酶(PAL)和4-香豆酸辅酶A连接酶(4CL)是整个代谢途径的关键酶和限速酶,PAL在苯丙素生物合成途径的前端催化苯丙氨酸转化为肉桂酸,4CL控制苯丙氨酸走向不同的代谢途径[38]。另外,C4HCCRCAD等基因在木质素的合成过程中也起着极为重要的调控作用,有的还参与缓解相关生物或非生物胁迫[3940]。本试验发现16个关键酶基因在柳枝稷叶片和根系的苯丙素生物合成途径中呈现差异化表达。其中,PAL注释到3个基因,LOC120642393基因主要在柳枝稷叶片中表达,而LOC120709328LOC120683054基因主要在根系中表达。CCRCAD基因在叶片P20处理中表达量较高,这与低磷胁迫下叶片抗氧化酶活性显著提高相符合。C4H基因和4CL基因在叶片和根系中的表达量没有明显差异,表明其在柳枝稷整个植物体中都是苯丙素生物合成的重要调控点。烟草 (Tobacco)[41]、百日草 (Zinnia)[42]等植物中证实了Class Ⅲ型植物过氧化物酶 (CIII Prxs) 参与木质素合成。在CIII Prxs催化下,各种木质素单体被氧化形成木质素聚合物而使细胞壁硬化,或者通过其生长素氧化酶活性控制细胞伸长[4344]。Vance等[45]认为,磷胁迫将促进防御代谢物的生物合成,如细胞壁木质化的增加。本研究中,CIII Prxs基因在叶片中的表达量高于根系,同时发现供磷水平会降低CIII Prxs的表达量,这表明磷的供应水平可能通过调节关键酶基因的表达来影响柳枝稷的木质素合成和细胞壁结构。

    在高等植物中,磷以无机磷酸盐 (Pi,即PO43−、HPO42−和H2PO4) 的形式从土壤中通过一个依赖于H+的共转运过程获得[46]。相关研究表明,PHT1PHT4基因编码磷酸盐转运体,使磷酸盐可以在植物组织和细胞器内进行转运[47]TPT基因编码磷酸三糖/Pi转运蛋白,催化TP、3-PGA和Pi在叶绿体包膜上进行严格的1∶1交换,在光合作用中发挥着至关重要的作用[4849]PPT基因编码PEP/Pi转运蛋白,可以将PEP输入质体[50]GPT2基因编码质体G6P/Pi转运体,可以通过叶绿体和细胞质之间的G6P分配信号以及光合作用相关蛋白质组的重置等机制,使光合作用适应辐照度增加[5152]。据前人研究结果,PHT2;1活性影响植物体内的Pi分配,并调节Pi饥饿反应,包括Pi饥饿反应基因的表达和Pi在叶片内的转运[53]。提高磷酸盐或外源糖供应会上调GPT2表达水平并促进淀粉积累[5455]。本研究中,P20处理降低了GPT2PHT2;1TPTPPT1PPT2PPT3基因在叶片的中表达,在P100~P500处理下基因表达量远高于根系。PiC作为一种膜嵌入蛋白,主要负责将Pi从细胞质转运到线粒体基质中,以支持ATP合成过程中的Pi需求[56]。在本试验中,PiC3APC2基因在根系中的表达量则高于叶片。以上表明,叶片和根系在磷的吸收和转运机制上存在明显的组织特异性,通过调控不同基因在叶片和根系中的差异表达,进而调节Pi 转运系统,以应对磷胁迫,保障柳枝稷幼苗在低磷环境下的磷吸收和转运。

    磷水平对柳枝稷根系表型和根系活力具有显著影响。供磷不足条件下,柳枝稷可通过激活叶片SOD、POD和CAT活性,增强其抗氧化能力。适宜磷水平可提高柳枝稷叶片和根系的酸性磷酸酶活性、磷含量。供磷水平变化会引起糖酵解途径、苯丙素生物合成途径和磷吸收转运相关基因(PGMHKPFKPAL4CLTPTPPT基因家族等)在叶片和根系中的差异表达。

  • 图  1   不同供磷水平下柳枝稷的生理和表型指标

    注:P20、P100、P200、P500分别代表KH2PO4供应水平为20、100、200和500 μmol/L。柱上或柱下不同小写字母表示处理间差异显著 (P<0.05)。

    Figure  1.   The biological and phynotype indicator values of switchgrass as affected by KH2PO4 supply levels

    Note: P20, P100, P200 and P500 stand for KH2PO4 supply levels of 20, 100, 200 and 500 μmol/L, respectively. Different lowercase letters above or below the bars indicate significant difference among treatments (P<0.05).

    图  2   柳枝稷叶片和根系磷含量与生化和根系表型指标的相关性及主控因子分析

    Figure  2.   Correlation of switchgrass leaf and root phosphorus content with the biological and phynotype indicators and the random forest analysis for the main driving factors

    Note: *—P<0.05, **—P<0.01.

    图  3   叶片和根系不同磷水平相比于P20处理的差异表达基因数

    注:P100、P200、P500分别代表KH2PO4供应水平为100、200和500 μmol/L。

    Figure  3.   Number of DEGs in leaves and roots under different phosphate levels compared to P20

    Note: P100, P200 and P500 stand for KH2PO4 supply levels of 100, 200 and 500 μmol/L, respectively.

    图  4   与P20处理相比P100处理下叶片和根系差异表达基因在生物代谢(BP)和分子功能(MF)调节过程的GO富集分析

    注:P20和P100分别代表KH2PO4供应水平为20和100 μmol/L。

    Figure  4.   GO analysis on the enrichment of differentially expressed genes (DEGs) in the biosynthetic process (BP) and molecular function (MF) regulation of leaf and root in P100 compared to P20

    Note: P20 and P100 stand for KH2PO4 supply levels of 20 μmol/L and 100 μmol/L, respectively.

    图  5   与P20处理相比P200处理下叶片和根系差异表达基因在生物代谢(BP)和分子功能(MF)调节过程的GO富集分析

    注:P20和P200分别代表KH2PO4供应水平为20与200 μmol/L。

    Figure  5.   GO analysis on the enrichement of differentially expressed genes (DEGs) involved in the biosynthetic process (BP) and molecular function(MF) regulation under P200, compared to P20

    Note: P20 and P200 stand for KH2PO4 supply levels of 20 μmol/L and 200 μmol/L, respectively.

    图  6   与P20处理相比P500处理下叶片和根系差异表达基因在生物代谢(BP)和分子功能(MF)调节过程的GO富集分析

    注:P20和P500分别代表KH2PO4供应水平为20与500 μmol/L。

    Figure  6.   GO analysis on the enrichment of differentially expressed genes (DEGs) in the biosynthetic process (BP) and molecular function (MF) regulation under P500, compared to P20

    Note: P20 and P500 stand for KH2PO4 supply levels of 20 μmol/L and 500 μmol/L, respectively.

    图  7   叶片及根系差异表达基因KEGG富集分析

    注:P20、P100、P200、P500分别代表KH2PO4供应水平为20、100、200和500 μmol/L。EIP、GIP和MET是KEGG富集分析的生物学分类,分别代表环境信息处理、遗传信息处理和代谢。

    Figure  7.   KEGG enrichment analysis of differentially expressed genes (DEGs) in leaves and roots

    Note: P20, P100, P200 and P500 stand for KH2PO4 supply levels of 20, 100, 200 and 500 μmol/L, respectively. EIP, GIP and MET are the biological classifications analyzed for KEGG enrichment, representing environmental information processing, genetic information processing and metabolism, respectively.

    图  8   叶片和根系糖酵解途径及苯丙素生物合成途径不同磷水平下的差异表达基因表达水平

    注:虚线代表多步反应,实线代表一步反应。热图是不同处理间差异表达基因的 FPKM 值,颜色越红,数值越高。FPKM—每千个碱基的转录每百万映射读取的fragments。从左往右处理依次为叶Leaf_P20、叶Leaf_P100、叶Leaf_P200、叶Leaf_P500、根Root_P20、根Root_P100、根Root_P200、根Root_P500。P20、P100、P200、P500分别代表KH2PO4供应水平为20、100、200和500 μmol/L。

    Figure  8.   Expression levels of differentially expressed genes (DEGs) in glycolysis and phenylpropyl biosynthesis pathway of leaves and roots as affected by P treatments

    Note: The dashed line represents a multi-step reaction, and the solid line represents a one-step reaction. The heatmap shows the FPKM values of the differential genes between treatments, the redder the colour, the higher the value. FPKM is fragments per kilobase of exon model per million mapped fragments. From left to right treatments are Leaf_P20、Leaf_P100, Leaf_P200, Leaf_P500, Root_P20, Root_P100, Root_P200, Root_P500. P20, P100, P200 and P500 stand for KH2PO4 supply levels of 20, 100, 200 and 500 μmol/L, respectively.

    图  9   叶片及根系磷吸收、转运及调控差异表达基因

    注:P20、P100、P200、P500分别代表KH2PO4供应水平为20、100、200和500 μmol/L。热图是不同处理间差异表达基因的 FPKM 值,颜色越红,数值越高。FPKM—每千个碱基的转录每百万映射读取的fragments。

    Figure  9.   Leaf and root phosphorus uptake, transport and regulation of DEGs

    Note: P20, P100, P200 and P500 stand for KH2PO4 supply levels of 20, 100, 200 and 500 μmol/L, respectively. The heatmap shows the FPKM values of the differentially expressed genes (DEGs) between treatments, the redder the colour, the higher the value. FPKM—Fragments per kilobase of exon model per million mapped fragments.

    图  10   糖酵解和苯丙素生物合成途径6个差异表达基因的qRT-PCR验证

    注:P20、P100、P200、P500分别代表KH2PO4供应水平为20、100、200和500 μmol/L。FPKM—每千个碱基的转录每百万映射读取的fragments。

    Figure  10.   qRT-PCR verification of 6 differentially expressed genes involved in glycolysis and phenylpropanoid biosynthesis pathways

    Note: P20, P100, P200 and P500 stand for KH2PO4 supply levels of 20, 100, 200 and 500 μmol/L, respectively. FPKM—Fragments per kilobase of exon model per million mapped fragments.

    表  1   qRT-PCR引物信息

    Table  1   Information of qRT-PCR primers

    基因名称 Gene name 正向引物 Forward primer (5′→3′) 反向引物 Reverse primer (5′→3′)
    CAD GACATCCACCAGGCCAAGAA GAGCTCACTAGGTACGCGTC
    PAL ATGTCCAGAGTGCCGAACAG GAATCGGTAGAGCGGGTACG
    PGK CCCCACCATCAAGTTCCTCC CTTCGCCAGCAGAGACTCAA
    PGM TCTGCTGCTGTTGACTCCAG GCCCTGAAACTCTTACGCCT
    PDC ACTTCAACCTCACGCTGCTT GCTTCACGGCCTTGTTCAAG
    ACS ATGATCACCCCTTTGCCTGG GGCGCCCAATGAATCTTGTC
    Actin CCACGTGCTGTTTTCCCAAG AAAGAGTAGCCCCTCTCCGT
    下载: 导出CSV
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  • 收稿日期:  2024-04-14
  • 录用日期:  2024-07-25
  • 网络出版日期:  2024-11-10
  • 刊出日期:  2024-11-24

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