The Guanylate Kinase OsGK1 is Essential for Seed Development in Rice

doi:10.16819/j.1001-7216.2018.8003

Abstract

Abstract:

【Objective】In this study, the phenotype of the floury and shrunken endosperm mutant fse2 was analyzed. Isolation of the responsible gene will lay a foundation for elucidating the mechanism underlying starch synthesis and embryo development in rice. 【Method】fse2 was obtained from the mutant library of japonica cultivar Dianjingyou 1 induced with N-Nitroso-N-methylurea. In this study, the physiochemical properties of fse2 endosperm were investigated and the structure of starch grains was observed. An F₂ population derived from fse2 and N22 was constructed, then the underlying gene was determined by map-based cloning and complementation tests. qRT-PCR and GUS staining were used to analyze the expression of FSE2. Western blotting was performed to analyze the protein levels of starch synthesis related genes and mitochondrial genes in the mutant. 【Result】Compared with the transparent endosperm of wild type, fse2 displayed a floury and shrunken endosperm, significantly declined 1000-grain weight, smaller and loosely packed irregular compound starch grains. Total starch and amylose content decreased significantly, while the lipid content increased obviously in fse2, and the gelatinization characteristics of fse2 were changed notably. FSE2 encodes a guanylate kinase named OsGK1, which is dual-targeted to both mitochondria and plastids. OsGK1 was constitutively expressed in various organs with the highest level in developing endosperm at 6th day after flowering. The protein levels of most of the starch synthesis related genes in the mutant endosperm were significantly decreased, especially AGPS2b and PHOI. In addition, the homozygous seeds of fse2 were lethal and the development of fse2 embryos was severely arrested. The accumulation of mitochondria AOX was notably elevated, while almost undetectable in the wild type, indicating the mitochondrial respiratory chain was impaired. 【Conclusion】Due to the functional defects of OsGK1, the development of mitochondria and amyloplasts are abnormal, leading to the embryo lethality and a floury and shrunken endosperm.

Key words: rice (Oryza sativa L.), floury and shrunken endosperm, guanylate kinase, seed development, starch synthesis

摘要：

【目的】对水稻粉质皱缩突变体fse2进行表型分析及基因克隆,为阐明水稻淀粉合成机制以及胚的发育奠定基础。【方法】fse2来自粳稻品种滇粳优1号的MNU(N-甲基-N-亚硝基脲)诱变突变体库。本研究考查了突变体fse2籽粒的理化性状,利用扫描电镜和半薄切片观察了淀粉颗粒的结构;构建了fse2与N22的F₂群体,通过图位克隆及转基因互补验证确定目标基因;通过qRT-PCR以及GUS活性染色对FSE2进行组织表达分析;免疫印迹分析了突变体中淀粉合成相关基因以及线粒体基因的蛋白变化。【结果】fse2籽粒粉质皱缩,千粒重显著下降;胚乳中淀粉颗粒变小变圆,排列松散,不能形成正常的复合淀粉颗粒;突变体中总淀粉、直链淀粉含量均显著下降,脂肪含量显著上升,突变体淀粉的糊化特性发生明显改变。FSE2编码一个线粒体和质体双定位的鸟苷酸激酶(guanylate kinase),命名为OsGK1。OsGK1在各器官中组成型表达,并在花后6 d的胚乳中表达水平最高。突变体胚乳中淀粉合成相关蛋白水平显著降低,尤其是AGPS2b和PHOI。此外,突变体fse2的胚发育严重受损,导致种子纯合致死;线粒体定位的AOX积累显著增强,而野生型中几乎检测不到,表明线粒体呼吸途径受损。【结论】由于OsGK1的功能缺陷,导致水稻种子中线粒体和造粉体发育异常,进而产生了胚致死以及胚乳粉质皱缩的表型,因此OsGK1对水稻种子的发育至关重要。

关键词: 水稻, 粉质皱缩胚乳, 鸟苷酸激酶, 种子发育, 淀粉合成

CLC Number:

Jingfang LI, Yunlu TIAN, Xi LIU, Shijia LIU, Liangming CHEN, Ling JIANG, Wenwei ZHANG, Dayong XU, Yihua WANG, Jianmin WAN. The Guanylate Kinase OsGK1 is Essential for Seed Development in Rice[J]. Chinese Journal OF Rice Science, 2018, 32(5): 415-426.

李景芳, 田云录, 刘喜, 刘世家, 陈亮明, 江玲, 张文伟, 徐大勇, 王益华, 万建民. 鸟苷酸激酶OsGK1对水稻种子发育至关重要[J]. 中国水稻科学, 2018, 32(5): 415-426.

Figures/Tables 8

Fig. 1. Phenotypic comparison of mature seeds of wild type and fse2 mutant. A, B, D, Mature seeds of Dianjingyou 1(DJY) and fse2. A, DJY (left), fse2 (right). B, D, DJY (upper), fse2 (lower). C, Cross-sections of mature seeds of DJY(left) and fse2 (right). E, F, Scanning electron microscopic (SEM) analysis of cross-sections of mature seeds of DJY (E) and fse2 (F). G-J, Semi-thin sections of DJY (G, H) and fse2 (I, J) endosperm at 10 days after pollination (DAP) stained with I2-KI. K, The 1000-grain weight of DJY and fse2. n=3. L, Grain length, width and thickness in DJY and fse2. n=20. All values are expressed as mean±SD. **means significant difference at 0.01 level (Student’s t-test).

Fig. 2. Physicochemical characteristics of mature seeds of fse2 and its wild type. A~C, The contents of total starch (A), amylose (B), and lipid (C) in the endosperm of Dianjingyou(DJY) and fse2. n=3, Values are means±SD, Student’s t-test, **P<0.01. D, Amylopectin chain length distributions of DJY and fse2. E, Analysis of RVA characteristic of starch in DJY and fse2. F, The swollen volume of DJY and fse2 starch in urea solution (n=3). G, Starch expansion of DJY and fse2 in urea solutions.

Table 1 Analysis of RVA characteristic values of starch in wild type and fse2 mutant.

试材 Test material	最高黏度 Peak viscosity	热浆黏度 Hot pasting viscosity	崩解值Breakdown viscosity	冷胶黏度 Cool pasting viscosity	消减值 Setback viscosity	峰值时间 Peak time / min	糊化温度 Gelatinization temperature /℃
DJY	3197	1677	1520	3225	28	5.67	76.00
fse2	293	34	259	83	-210	3.80	75.95

Table 2 Genetic analysis of fse2.

年份 Year	透明种子数 Number of normal seeds	粉质皱缩种子数 Number of floury and shrunken seeds	χ²(3:1)
2017	681	208	1.508
2016	573	172	1.612

Fig. 3. Map-based cloning of FSE2. A, Map-based cloning of the FSE2 locus. The FSE2 locus was mapped to a 106 kb region by markers LJF3-16 and LJF3-17 on the short arm of chromosome 3, which contains 11 predicted open reading frames (ORFs). B, The structure of Os03g0320900 and the mutation site. Three nucleotide substitutions (red triangles) and a 70 bp deletion (red box) in the sequence are indicated. The genetic background was identified with the primer pair F/R in transgenic lines. C, PCR analysis of the 70 bp deletion in the genomic region of Os03g0320900. D, Target peptides and functional domain of FSE2 protein. A total of 68 amino acids in fse2, and only the first 20 amino acids in the N-terminus are consistent with the wild type (left of the dotted line). mTP, Mitochondrial transit peptide; cTP, Chloroplast transit peptide; aa, amino acids. E, The relative expression levels of developing seeds (10 days post-pollination) in DJY and fse2. F, Positive transgenic seeds with homozygous fse2 background (CP-1 #3 and CP-2 #7) showed transparent endosperm. CP-1 and CP-2 are two positive transgenic lines with heterozygous background. Bar=1 cm. G-H, PCR analyses of the transparent seeds from CP-1 and CP-2 lines. The upper panels in G and H represent positive transgenic individuals. The lower panels of G and H show the backgrounds of these positive transgenic individuals. The red asterisks indicate the positive transgenic seeds with homozygous fse2 background. DJY, Dianjingyou 1.

Fig. 4. Protein sequence alignment of FSE2 and its homologs. The red box indicates the catalytic site of GK; GenBank protein accession number are as follows O. sativa, XP_051628708.1; A. thaliana, NP_566276.1; Z. mays, NP_001149581.1; B. distachyon, XP_003561683.1; P. trichocarpa, XP_006380050.1; V. vinifera, XP_002279802.1; G. max, XP_003542897.2.

Fig. 5. The expression patterns of FSE2. A, Expression levels of FSE 2 in leaf, panicle, sheath and root of wild type. B, Expression levels of FSE2 in the developing endosperms of 3, 6, 9, 12, 15 and 18 days after flowering. C-F, GUS staining patterns in root (C), leaf (D), sheath (E) and panicle (F); G-J, GUS staining patterns in developing endosperm of 6 (G), 9 (H), 12 (I) and 15 days after flowering (J).

Fig. 6. The abnormal development of embryo and endosperm in the fse2 mutant. A, Comparison of the seed of wild type and fse2 after germinating for two days. B,C, Embryos of wild type and fse2 mutant seeds after imbibition (30℃, 9 h). Bars=1 mm. D, E, Developing embryos of 15 DAP seeds of wild type and fse2. Bars=1 mm. F, The determination of seed viability by TTC staining. G, Western blotting analysis of mitochondrial proteins in mature seeds of wild type and mutant. CytC, Cytochrome c biogenesis C; COX2, Cytochrome c oxidase subunit 2; NADH9, NADH dehydrogenase subunit 9; ATPβ, ATP synthase F0 subunit 6; AOX, Alternative oxidase. H, Western blotting analysis of starch synthesis enzymes in mature seeds of wild type and mutant. AGPL2, ADP-glucose pyrophosphorylase large subunit 2; AGPS2b, ADP-glucose pyrophosphorylase small subunit 2b; PPDKB, Cytosolic pyruvate orthophosphate dikinase B; PHOⅠ, Plastid phosphorylaseⅠ; BEⅠ, Starch-branching enzyme; BEⅡb, Starch-branching enzymeⅡb; SSⅡa, Starch synthaseⅡa. ACTIN antibody was used as a loading control in G and H. DJY, Dianjingyou 1.

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