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作者简介:

林立(1985—),博士,高级实验师,研究方向为观赏植物开发与利用,(E-mail)linli851111@163.com。

中图分类号:Q943

文献标识码:A

文章编号:1000-3142(2024)07-1319-18

DOI:10.11931/guihaia.gxzw202312021

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目录contents

    摘要

    为揭示赤皮青冈叶色黄化变异机制,该研究以赤皮青冈叶色变异植株和正常植株的叶片为试验材料,采用超高效液相色谱串联质谱法和高通量RNA测序技术分别进行代谢组和转录组分析。结果表明:(1)代谢组在正离子(POS)、负离子(NEG)模式下分别检测出正常植株和突变体之间存在257个和357个显著差异代谢物(SCMs),其中槲皮素、白矢车菊素、杨梅素等多种黄酮类化合物及其糖苷衍生物(吡喃酮啡肽A、异鼠李素3-葡糖苷酸等)在突变体中显著上调,而叶绿素a、叶绿素b、类胡萝卜素等色素含量则显著下降。(2)转录组测序检测出4146个差异表达基因(DEGs),其中1711个基因上调表达,2435个基因下调表达。(3)KEGG富集分析表明,SCMs和DEGs显著富集到光合作用、卟啉与叶绿素代谢、类黄酮生物合成等途径。综上表明,突变体叶色黄化可能是受到叶绿素合成受阻、叶绿体发育异常及黄酮物质合成增加等因素的综合影响。此外,MYBbHLH家族基因在突变体中显著上调,证实该两类转录因子参与调控类黄酮生物合成。该研究结果为植物黄化突变的分子机制研究提供了新的见解,也为叶色功能基因挖掘与园林植物育种工作提供了参考。

    Abstract

    In order to reveal the etiolation mechanism of gold-coloured mutant leaves of Quercus gilva, a naturally-occurring leaf-color mutant was used as experimental materials, and the metabolome and transcriptome of mutant leaves and normal green leaves were analyzed by ultra-high performance liquid chromatography-Q (UHPLC-Q) Exactive HF-X and high-throughput RNA sequencing, respectively. The results were as follows: (1) A total of 257 and 357 significantly changed metabolites (SCMs) were respectively identified under the positive ion (POS) mode and the negative ion (NEG) mode. Compared with green leaves, the content of some flavonoids such as quercetin, leucocyanidin, myricetin and their glucoside derivatives (pyranodelphinin A, isorhamnetin 3-glucuronide, etc. ) increased significantly in mutant leaves, but the content of chlorophyll a, chlorophyll b, and carotenoids decreased significantly. ( 2 ) A total of 4146 differentially expressed genes (DEGs) were detected, of which 1711 were up-regulated and 2435 were down-regulated. ( 3 ) KEGG enrichment analysis showed that SCMs and DEGs were mainly enriched in photosynthesis, porphyrin and chlorophyll metabolism, and flavonoid biosynthesis. In conclusion, the inhibition of chlorophyll synthesis, chloroplast developmental abnormalities and promotion of flavonoid synthesis were the main factors driving the leaf etiolation in the mutant Q. gilva. In addition, the genes of the MYB and bHLH families were significantly up-regulated in mutant leaves, confirming these two types of transcription factors were involved in regulating flavonoid biosynthesis. This study provides new molecular insights for the phenomenon of leaf etiolation, and also provides the reference for exploring leaf color-related functional genes and breeding of landscape plant.

  • 叶色突变是指植物在生长发育过程中发生的叶色变化现象(刘新亮等,2017)。叶色突变易于鉴别,Gustafsson最早将其分为条纹、斑点、浅绿、黄化和白化5类(Gustafsson,1942),此后Manjaya 和 Nandanwar(2007)又进一步细分出黄绿、紫叶、类病斑等类型。黄化是其中一种重要的突变类型,黄化突变体常被用于植物光合生理(张晨禹等,2019)、叶绿体超微结构(李淑培等,2023)、叶绿素生物合成(Zhu et al.,2014)等研究。此外,黄化突变在育种领域也有重要应用,许多“金叶”类园林植物品种都来自黄化突变,如‘金叶国槐’(Sophora japonica ‘Aurea’),‘中华金叶榆’(Ulmus pumila ‘Jinye’),‘金叶鸡爪槭’(Acer palmatum ‘Aurea’)等。目前,有关植物叶色黄化的研究集中于少数模式植物和一些重要的农作物,比如拟南芥(Arabidopsis thaliana),水稻(Oryza sativa),黄瓜(Cucumis sativus)等(Li et al.,2012;Maekawa et al.,2015;熊兴伟等,2023)。在园林植物中,仅在黄山栾树(Koelreuteria bipinnata var. integrifoliola)(Lyu et al.,2017),银杏(Ginkgo biloba)(Li et al.,2019),杂交构树(Broussonetia kazinoki × B. papyrifera)(Wang et al.,2022)等少数植物中有过报道。

  • 近年来,高通量测序技术被广泛用于生物组学研究,能够从分子层面揭示生物现象和生物过程的发生机制(熊兴伟等,2023)。并且,通过多组学的联合应用,可以更加直观地反映机体的变化水平,为生物体静态和动态的变化提供更为深入和广阔的研究视野(陆小雨等,2020)。Lyu等(2017)通过高通量测序技术,从黄山栾树品种‘金焰彩栾’(Koelreuteria bipinnata var. integrifoliola ‘Jinyan’)中检测出9个叶绿素代谢和14个类胡萝卜素生物合成的关键基因。Wang等(2022)采用多组学方法对杂交构树的黄叶突变体进行研究,揭示了金黄叶色表型的形成与光合色素的含量与比例变化以及叶绿体结构和功能缺陷有关。Yamashita等(2021)对茶树黄化品种(Camellia sinensis ‘Koganemidori’)的叶绿素代谢和氨基酸积累机制进行了探究,表明供试品种的黄化表型是由于叶绿体发育和叶绿素合成相关基因缺乏所致,高氨基酸则源于代谢相关基因的上调表达所引起的广泛蛋白质降解。Luo等(2022)采用转录组和代谢组联用技术,揭示了甘蔗叶色黄化现象与叶绿素合成途径减少、光合基因表达下降、金属离子调控功能障碍以及次生代谢物质改变等因素有关。总而言之,植物叶色黄化与叶绿素代谢相关基因的突变或表达受阻有重要关系。此外,叶色黄化也会受到花青素、类胡萝卜素等其他色素代谢变化的综合作用(林馨颖等,2022)。

  • 赤皮青冈(Quercus gilva)为壳斗科(Fagaceae)栎属(Quercus)常绿乔木,主要分布于我国南方海拔300~1 500 m的山林地带(中国科学院中国植物志编辑委员会,1998)。其木材质地坚硬,纹理细腻,是优良的硬木用材树种,也可用于低山丘陵混交造林、林下补植和园林绿化(欧阳泽怡等,2021;秦之旷等,2023)。本研究团队于2016年在宁波海曙溪下育苗基地发现一株赤皮青冈播种变异株,其成熟叶片为黄色,新叶金黄色,枝干明黄色,并于2018年底通过嫁接繁殖多株材料,其特异性状已连续4年表现稳定。本研究以赤皮青冈黄化突变植株和正常植株的叶片为研究材料,采用代谢组和转录学联用技术同时结合生理生化测定,拟探讨:(1)赤皮青冈变异植株金黄叶色表型形成的生理机制;(2)变异植株叶色黄化的转录调控机制。本研究可为金叶系园林植物的选育和遗传改良提供参考依据。

  • 1 材料与方法

  • 1.1 试验材料

  • 以赤皮青冈黄化(突变体)植株(yellow leaf,YL)和正常植株(no yellow leaf,NYL)为研究材料,两种材料均种植于宁波市农业技术推广总站林特基地(121°42′24″ E、29°48′46″ N)。2021年8月采集两种植株叶片样品(图1),置于液氮速冻后置于-80℃冰箱中保存备用。

  • 1.2 代谢组分析

  • 1.2.1 代谢物提取

  • 称取50 mg样品至离心管,加入400 μL的甲醇∶乙腈(V/V=1∶1,含内标)提取液。用冷冻组织研磨仪(Wonbio-96c,上海万柏生物科技有限公司)进行研磨,提取液静置后在4℃条件下13 000 g离心15 min,之后取上清液进行上机分析。YL组和NYL组各设置6个生物学重复。每个样品取20 μL上清液,混合后用于质控分析。

  • 1.2.2 代谢物检测

  • 利用Thermo Fisher Scientific超高效液相色谱(ultra-high performance liquid chromatography,UHPLC)串联傅里叶变换质谱(Q Exactive HF-X)进行LC-MS分析。色谱柱:ACQUITY UPLC HSS T3(100 mm × 2.1 mm,1.8 μm;Waters,USA)。液相条件:流动相A为95%水 + 5%乙腈(含0.1%甲酸),流动相B为47.5%乙腈 + 47.5%异丙醇 + 5%水(含0.1%甲酸)。洗脱梯度如下。正离子模式:0 min,A/B(V/V)为100∶0;3.0 min,为80∶20;4.5 min,为65∶35;5.0 min,为0∶100;6.3 min,为0∶100;6.4 min,为100∶0;8.0 min,为100∶0。负离子模式:0 min,A/B(V/V)为100∶0;1.5 min,为95∶5;2.0 min,为90∶10;4.5 min,为70∶30;5.0 min,为0∶100;6.3 min,为100∶0;6.4 min,为100∶0;8.0 min,为100∶0。柱温40℃;进样量3 μL;流速0.4 mL·min-1。质谱条件:电喷雾离子源(ESI),喷雾电压3 500 V(正模式)和-3 500 V(负模式);加热温度425℃;毛细管温度325℃;鞘气流速50 Arb;辅助气流速13 Arb;扫描范围70~1 050 m/z;一级分辨率60 000;二级分辨率7 500;碰撞能分别为20、40、60 eV;采用质谱连续扫描采集数据。

  • 1.2.3 数据分析

  • 将原始数据导入Progenesis v2.2软件(Waters,USA),通过与HMDB、METLIN等代谢数据库进行比对鉴定代谢物。利用R软件包进行代谢组数据分析和热图制作,对两组样品进行主成分分析(principal component analysis,PCA)和正交偏最小二乘判别分析(orthogonal partial least squares discriminant analysis,OPLS-DA),得到模型的变量权重值(variable important in projection,VIP),以VIP > 1,P<0.05且FC(fold change)>1.10或FC < 0.91为标准筛选组间的显著差异代谢物(significantly changed metabolites,SCMs),通过与KEGG数据库进行代谢途径富集。

  • 1.3 转录组测序与数据处理

  • 1.3.1 转录组测序及文库构建

  • 采集YL和NYL的新鲜叶片,参考陆小雨等(2020)方法进行RNA提取和质控处理。采用TruseqTM RNA sample prep Kit(Illumina)的方法构建文库。用带Oligo(dT)的磁珠分离mRNA;将mRNA随机打断,在SMART逆转录酶(SMARTScribeTM Reverse Transcriptase)作用下合成cDNA第1条链,利用RNaseH将RNA链降解,合成cDNA第2条链;通过End-Repair Mix将双链cDNA补齐,其3′末端加A处理后连接测序接头;将cDNA进行PCR富集,利用AMPure XP beads纯化产物;经过TBS-380(Picogreen)定量,按数据比例混合上机;测序时通过cBot平台完成桥式PCR扩增,生成簇(clusters),利用IIlumina Noveseq 6000平台完成转录组测序,构建Illumina PE文库(读长2 bp × 150 bp)。

  • 1.3.2 数据处理

  • 将测序得到的图像信号经CASAVA碱基识别转换为原始数据(raw data),经过质控获得高质量的干净数据(clean data)。采用无参考基因组的转录组分析,通过Trinity v2.8.5软件(Grabherr et al.,2011)将Clean reads拼接成重叠群(contig)和单一序列( singleton)。此后,利用TransRate v1.0.3(Smith-unna et al.,2016)和CD-hit v4.5.7(Li &Godzik,2006)对初始组装序列进行优化过滤,并利用BUSCO v3.0.2软件(Simão et al.,2015)再次评估,得到后续分析的最终转录本。

  • 将转录本与6大数据库(NR、Swiss-Prot、Pfam、eggNOG、GO和KEGG)比对以获取注释信息。通过RSEM v1.3.1软件(Li &Pewey,2011)计算基因的TPM值(transcripts per million reads,每百万读段中来自于某转录本的读段数),利用DESeq2 v1.24.0软件(Michael et al.,2014)筛选2个组的差异表达基因(differentially expressed genes,DEGs),将阈值差异倍数FC ≥ 2且修正P值(P adjust)< 0.05作为筛选标准。以P adjust < 0.05为标准,采用Goatools v0.6.5软件(Klopfenstein et al.,2018)进行GO富集分析和KEGG通路富集分析,筛选参与叶色变化的关键基因。

  • 1.3.3 代谢物与基因相关性分析

  • 采用Prism 8.0(GraphPad,USA)软件进行SCMs与DEGs的皮尔森(Pearson)相关性分析。以相关性系数|r| ≥ 0.8且P < 0.05为阈值,得到SCMs与DEGs的分子间相互作用关系数据。

  • 1.3.4 RT-qPCR验证

  • 选用8个DEGs进行RT-qPCR来检验转录组数据的可靠性,利用Primer Premier 5.0软件设计PCR引物(表1)。以CACs(Accession number:ID6728500)为内参基因(Marum et al.,2012)。设置3个生物学重复。将荧光定量表达水平进行Log2转换,与对应基因的转录组表达丰度进行比较。

  • 1.4 叶绿素含量测定

  • 取YL和NYL的新鲜叶片,经过称量、研磨和过滤后,参照张丽霞等(2021)的方法来测定两组叶片中的叶绿素含量。设置3个生物学重复。

  • 1.5 光合参数测定

  • 采用CI-340便携式光合作用测定仪(CID,USA)对YL突变体和NYL植株叶片的净光合作用、气孔导度、胞间CO2浓度、蒸腾速率等光合参数进行测定。设置3个生物学重复。

  • 2 结果与分析

  • 2.1 代谢组测序结果与分析

  • 2.1.1 代谢组的多元统计分析

  • 对YL和NYL两组样本在正、负离子模式下的UHPLC-Q Exactive HF-X数据进行代谢物组成的多元统计分析。PCA分析结果显示,正离子模式下第1主成分(PC1)和第2主成分(PC2)分别占总变量的40.60%和19.10%,负离子模式下第1主成分(PC1)和第2主成分(PC2)分别占总变量的37.30%和24.10%(图2:A,B)。OPLS-DA分析结果(图2:C,D)显示,两种离子模式下差异代谢物R2值均高于Q2值,并且Q2Y轴截距小于0,说明样本数据描述良好。

  • 在正、负离子模式下共鉴定出614个SCMs,其中,正离子模式下257个,上调的有148个,下调的有109个,负离子模式下357个,上调的有147个,下调的有210个(图2:E,F)。

  • 2.1.2 主要的SCMs分析

  • 从614个SCMs中筛选出相对含量前30的差异代谢物。由表2可知,黄酮类化合物有9种,均在YL组表达上调,以吡喃酮啡肽A上调最为显著,上调了2.28倍,槲皮素3-O-(6″-乙酰葡萄糖苷)也上调了1.55倍;5种核苷及其衍生物在YL中有差异积累,其中2种嘧啶核苷显著上调,3种嘌呤核苷显著下调;9种脂类代谢物中,有5种在YL中上调,主要为异戊烯醇脂类;2种有机酸中,1种显著上调,另1种显著下调。

  • 2.1.3 SCMs的KEGG通路分析

  • 将YL和NYL的SCMs进行KEGG富集分析,共富集到66条通路,主要包含甘油磷脂代谢、辅助因子生物合成、类黄酮生物合成、ABC转运蛋白、异黄酮生物合成、氨基糖和核苷酸糖代谢等13条代谢通路(图3)。其中,黄酮类生物合成富集的SCMs最多,包括类黄酮生物合成富集12个、异黄酮生物合成富集7个、黄酮和黄酮醇生物合成富集4个,说明黄酮类物质可能与YL叶色黄化有关。此外,甘油磷脂代谢、辅酶合成代谢、ABC转运蛋白、氨基糖和核苷酸糖代谢、苯丙烷生物合成分别富集了17、12、7、6、4个代谢物。

  • 2.2 转录组测序结果与分析

  • 2.2.1 测序质量分析

  • 对NYL和YL材料的RNA进行转录组测序,质控后共获得44.26 Gb干净数据(clean data)(表3)。各样品的干净序列(clean read)条数在43 864 122和56 594 944之间,Q20值在97.61%和97.79%之间,Q30值在93.03%和93.49%之间,GC碱基占比在44.49%~44.99%之间,满足于后续分析要求。

  • 2.2.2 差异表达基因统计

  • 从YL和NYL的样品中分别鉴定了46 391个和48 018个表达基因,其中16 699个和15 072个基因分别在YL和NYL中特异表达(图4:A)。以NYL为对照组,在YL中共检测到4 146个DEGs,其中1 711个上调,2 435个下调(图4:B)。

  • 图1 赤皮青冈黄化植株(A)和叶色对比图(B)

  • Fig.1 Etiolated plant ( A) and leaf color contrast ( B) of Quercus gilva

  • 表1 qPCR引物序列

  • Table1 Primer sequences for qPCR

  • 2.2.3 DEGs的GO功能分析

  • GO分析表明,YL和NYL的3 612个DEGs(P < 0.05)显著富集到12个生物学过程、14个细胞组分和2个分子功能条目(图5)。在生物学过程中,富集基因最多的是前体代谢产物和能量产生,其次为光合作用、色素生物合成过程、色素代谢过程、卟啉化合物生物合成过程等,涉及过程多与光合作用或叶绿素合成有关,以下调基因为主,也有少数与磷酸脱氢酶、质体丙酮酸激酶、葡萄糖基转移酶及二氢黄酮醇还原酶相关的代谢途径发生了不同程度上调。在细胞组分中,富集最多的GO条目是质体和叶绿体,其次为类囊体膜、光合膜、叶绿体类囊体膜等,均以下调基因的数目较多。在分子功能中,存在显著差异的代谢途径最少,仅有四吡咯结合和叶绿素结合,均以下调基因居多,其中叶绿素结合途径当中的下调基因占总数的90%以上。GO功能富集分析表明,YL的叶色黄化突变与光合作用、叶绿素代谢等过程有密切关系。

  • 2.2.4 差异代谢途径的KEGG富集分析

  • KEGG富集分析结果显示,共有744条DEGs被注释到KEGG通路,其中192条基因显著富集到植物-病原体相互作用、光合作用、乙醛酸及二羧酸代谢等12个过程(表4)。与光合作用相关的途径有5种,具体如下:光合作用-天线蛋白质富集因子最高,为0.666 7,该通路注释到10个基因,全部为捕光叶绿素蛋白复合体(light-harvesting chlorophyll protein complex,LHC)编码基因;富集到光合作用的基因有23个,主要涉及光系统的反应中心复合物(reaction-center complex,RCC);富集到光合生物碳固定途径的有16个,包括磷酸丙糖异构酶(triosephosphate isomerase,TIM)、3-磷酸甘油醛脱氢酶(glyceraldehyde3-phosphate dehydrogenase,GAPA)等关键酶的编码基因;富集到卟啉和叶绿素代谢的有12个;富集到乙醛酸及二羧酸代谢的有22个基因。此外,一些DEGs也参与了类黄酮生物合成、花青素生物合成、氨基酸代谢、苯丙烷生物合成等途径。

  • 图2 黄化植株(YL)和正常植株(NYL)的PCA得分图(A、B)、OPLS-DA得分图(C、D)和差异代谢物火山图(E、F)

  • Fig.2 PCA score plots ( A, B) , OPLS-DA score plots ( C, D) and volcano plots ( E, F) of differential metabolites between YL and NYL

  • 表2 YL和NYL组中相对含量前30的SCMs

  • Table2 Top 30 SCMs of relative contents between YL and NYL

  • 续表2

  • 图3 黄化植株(YL)和正常植株(NYL)差异代谢物的富集图

  • Fig.3 Enrichment diagram of different metabolites between YL and NYL

  • 2.2.5 差异转录因子鉴定与分析

  • 转录因子因与功能基因调控区域结合而影响基因表达,进而会对许多生物学过程产生影响(刘玉飞等,2022)。对4 146个DEGs中的转录因子基因进行鉴定,共筛得39个转录因子相关基因,属于15个家族(图6)。其中,差异基因数目最多的是MYB家族(8个DEGs),AP2/ERF和bHLH次之,分别有5个和4个,WRKY、bZIP和SBP则都有3个。15个转录因子家族中, WRKY、GRAS、AP2/ERF和C2H2以下调基因居多,其他则以上调为主。

  • 表3 赤皮青冈转录组测序质量分析

  • Table3 Quality analysis of transcriptome sequencing of Quercus gilva

  • 图4 YL和NYL组间差异表达基因的韦恩图(A)和差异表达基因上/下调统计图(B)

  • Fig.4 Venn diagram ( A) and statistical chart for DEGs up/down regulation ( B) between YL and NYL

  • 2.2.6 DEGs的RT-qPCR验证分析

  • 选取8个DEGs进行qPCR验证,结果(图7)表明,荧光定量表达与转录组基因表达的变化趋势一致,证实转录组数据可靠。

  • 2.3 YL的DEGs和SCMs关联分析

  • 2.3.1 色素代谢相关DEGs和SCMs的关联分析

  • 植物叶片颜色主要由叶绿素、类胡萝卜素、花青素等植物色素决定(林馨颖等,2022)。通过测定赤皮青冈YL和NYL的叶绿素和类胡萝卜素含量,发现YL中叶绿素a(0.35 mg·g-1)、叶绿素b(0.31 mg·g-1)、总叶绿素(0.66 mg·g-1)以及类胡萝卜素(0.05 mg·g-1)的含量都较NYL(分别为2.38、1.49、3.87、0.44 mg·g-1)显著下降。此外,叶绿素a/b的比值也显著降低(图8),表明YL可能受到环境胁迫(Sun et al.,2022)。该比值的变化会造成叶绿素a对不同波长吸收的改变,进而对叶色产生影响(李丽菁等,2022)。

  • 在叶绿素合成途径中,谷氨酸-tRNA还原酶(glutamyl-tRNA reductase,HemA)、尿卟啉原脱羧酶(uroporphyrinogen decarboxylase,HemE)、原卟啉原氧化酶(protoporphyrinogen oxidase,HemF)、原叶绿素酸酯氧化还原酶(protochlorophyllide oxidoreductase,POR)等9种酶的相关DEGs都出现下调表达,其中编码HemE的基因下降80%(4/5),编码POR的2个基因表达量下调超过66.7%(2/3),编码HemA、HemB(δ-aminolevulinic acid dehydratase)、DVR(divinyl protochlorophyllide a8-vinyl-reductase)和ChlI(Mg-chelatase subunit ChlI-1)的基因表达下调都均约50%(图9:A)。

  • 类胡萝卜素也是参与植物光合作用的主要色素之一。YL中类胡萝卜素含量[(0.056 1±0.008 9)mg·g-1]较NYL(0.441 5±0.083 0 mg·g-1)显著减少。在类胡萝卜素生物合成途径中(图9:B),八氢番茄红素合酶(phytoene synthase,PSY)是该途径的第1个合成酶,编码该酶基因的表达量下降了80%(4/5),其显著下调会对后续通路产生重要影响。番茄红素ε环化酶(lycopene ε-cyclase,LCYE)是叶黄素代谢途径中的关键酶,催化线性的番茄红素环化形成胡萝卜素,再进一步合成叶黄素。LCYE基因的表达量也出现显著下降,下调约80%(4/5),会对叶黄素生物合成产生影响,进而影响突变体的叶色表现。

  • 图5 YL组和NYL组差异表达基因GO富集分析

  • Fig.5 GO enrichment analysis on DEGs in YL and NYL

  • 类黄酮合成的起始代谢物为对香豆酰辅酶A(p-coumaroyl-CoA),通过苯丙烷生物合成途径产生。在YL组中,编码4-香豆酸辅酶A连接酶(4-coumarate-CoA ligase)的基因 4CL表达上调(图9:C),但并未对香豆酰辅酶A的含量形成显著影响。在类黄酮生物合成初期阶段,编码查尔酮合酶(chalcone synthase,CHS)和查尔酮异构酶(chalcone isomerase,CHI)的基因表达显著上调,导致柚皮素查尔酮(naringenin chalcone)和柚皮素(naringenin)在YL中积累增多(FC = 1.05)。在此后的步骤中,许多关键酶的基因表达水平也显著上调,其中黄酮醇合酶(flavonol synthase,FLS)的基因表达量上调了6.98倍,类黄酮3′-单加氧酶(flavonoid 3′-monooxygenase,F3′H)的基因表达上调了5.77倍,二氢黄酮醇-4-还原酶(dihydrofla-vonol-4-reductase,DFR)的基因表达水平也显著上调了4.44倍。这些基因的表达上调,与YL中槲皮素(quercetin)、白矢车菊素(leucocyanidin)、杨梅素 (myricetin) 在内的多种黄酮类化合物的积累具有较强的相关性(|r| ≥ 0.8)(图9:D;图10)。

  • 表4 YL和NYL间显著富集KEGG通路(修正P<0.05)

  • Table4 Significantly enriched KEGG pathways between YL and NYL ( P adjust<0.05)

  • 图6 差异表达基因中不同转录因子统计分析

  • Fig.6 Statistical analysis of transcription factors in DEGs

  • 图7 YL和NYL中8个差异表达基因的表达水平验证

  • Fig.7 Verification of expression levels of eight DEGs in YL and NYL

  • 图8 YL和NYL的叶绿素和类胡萝卜素含量比较

  • Fig.8 Comparison of chlorophyll contents and carotenoid contents between YL and NYL

  • 2.3.2 光合作用相关DEGs和SCMs的关联分析

  • 在光合作用途径中,共有28个编码PS I、PS Ⅱ和LHC核心蛋白的DEGs表达水平发生变化(图11)。其中,与PS I反应中心相关的8个DEGs,都在YL中表现下调。与PS Ⅱ反应中心相关的10个DEGs,只有编码PS Ⅱ反应中心D1蛋白(photosystem Ⅱ P680 reaction center D1 protein)的psbA基因上调,5个PS Ⅱ放氧增强蛋白(photosystem Ⅱ oxygen-evolving enhancer protein)和4个不同分子量蛋白的DEGs都表现为下调。此外,编码叶绿素a/b结合蛋白的10个基因(LHCA 1、LHCA 2-1、LHCA 2-2、LHCA 4、LHCB 1-1、LHCB 1-2、LHCB 2、LHCB 4、LHCB 6、LHCB 7)在YL叶片中下调表达。综上结果表明,下调的与光合作用有关的编码基因对YL叶片中叶绿体的发育有影响,这与YL组叶片叶绿素含量降低的情况相符。叶绿素含量减少,引起YL组光合速率下降(表5)。

  • 3 讨论

  • 3.1 叶绿素合成受阻和叶绿体发育异常导致YL叶色黄化

  • 叶色是植物的一个重要性状,其形成涉及较多复杂的生物学过程(Wang et al.,2022)。在高等植物中,叶色表现主要决定于叶绿素代谢过程(吴砚农等,2021),通常是因基因突变引起的叶绿素合成受阻或降解加速,导致植株叶色黄化(朱美玉,2020)。在YL突变体的叶绿素合成过程中,共有HemA、HemB、HemE、ChlI、POR等9种酶的基因下调表达,涉及从L-谷氨酰-tRNA到叶绿素酸酯合成的大多数过程。其中, HemA催化L-谷氨酰-TRNA(L-glutamate-TRNA)还原,是调控叶绿素合成的第一个限速反应(Zhao et al.,2014)。HemB则参与δ-氨基乙酰丙酸(δ-aminolevulinate)向胆色素原转化,也是叶绿素合成的关键步骤(Yang et al.,2015)。HemAHemB基因在YL中的表达量均下调约50%,推测是由于该两个前端基因的显著下调导致叶绿素合成的前体物质减少,引起后续反应受阻,进而使叶片整体变色。若突变发生于叶绿素合成的后期基因,则突变体通常形成条纹或斑点状(Sakuraba et al.,2015)。例如,HemE基因突变会导致玉米(Zea mays)叶片出现病斑(Hu et al.,1998),HemF基因突变则会引起玉米叶片黄化坏死(Williams et al.,2006)。此外,ChlI基因表达下调也可能引起YL叶色黄化。ChlI是镁离子螯合酶(Mgch)的三个亚基之一,Mgch则是叶绿素合成途径(镁分支)中的第一种酶,也是一种关键的限速酶(罗莎等,2015)。已有研究表明,Mgch亚基编码基因ChlIChlD的表达会受该酶底物浓度的影响(Zhang et al.,2006)。在YL黄化突变体中,ChlI基因表达量下调约50%,推测可能是由于其前体—原卟啉IX(protoporphyrin IX)含量降低所致。并且,该过程也可能进一步抑制后续反应,最终导致YL组叶绿素含量显著减少。此外,YL中类胡萝卜素含量的显著下降,对突变体黄化现象的形成也有一定影响。但是,突变体中叶绿素与类胡萝卜素的比值(Chl/Car)未发生显著改变,两组比值分别为8.21和8.31,说明Chl/Car比值对YL的黄叶表型影响不大。

  • 图9 YL组和NYL组叶绿素、类胡萝卜素和类黄酮生物合成通路分析

  • Fig.9 Analysis of chlorophyll, carotenoid and flavonoid biosynthesis pathways in YL and NYL

  • 图10 YL和NYL中SCMs与DEGs的互作网络图

  • Fig.10 Interaction network diagram between SCMs and DEGs in YL and NYL

  • 图11 YL组和NYL组叶绿素、类胡萝卜素和类黄酮生物合成关联分析

  • Fig.11 Correlation analysis of chlorophyll, carotenoid and flavonoid biosynthesis in YL and NYL

  • 表5 YL和NYL的光合作用参数

  • Table5 Photosynthetic parameters of YL and NYL

  • 叶绿体结构异常也可能导致植株叶色黄化(江新凤,2021)。在YL突变体中,有33条与光合作用相关的DEGs表达水平发生变化,其中28条为PS I、PS Ⅱ的RCC和LHC的编码基因。PsaPsb家族中许多基因的下调表达,可能导致PS Ⅰ和PS Ⅱ中相关蛋白的功能受阻。已有研究证实,PS Ⅱ蛋白复合物的显著下调会引起不良基粒的堆叠(熊兴伟等,2023)。LHC与色素结合起到吸收和传递光能的作用,并能实现光保护,其表达下降会导致叶绿体中基粒堆积异常,引起黄叶表型(Kim et al.,2009)。在对水稻黄叶突变体的研究中,Wu等(2007)发现叶绿体发育相关基因以及PS Ⅱ蛋白复合体基因的表达都会受叶绿素合成中间产物的调控。在拟南芥研究中,LHCB基因的表达受到叶绿素合成相关酶(如Mgch)的反馈调节(Mochizuki et al.,2001)。由此推测,叶绿素合成途径受阻,其中间产物和相关酶的含量发生变化,从而抑制叶绿体光系统中RCC和LHC的基因表达,影响类囊体膜结构的形成,进而导致叶绿体发育异常、叶色黄化。此外,叶绿素缺乏与叶绿体发育异常,也导致YL突变体捕光能力下降,对光合作用产生不良影响。

  • 3.2 类黄酮物质合成是YL叶色黄化的物质基础

  • 采用非靶向代谢组(LC-MS)分析,在YL组中共检测到614个SCMs,主要涉及黄酮类、氨基酸、氨基糖或核苷酸糖等。黄酮类物质中的SCMs数量最多,有23个,包括白矢车菊素、杨梅素、槲皮素等类黄酮化合物及其糖苷衍生物。其中,吡喃酮啡肽A在YL突变体中上调倍数最大,达2.28倍。该物质最早发现于黑加仑种子提取物中(Lu et al.,2000),目前关于其合成途径和形成机制尚不清楚。黄酮类等含氮化合物中形成SCMs的积累,表明YL突变体中与碳和氮相关的通路发生了代谢重编。在YL中,参与叶绿素合成、碳固定以及光合作用的DEGs主要表现为下调表达(HEMATIMGAPALHC等),会造成光合作用受阻,进而导致下游的糖酵解、TCA循环等碳代谢过程受到抑制(宋建民等,1998)。糖酵解和TCA循环的中间体是黄酮类物质碳骨架的主要来源(刘健伟,2016),代谢速率下降会影响类黄酮的生物合成。然而,代谢组数据分析表明,两个代谢通路的中间产物及相关酶的含量在YL中无显著变化。并且,YL组中糖酵解途径的丙酮酸激酶(pyruvate kinase)编码基因表达上调了3.14倍,TCA循环中苹果酸合酶(malate synthase)编码基因表达上调达20.42倍,说明黄化突变促进了叶片中糖酵解和TCA循环的代谢。相同现象也在赤霞珠葡萄的黄化突变体中被发现,原因可能是黄化叶片对碳源和氮源的征调能力更强(陈迎春,2011),使得黄酮类物质合成的碳骨架供应充足。此外,由于叶绿素合成受阻,减少了对氮的消耗,因此氮积累也可能在一定程度上促进了黄酮类物质的合成与积累,并为YL叶色黄化表现的形成提供了物质基础(江新凤,2021)。

  • 3.3 转录因子参与YL叶色黄化过程

  • 转录因子对生物的生长发育具有重要的调节作用,许多种类还参与对非生物胁迫的应答(Sun et al.,2022)。在YL中,转录因子富集最多的DEGs为MYBAP2/ERFbHLH。An等(2017)研究表明,MYB家族转录因子能够介导类黄酮合成途径中很多关键酶的转录,促进黄酮类物质产生。bHLH转录因子则被证实参与对环境胁迫的应答,并能协同MYB调控类黄酮生物合成(Liu et al.,2018;Wang et al.,2018)。在YL组中,CHS、CHI、F3H、FLS等类黄酮合成关键酶的基因表达上调,可能是受MYBbHLH转录因子的调控。此外,AP2/ERF家族4个DEGs表达发生显著改变,该基因家族主要参与强光照、高温、强光等非生物胁迫的应激反应(Wu et al.,2015)。WRKYHSF等与逆境胁迫相关的DEGs也出现富集,表明YL黄化突变体可能受到环境胁迫。进一步分析胁迫条件发现,其可能源于强光或水分缺失。黄化突变体因缺少叶绿素而更容易遭受强光胁迫。江新凤(2021)研究证实强光胁迫能诱导MYB基因上调并参与植株黄化过程。此外,更快的蒸腾速率可能使YL遭受水分胁迫。YL的蒸腾速率是NYL的1.42倍,蒸腾速率过快易导致植株缺水(张丽霞等,2021)。因此推测,YL受到强光或缺水胁迫,诱导MYBbHLH等转录因子表达上调并参与类黄酮的生物合成,促进黄酮类化合物的产生。

  • 4 结论

  • 本研究通过代谢组和转录组联用技术探索赤皮青冈叶色黄化的形成机制。结果表明,YL金黄叶色可能是受到叶绿素合成受阻、叶绿体发育异常以及黄酮类物质合成加强等因素的综合作用。此外,在YL组还发现MYBbHLH家族的基因表达水平显著上调,证实了该两类转录因子参与调控类黄酮的生物合成。本研究拓展了对赤皮青冈黄叶表型形成机制的认识,为更多“金叶”类型园林植物资源的选育工作提供了理论依据。

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    • ZHU LX, ZENG XH, CHEN YL, et al. , 2014. Genetic characterisation and fine mapping of a chlorophyll deficient mutant (BnaC. ygl) in Brassica napus [J]. Mol Breed, 34(2): 603-614.

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