水稻细胞分裂素氧化酶9参与调控水稻叶夹角发育

doi:10.11983/CBB22226

摘要/Abstract

摘要： 叶片直立是决定作物株型、光合效率和产量的重要农艺性状之一。细胞分裂素(CTK)是调控农作物株型、抗逆和产量的重要激素, 但其在水稻(Oryza sativa)叶片直立生长中的作用仍不清晰。该文报道了水稻细胞分裂素氧化酶/脱氢酶9 (OsCKX9)影响叶枕发育并正调控叶夹角。组织学切片显示, osckx9突变体叶夹角的变化是由于叶枕近轴端和远轴端细胞的不对称分裂所致。qRT-PCR检测表明, OsCKX9在叶枕中的表达量较高。激素处理表明, OsCKX9能被tZ、iP、cZ、6-BA和eBL等诱导表达。激素测定显示, osckx9突变体的叶枕处积累了大量的CTK, 且其对eBL的敏感度显著低于野生型。综上, OsCKX9正调控水稻叶夹角, 该研究为解析水稻叶夹角的遗传基础和培育理想株型提供了基因资源。

关键词: 水稻, 叶夹角, 细胞分裂素, 细胞分裂素氧化酶9

Abstract: The erection of rice leaf is one of the important agronomic traits that determine plant architecture, photosynthetic efficiency, and crop yield. Cytokinin is one of the most important plant hormones that regulate crop morphology, stress resistance and yield, but its role in the lamina joint development and leaf angle is still need to be further studied. Here, we report that rice CYTOKININ OXIDASE/DEHYDROGENASE9 (OsCKX9) controls lamina joint development and positively regulates leaf angle. Histological sections indicated that the leaf inclination changes in the WT and osckx9 resulted from the asymmetric proliferation of cells and vascular bundles in lamina joint. qRT-PCR showed that OsCKX9 was highly expressed in lamina joint. Quantification of cytokinin content in osckx9 mutant lamina joint showed that there were a mass of cytokinin accumulated. Moreover, the osckx9 showed insensitive to eBL. Therefore, our results revealed that OsCKX9 played a positive role in regulating leaf erectness, which provides genetic resources for analyzing the genetic basis of leaf angle and molecular-breeding of the ideal plant architecture rice.

Key words: rice, leaf angle, cytokinin, OsCKX9

朱宝, 赵江哲, 张可伟, 黄鹏. 水稻细胞分裂素氧化酶9参与调控水稻叶夹角发育. 植物学报, 2024, 59(1): 10-21.

Bao Zhu, Jiangzhe Zhao, Kewei Zhang, Peng Huang. OsCKX9 is Involved in Regulating the Rice Lamina Joint Development and Leaf Angle. Chinese Bulletin of Botany, 2024, 59(1): 10-21.

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链接本文: https://www.chinbullbotany.com/CN/10.11983/CBB22226

https://www.chinbullbotany.com/CN/Y2024/V59/I1/10

图/表 6

表1 引物序列

Table 1 The primers used in this study

Primer name	Primer sequence (5′-3′)
OsCKX9 F	CTATCCTCAGCACTTGGCCC
OsCKX9 R	AAATGGGACTGCCACTCCTG
OsUBQ5 F	GCACAAGCACAAGAAGGTGA
OsUBQ5 R	CCAAAGAACAGGAGCCTACG
OsCYC U4;1 F	CGACGACATATGCTACAACAATGC
OsCYC U4;1 R	CCAAAGAGGAAGTCCACCTCAAG

图1 osckx9突变体叶夹角变小 (A) 使用CRISPR/Cas9技术对OsCKX9进行敲除,“target”表示敲除靶点位置(蓝色方框表示外显子, 黑色横线表示非翻译区; PAM: 原间隔序列邻近基序); (B) 基因敲除株的测序验证; (C) 野生型(WT)、osckx9-1以及osckx9-2的7-DAG (萌发后天数)幼苗叶夹角表型(bar=1 cm); (D) WT、osckx9-1和osckx9-2的110-DAG植株形态表型(bar=15 cm); (E) 110-DAG的WT、osckx9-1以及osckx9-2剑叶叶夹角表型(bar=1 cm); (F) 110-DAG的WT、osckx9-1和osckx9-2第2叶叶夹角表型(bar=1 cm); (G) 量化(C)中所示植株的叶夹角(平均值±标准差, n=24); (H), (I) 量化(E), (F)中所示110-DAG植株的剑叶(H)和第2叶(I)叶夹角(平均值±标准差, n=16)。使用Student’s t-test进行统计分析。*** P<0.001

Figure 1 osckx9 mutants show smaller leaf angle (A) OsCKX9 was knocked out using CRISPR/Cas9 technique, “target” means knockout target positions (blue boxes represent exons, black horizontal lines represent untranslated regions; PAM: Primitive interval sequence adjacent to the motif); (B) Sequencing verification of gene knockout strains; (C) Morphological phenotypes of the 7-DAG (days after germination) seedlings of wild type (WT), osckx9-1 and osckx9-2 (bar=1 cm); (D) Morphological phenotypes of the adult plants of WT, osckx9-1 and osckx9-2 at 110-DAG (bar=15 cm); (E) Flag leaf phenotype of 110-DAG of WT, osckx9-1 and osckx9-2 (bar=1 cm); (F) The second leaf phenotype of 110-DAG of WT, osckx9-1 and osckx9-2 (bar=1 cm); (G) Quantification of the leaf angle of the plants shown in (C) (means±SD, n=24); (H), (I) Quantification of the flag leaf angle (H) and the second leaf angle (I) of the 110-DAG plants shown in (E), (F) (means±SD, n=16). Statistical analyses were performed by Student’s t-test. *** P<0.001

图2 OsCKX9影响叶枕远轴端细胞增殖和维管束数量 (A), (B) 野生型(WT)和osckx9-1叶枕的横截面(红框代表叶枕的近轴端和远轴端(bars=250 μm)); (C), (D) WT和osckx9-1叶枕近轴端的高放大倍率图像(bars=100 μm); (E), (F) WT和osckx9-1叶枕远轴端的高放大倍率图像(bars=250 μm); (G) 叶枕D1区和D2区长度(如(C)-(F)所示) (平均值±标准差, n=15); (H) D1区中薄壁细胞层和D2区中厚壁细胞层的定量(如(D)至(F)所示) (平均值±标准差, n=15); (I) 上下和同轴侧的维管束定量(平均值±标准差, n=15); (J) qRT-PCR检测OsCYC U4;1在WT、osckx 9-1和osckx 9-2中的表达量(平均值±标准差, n=3); (K) qRT-PCR检测OsCYC U4;1在Mock和tZ处理时的表达量(平均值±标准差, n=3)。D1: 近轴端表皮与近轴端中央维管束之间的区域; D2: 远轴端表皮与厚壁组织之间的区域; tZ: 反式玉米素。使用Student’s t-test进行统计分析。* P<0.05; ** P<0.01; *** P<0.001

Figure 2 OsCKX9 affects the cell proliferation and vascular number in the abaxial of lamina joint (A)-(B) Transverse section of the lamina joints of wild type (WT) and osckx9-1 (red box represent the adaxial and the abaxial side of the lamina joint) (bars=250 μm); (C)-(D) High magnification images of the adaxial side of the lamina joints of WT and osckx9-1 (bars=100 μm); (E)-(F) High magnification images of the abaxial side of the lamina joints of WT and osckx9-1 (bars=250 μm); (G) Lengths of the D1 and D2 of the lamina joints shown in (C) to (F) (means±SD, n=15); (H) Quantification of the parenchyma cell layers in D1 and sclerenchyma cell layers in D2 shown in (D) to (F) (means±SD, n=15); (I) Quantification of vascular bundles on the abaxial and adaxial sides (means±SD, n=15). (J) Expression analysis of OsCYC U4;1 in WT, osckx9-1 and osckx9-2 by qRT-PCR (means±SD, n=3); (K) Expression analysis of OsCYC U4;1 in Mock and tZ treatment by qRT-PCR (means±SD, n=3). D1: The region between the adaxial epidermis and the adaxial central vascular bundle; D2: The region between the abaxial epidermis and the sclerenchyma; tZ: Trans-zeatin. Statistical analyses were performed by Student’s t-test. *P<0.05; **P<0.01; ***P<0.001

图3 OsCKX9的表达谱分析 (A) 通过qRT-PCR分析抽穗期野生型(WT)水稻根、茎、叶枕和叶中OsCKX9的表达量; (B) OsCKX9在tZ (反式玉米素)、iP (异戊烯基腺嘌呤)、cZ (顺式玉米素)、6-BA和eBL (表油菜素内酯)处理3小时的7-DAG幼苗中的相对表达量, OsUBQ5为内参基因。数值为平均值±标准差, n=3; 使用Student’s t-test进行统计分析。** P<0.01; *** P<0.001

Figure 3 The expression pattern of OsCKX9 (A) Expression analysis of OsCKX9 in various rice tissues (root, stem, lamina, and leaf) in the heading stage at the wild type (WT) by qRT-PCR; (B) Relative expression levels of OsCKX9 in 7-DAG seedlings treated by tZ (trans-zeatin), iP (isopentenyladenine), cZ (cis-zeatin), 6-BA and eBL (epibrassinolide) for 3 h. OsUBQ5 was used as an internal control. Values are means±SD, n=3; statistical analyses were performed by Student’s t-test. ** P<0.01; *** P<0.001

图4 osckx9叶枕处细胞分裂素(CTK)含量测定 (A)-(F) 野生型(WT)、osckx9-1和osckx9-2叶枕中内源性CTK含量定量。tZR: 反式玉米素核苷; iPR: 异戊烯基腺嘌呤核苷; cZR: 顺式玉米素核苷。tZ、iP和cZ同图3。数值为平均值±标准差, n=3; 使用Student’s t-test进行统计分析。* P<0.05; ** P<0.01

Figure 4 Quantification of cytokinin (CTK) content in osckx9 lamina joint (A)-(F) Quantification of endogenous CTK contents in the lamina joint of wild type (WT), osckx9-1 and osckx9-2. tZR: Trans-zea tin riboside; iPR: Isopentenyladenine riboside; cZR: Cis-zeatin riboside; tZ, iP, and cZ are the same as shown in Figure 3. Values are means±SD, n=3; statistical analyses were performed by Student’s t-test. * P<0.05; ** P<0.01

图5 osckx9对BR不敏感 (A) 野生型(WT)、osckx9-1和osckx9-2的7-DAG幼苗叶夹角对Mock和eBL (表油菜素内酯)处理的响应(bar=1 cm); (B) Mock和eBL处理的叶夹角测量(黑色为Mock叶夹角, 橙色为eBL处理后增加的角度) (数值为平均值±标准差, n=20; 使用Student’s t-test进行统计分析。*** P<0.001); (C) WT、osckx9-1和osckx9-2的7-DAG幼苗叶夹角对Mock、ABA和MeJA (茉莉酸甲酯)处理的响应(bar=1 cm); (D) Mock、ABA和MeJA处理的叶夹角测量(数值为平均值±标准差, n=20; 使用One-way ANOVAL of the LSD test对数据进行显著性分析, 不同小写字母表示对照组与处理组间在P<0.05水平差异显著)。

Figure 5 osckx9 shows insensitivity to BR (A) Wild type (WT), osckx9-1 and osckx9-2 plants responded with 7-DAG seedling leaf angles using Mock and eBL (epibrassinolide) treatment (bar=1 cm); (B) Measurement of leaf angles treated with Mock or eBL (black is the angle of the Mock leaf, orange is the angle added after eBL treatment (values are means±SD, n=20; statistical analyses were performed by Student’s t-test. *** P<0.001); (C) WT, osckx9-1 and osckx9-2 plants responded with 7-DAG seedling leaf angles using Mock, ABA and MeJA (jasmonates) treatments (bar=1 cm); (D) Mock-, ABA- and MeJA-treated measurement of leaf angles (values are means±SD, n=20; data were analyzed statistically using the One-way ANOVAL of the LSD test, and the different lowercase letters indicate significant differences in the Mock group and the treatment group at P<0.05 level).

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