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2024 Volume 46 Issue 2
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FENG Ping, LIU Yang, YANG Jie, et al. The Class Ⅲ Peroxidase Gene IPH1 Regulates Plant Height in Rice[J]. Journal of Southwest University Natural Science Edition, 2024, 46(2): 24-33. doi: 10.13718/j.cnki.xdzk.2024.02.003
Citation: FENG Ping, LIU Yang, YANG Jie, et al. The Class Ⅲ Peroxidase Gene IPH1 Regulates Plant Height in Rice[J]. Journal of Southwest University Natural Science Edition, 2024, 46(2): 24-33. doi: 10.13718/j.cnki.xdzk.2024.02.003
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The Class Ⅲ Peroxidase Gene IPH1 Regulates Plant Height in Rice

  • Rice Research Institute, Southwest University/Academy of Agricultural Sciences/Chongqing Key Laboratory of Transgenic Plant and Safety Control, Chongqing 400715, China

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  • Corresponding author: SHEN Wenqiang
  • Received Date: 15/07/2023
    Available Online: 20/02/2024

  • MSC: S511

  • Peroxidase (PRX) is a marker enzyme of peroxisome, which can protect cells from oxidative damage and relieve the toxic effect of H2O2. It can also enhance the activity of natural killer cells and regulate cell proliferation, differentiation and apoptosis. The class Ⅲ peroxidase (CⅢ PRXs) is a unique peroxidase family in plants, which plays an important role in plant immunity by scavenging reactive oxygen species (ROS). However, the function of CⅢ PRXs in the establishment of plant architecture remains unclear in rice. In this study, two different forms of knockout homozygous mutants iph1-1 and iph1-2 of CⅢ PRXs gene (LOC_Os12g09460) were obtained by CRISPR/Cas9. The plant height of the iph1 mutant was significantly higher than that of the wild type. Except for the length of the last internode, the lengths of other internodes were significantly or extremely significantly longer than that of the wild type. The investigation of agronomic traits showed that there were no significant differences in main traits such as panicle length and seed setting rate. The expression pattern analyzed by RT-qPCR showed that IPH1 was expressed in roots, stems, leaves, sheaths and panicles, and was relatively highly expressed in stems and sheaths. Subcellular localization analysis showed that IPH1 was mainly located in peroxisomes. Through further physiological analysis, it was found that the peroxidase activity in iph1 mutant was significantly reduced, while the H2O2 content was increased. These results preliminarily confirmed that IPH1 regulates the formation of plant height through the hydrogen peroxide pathway in rice, which can provide favorable gene resources for enriching the plant height regulation network and further lay a good foundation for plant architecture related biological breeding.

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The Class Ⅲ Peroxidase Gene IPH1 Regulates Plant Height in Rice

    Corresponding author: SHEN Wenqiang
  • Received Date: 15/07/2023
  • Available Online: 20/02/2024

Abstract: 

Peroxidase (PRX) is a marker enzyme of peroxisome, which can protect cells from oxidative damage and relieve the toxic effect of H2O2. It can also enhance the activity of natural killer cells and regulate cell proliferation, differentiation and apoptosis. The class Ⅲ peroxidase (CⅢ PRXs) is a unique peroxidase family in plants, which plays an important role in plant immunity by scavenging reactive oxygen species (ROS). However, the function of CⅢ PRXs in the establishment of plant architecture remains unclear in rice. In this study, two different forms of knockout homozygous mutants iph1-1 and iph1-2 of CⅢ PRXs gene (LOC_Os12g09460) were obtained by CRISPR/Cas9. The plant height of the iph1 mutant was significantly higher than that of the wild type. Except for the length of the last internode, the lengths of other internodes were significantly or extremely significantly longer than that of the wild type. The investigation of agronomic traits showed that there were no significant differences in main traits such as panicle length and seed setting rate. The expression pattern analyzed by RT-qPCR showed that IPH1 was expressed in roots, stems, leaves, sheaths and panicles, and was relatively highly expressed in stems and sheaths. Subcellular localization analysis showed that IPH1 was mainly located in peroxisomes. Through further physiological analysis, it was found that the peroxidase activity in iph1 mutant was significantly reduced, while the H2O2 content was increased. These results preliminarily confirmed that IPH1 regulates the formation of plant height through the hydrogen peroxide pathway in rice, which can provide favorable gene resources for enriching the plant height regulation network and further lay a good foundation for plant architecture related biological breeding.

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  • 株高是水稻的重要农艺性状之一，也是构建理想株型的一个重要因素. 株高与水稻光合效率、抗倒伏以及产量有着密切的联系. 20世纪的绿色革命在矮化株型育种中取得了巨大成功，然培育兼顾产量和抗倒伏的适宜株高仍是水稻、玉米和小麦等大多数作物的首要育种目标，因此水稻株高的遗传及分子机制一直是研究的热点.

    水稻株高主要由节间细胞的数目和大小决定. 节间细胞的增殖及扩张受多重因素的影响，包括植物激素、细胞周期、细胞壁合成、表观遗传学及转录因子等，同时也受到外界环境因素的影响，如光照、温度、物种间竞争等. 植物激素作为植物中重要的调节因子，参与植物的整个生长发育过程，其中赤霉素(GA)和油菜素内酯(BR)是水稻株高的重要决定因子[1-2]. SD1编码赤霉素生物合成的关键酶GA20ox，突变后导致GA20ox失活，造成株高降低[3-4]. EUI1编码细胞色素P450单加氧酶CYP714D1，eui1突变体及eui1 RNAi植株赤霉素含量升高，倒1节显著伸长，株高增加，而过表达植株中赤霉素含量降低，导致株高降低，因此EUI1负调控赤霉素介导的细胞伸长[5-6]. D35/OsKO2编码贝壳杉烯氧化酶，参与催化赤霉素生物合成的早期步骤，其功能缺失导致d35株高严重降低[7]. SLR1编码1个DELLA蛋白，是赤霉素信号传导的负调控因子，其功能是导致突变体株高降低. GID1编码1个可溶的赤霉素受体，类似于激素敏感脂肪酶，并且对具有生物活性的GAs具有高度的亲和性，GID1过量表达表现出GA高敏感性表型. GID1与SLR1相互作用，从而形成GID1-GA-SLR1复合体，阻止赤霉素信号向下传递[8-11].

    油菜素内酯能够促进植物生长，在细胞伸长及分裂中也具有重要作用. BRD1，BRD2和D2均参与油菜素内酯的生物合成，分别编码C-6氧化酶、氧化还原酶和细胞色素P450，这些基因的突变导致油菜素内酯生物合成受阻，突变体中油菜素内酯含量降低，表现出严重矮化[12-14]. BRI1和BZR1参与油菜素内酯信号转导，分别编码油菜素内酯受体激酶和下游信号分子. bri1第2节不伸长，株高矮化，并且对外源油菜素内酯不敏感；BZR1能够调控下游油菜素内酯响应基因，干涉植株表现为矮杆[15-16].

    过氧化物酶是一大类催化各种底物发生氧化的生物体保护酶类家族，在植物的生长发育中具有重要作用. 根据功能不同，将过氧化物酶分为3类：Ⅰ类(抗坏血酸)过氧化物酶、Ⅱ类(木质素)过氧化物酶和Ⅲ类(分泌型)过氧化物酶[17]. Ⅲ类过氧化物酶(CⅢ PRXs)广泛分布于植物界并且是植物中特有的一类过氧化物酶，参与植物激素分解代谢、木质素生物合成、种子萌发、细胞伸长和防御病原体等多种生物生理过程. CⅢ PRXs基因编码含血红素的酶，接受来自不同分子供体的电子，催化H2O2的代谢，产生活性氧(ROS，即OH-或O2-). 这些特性使得CⅢ PRXs能够在过氧化循环过程中调控损伤植物细胞的ROS产生[18]. CⅢ PRXs酶活性由ROS触发，ROS的产生由不同的环境刺激诱导，以抵御潜在的威胁[19].

    Ⅲ类PRX家族较为庞大，在水稻和拟南芥中分别包含138个和73个成员，其中大部分的生物学功能并不清楚[20-21]. 拟南芥中研究表明过氧化物酶与植物生长呈负调控，AtPRX71通过在外质体中积累H2O2抑制细胞扩张，atprx71突变体莲座叶变大、生物量显著增加，而过表达植株生长缓慢莲座叶变小、生物量显著下降，并且AtPRX71表达水平的升高会促进ROS的积累导致细胞壁成分变化[22]. AtPRX37过表达植株生长迟缓，表现为矮化[23]，因此AtPRX71和AtPRX37均是拟南芥生长的负调控因子. AtPRX72，AtPRX52和AtPRX4参与拟南芥木质素的生物合成，atprx72生长缓慢，叶片形态及角果数减少等. AtPRX52参与木质化过程中微管纤维的合成，突变体中木质素含量降低，木质素生物合成相关基因的表达下调[24-25]. OsPRX38在拟南芥中过表达后激活不同抗氧化系统信号网络，促进木质素生物合成，增强根部的木质化程度，导致过表达植株中砷积累减少[26]. AtPRX17调控木质化组织的形成，在营养生长向生殖生长的转变过程中受到AGL15的调控[27]. 在水稻中，OsPRX30通过调节过氧化物酶体(POD)的活性与ROS的含量来调节白叶枯病抗性，而OsPRX30的表达又受到含有AT-hook结构域的转录调节因子OsATH1的调控，进一步的研究揭示了OsATH1通过调节OsPRX30启动子区内的组蛋白H3乙酰化水平进而影响OsPRX30表达以调控白叶枯病抗性[28]. 目前为止，仅有少数Ⅲ类PRX基因生物学功能被揭示，还有大部分尚未报道，尤其在水稻中还未见Ⅲ类PRX基因参与调控株高的相关报道.

    本研究通过CRISPR/Cas9基因编辑技术，在粳稻中花11背景下创制的Ⅲ类过氧化物酶家族敲除突变体库中鉴定到1个株高变高的突变体，将其命名为iph1(increaseing plant height 1). 与野生型相比，iph1的节明显变长，导致株高增加. 本研究通过表型分析、农艺性状考察、表达模式分析、过氧化物酶活性分析、亚细胞定位等对IPH1进行功能分析，探究IPH1在调控水稻株高发育及株型形态建成中的作用，为利用该基因进行水稻株型分子设计育种奠定基础.

1.   材料与方法

    1.1.   试验材料

  • 水稻突变体iph1来源于以中花11为背景的敲除突变体库，经过多代自交，性状能够稳定遗传. 所有试验材料均种植于西南大学水稻研究所.

    • 1.2.   生物信息学分析

  • 通过NCBI数据库的Blast工具获取IPH1的结构域信息和同源蛋白序列，下载同源蛋白序列，并将其保存为FASTA文件格式，将FASTA文件导入MEGA5软件，根据贝叶斯最低分准则利用Neighbor-joining方法构建进化树，对IPH1进行进化分析.

    • 1.3.   RNA提取及RT-qPCR分析

  • 利用天根RNA提取试剂盒(TIANGEN生物公司)提取野生型和iph1突变体总RNA，并用宝生物PrimeScript试剂盒(TaKaRa)将RNA反转录成cDNA. 以水稻ACTIN为内参基因，使用PrimerPrimer5.0软件设计引物(表 1)[28]，用Bio-Rad实时荧光定量PCR仪(CFX Connect Realtime Reaction System)进行扩增，每个样品设置3个重复. 基因相对表达量计算用2-ΔΔCt法，并用t检验进行差异显著性分析.

    • 1.4.   亚细胞定位

  • 为了确定IPH1的亚细胞定位，使用引物pAN580-IPH1-F和pAN580-IPH1-R(表 1)从野生型中扩增不含终止密码子的IPH1编码序列，将该片段克隆到瞬时表达载体pAN580的XbalⅠ/Bam HⅠ位点上，构建IPH1-GFP载体. 经序列分析验证后，通过PEG介导法将pAN580-GFP和IPH1-GFP质粒转化水稻原生质体. 28 ℃孵育12~16 h后，用激光共聚焦显微镜(LSM800，Zeiss，德国蔡司)观察荧光信号并拍照.

    • 1.5.   过氧化物酶活性分析

  • 取8周龄水稻幼苗的新鲜叶片(300 mg)，在液氮中研磨成粉，并与4 mL 0.05 mol/L PBS(pH值为7.8)混合在5 mL试管中. 快速解冻后，将试管7 500 r/min离心15 min，收集上清液(含POD). 以愈创木酚为供氢体，采用分光光度计在405 nm处测定OD值，根据公式计算过氧化物酶活性[29].

    • 1.6.   过氧化氢(H2O2)质量分数测定

  • 将野生型和iph1突变体新鲜组织于液氮中充分研磨，加入9倍体积的生理盐水，冰水浴条件下机械匀浆，1 200 r/min离心10 min，取上清液待测. 每个样本设置3个重复，采用南京建成生物工程研究所的H2O2试剂盒测定H2O2质量分数.

2.   结果与分析

    2.1.   iph1敲除突变体的获得及鉴定

  • CRISPR/Cas9表达载体构建的靶点选择如图 1a，在基因(LOC_Os12g09460)的5′-UTR及编码区前端设计了2个靶点. 通过农杆菌介导转化中花11愈伤，对转基因植株靶点测序，比对发现2个独立的转基因株系在PAM(protospacer adjacent motif)附近发生了2种方式的编辑：iph1-1靶点1处发生了大片段替换，导致IPH1蛋白翻译的起始密码子缺失，无法正常翻译；iph1-2在目标位置缺失了7个碱基(ACCACGG)(图 1b). 进一步通过qRT-PCR分析，IPH1在两种编辑方式的突变体中表达均极显著下调(图 1c). 以上结果表明所构建的iph1突变材料为过氧化物酶基因(LOC_Os12g09460)敲除突变体，可用于后续的表型及生理生化等分析.

    • 2.2.   表型鉴定

  • 对iph1突变体植株经过多代全生育期的田间观察和统计，发现iph1突变株在苗期较野生型差异无统计学意义(图 2a)，在抽穗期至成熟期阶段株高出现差异，成熟期iph1突变体的株高显著高于野生型(图 2b)，iph1-1和iph1-2的株高分别增加了7.4%，14.6%(图 2d). 结果表明IPH1基因功能缺失主要影响株高，且遗传性状稳定.

    成熟期时，对野生型和iph1-1，iph1-2突变体的结实率、穗长和各节间长度进行了详细统计. 结果发现，iph1-1，iph1-2突变体的结实率、穗长以及倒1节(即第5节)节长较野生型差异无统计学意义(图 2e至2g)，iph1-1突变体的第4节节长较野生型显著增加，iph1-2突变体的第3节节长较野生型显著增加，iph1-1和iph1-2突变体位于茎基部的第1节和第2节节长较野生型均显著增加(图 2c，2h至2k). 该结果表明突变体株高变高主要由茎基部节长变化所致.

    • 2.3.   生物信息学分析

  • 通过NCBI数据库获取IPH1的结构域信息，发现IPH1是一个过氧化物酶基因，在氨基酸第36~120区间内含有1个分泌型过氧化物酶(secretory peroxidase)结构域，属于第Ⅲ类过氧化物酶家族(图 3a)；同时也获得了IPH1蛋白在其他物种的同源蛋白序列，将其保存为FASTA文件格式，根据贝叶斯最低分准则，利用MEGA5软件的Neighbor-joining方法构建进化树，分析发现IPH1编码的蛋白独立于一个分支，具有高度保守性(图 3b).

    • 2.4.   表达模式及亚细胞定位分析

  • 为了确定IPH1基因在水稻各组织中的表达情况，我们以野生型根、茎、叶、鞘和穗的cDNA作为模板，进行实时荧光定量表达分析. 结果显示IPH1转录本在不同部位均有表达(图 4a)，其中在茎和鞘中有较高表达.

    IPH1蛋白属于第Ⅲ类过氧化物酶家族蛋白，为了探究IPH1在细胞内部细胞器的定位情况，将CaMV35S-IPH1-GFP融合蛋白及过氧化物酶体CaMV35S-PTS1-mCherry融合蛋白共同转化水稻原生质体，同时将转化含CaMV35S-GFP的空载体pAN580作为对照，28 ℃避光过夜培养18 h，利用激光共聚焦显微镜观察荧光信号. 结果发现大部分绿色荧光都能与红色荧光重叠(图 4b)，表明IPH1主要定位于过氧化物酶体中.

    • 2.5.   过氧化物酶活性及H2O2质量分数分析

  • IPH1编码1个Ⅲ类过氧化物酶，但IPH1是否影响过氧化物酶的活性和H2O2质量分数尚不清楚，因此，采用分光光度法测定野生型及iph1突变体植株中的过氧化物酶活性和H2O2质量分数. 结果显示，iph1突变体的过氧化物酶活性显著降低，H2O2质量分数显著增加(图 5).

3.   讨论

    3.1.   IPH1编码1个含有分泌型过氧化物酶结构域的新基因

  • IPH1是位于水稻第12染色体上的一个新基因，目前还未见其生物学功能的相关报道. 蛋白序列分析发现IPH1包含1个分泌型过氧化物酶结构域，属于第Ⅲ类过氧化物酶(Class Ⅲ peroxidases，CⅢ PRXs)家族. CⅢ PRXs作为一类植物特异性氧化还原酶参与植物激素的分解代谢、木质素的生物合成、细胞伸长和病原菌防御等多种生物学过程[18]，水稻和拟南芥中CⅢ PRXs家族成员分别有138个和73个[21]. 目前在水稻中对CⅢ PRXs家族基因的功能研究较少，仅有1个家族基因OsPRX30被报道. 该基因编码1个主要定位于内质网的过氧化物酶前体蛋白，对细菌性黄单胞菌有反应，过表达OsPRX30会维持高水平的过氧化物酶(POD)活性和降低过氧化氢质量分数，从而增强植物对白叶枯病的敏感性. 进一步研究揭示含有AT-hook结构域的转录因子OsATH1通过调节OsPRX30启动子区内的组蛋白H3乙酰化水平进而调节OsPRX30表达，通过调控过氧化物酶体活性与ROS量调控白叶枯病抗性[30]. 本研究中，IPH1蛋白主要定位于过氧化物酶体内，在根、茎、叶、鞘、穗中均有表达，且在茎和鞘中表达相对较高.

    • 3.2.   IPH1可能通过过氧化氢途径调控水稻株高

  • 水稻株高是非常重要的一个农艺性状，对抗倒伏及生物量的产生具有重要意义. 株高受到植物激素、细胞周期、细胞壁合成、表观遗传学、转录因子以及光照、温度等多重因素的影响. 如SD1，EUI1，D35/OsKO2，SLR1，GID1等通过调控赤霉素的合成及信号传导等调控细胞分裂或细胞伸长[3-6, 8-9]. BRD1，BRD2，D2，BRI1和BZR1通过参与油菜素内酯的生物合成及信号转导调控水稻株高[12-16]. 此外，一些基因的异常表达也会影响株高，OsCD1，BC12，OsGLP1等影响水稻细胞伸长或分裂的基因异常表达，导致产生矮化或半矮化表型[31-33]. 本研究中，鉴定到1个编码Ⅲ类过氧化物酶的基因IPH1影响水稻株高. 有关CⅢ PRXs调节植物株高的报道相对有限，小麦新型矮杆突变体Rht-B13b中CⅢ PRXs在扩张组织中表达显著上调，CⅢ PRXs可以使用H2O2增加细胞壁交联，从而导致细胞扩增和生长减少，最终使得植株表现为矮化表型[34]. 本研究中，CⅢ PRXs家族基因IPH1不同方式的突变均导致植株高度增加；进一步研究发现，iph1突变体中过氧化物酶活性降低，而H2O2的质量分数显著增加，因此IPH1可能通过过氧化氢途径调控水稻株高的形成，但是具体的分子作用机理仍需进一步研究.

4.   结论

  • 本研究鉴定了1个在粳稻中花11背景下敲除的第Ⅲ类过氧化物酶家族突变体iph1. 与野生型相比，两种突变方式的iph1突变体株高变高的表型能够稳定遗传；进一步统计也发现iph1突变体的第1节和第2节节长较野生型显著增加. 生物信息学分析表明，IPH1基因包含1个分泌型过氧化物酶结构域，利用同源蛋白序列进行进化树分析，结果显示IPH1独立于1个分枝，具有高度保守性. IPH1基因在各个组织中均有表达且在茎秆中表达相对较高，可能基因在表达水平上会影响表型，亚细胞定位表明该蛋白主要定位于过氧化物酶体中. 生理分析发现突变体过氧化物酶活性降低，H2O2质量分数增加. 本研究为深入了解水稻株高遗传调控网络提供了基因资源，并进一步丰富了株高相关的生物育种材料.

Figure (5)  Table (1) Reference (34)

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