植物学报 ›› 2020, Vol. 55 ›› Issue (2): 126-136.DOI: 10.11983/CBB19242
收稿日期:
2019-12-16
接受日期:
2020-02-17
出版日期:
2020-03-01
发布日期:
2020-02-17
通讯作者:
王超
基金资助:
Yuting Yao,Jiaqi Ma,Xiaoli Feng,Jianwei Pan,Chao Wang()
Received:
2019-12-16
Accepted:
2020-02-17
Online:
2020-03-01
Published:
2020-02-17
Contact:
Chao Wang
摘要: FAB1/PIKfyve是介导PI(3,5)P2 (磷脂酰肌醇3,5-二磷酸)生物合成的磷酸肌醇激酶。在动物和酵母(Saccharomyces cerevisiae)中, PI(3,5)P2参与调控胞内膜运输, 但在植物中的研究较少。该文通过分析拟南芥(Arabidopsis thaliana) FAB1的T-DNA插入突变体的表型解析PI(3,5)P2的生物学功能。拟南芥FAB1基因家族包含FAB1A、FAB1B、FAB1C和FAB1D四个基因。研究发现, fab1a/b呈现雄配子体致死的表型。利用遗传杂交获得fab1b/c/d三突变体, 发现FAB1B、FAB1C和FAB1D功能缺失导致根毛相比野生型变短, 经FAB1特异性抑制剂YM201636处理后的野生型中也观察到相似的短根毛表型。此外, fab1b/c/d三突变体中DR5转录水平降低。同时, 外源施加生长素类似物2,4-D和NAA能部分恢复fab1b/c/d植株短根毛的表型, 但fab1b/c/d突变体对生长素转运抑制剂(1-NOA和TIBA)的敏感性与野生型相似。此外, FAB1B/C/D功能缺失使根毛中ROS的含量减少且影响肌动蛋白的表达。上述结果表明, FAB1B/C/D通过调控生长素分布、ROS含量和肌动蛋白的表达影响拟南芥根毛伸长。
姚玉婷,马家琦,冯晓莉,潘建伟,王超. 磷酸肌醇激酶FAB1调控拟南芥根毛伸长. 植物学报, 2020, 55(2): 126-136.
Yuting Yao,Jiaqi Ma,Xiaoli Feng,Jianwei Pan,Chao Wang. A Role of Arabidopsis Phosphoinositide Kinase, FAB1, in Root Hair Growth. Chinese Bulletin of Botany, 2020, 55(2): 126-136.
Primer name | Primer sequence (5'-3') | Purpose |
---|---|---|
F1 | GGCGAGGGATATTGA GTTCAG | Genotyping of fab1b-2 and RT-PCR |
R1 | GTCATACATGTGGGA TCACCG | Genotyping of fab1b-2 and RT-PCR |
F2 | TGGGAGAAAACAGCAA TGAAC | Genotyping of fab1c-2 and RT-PCR |
R2 | CACGACAACTTCCCCG AAGCACAA | Genotyping of fab1c-2 and RT-PCR |
F3 | AGGTTGGGATGAATGG TTTTG | Genotyping of fab1d-2 and RT-PCR |
R3 | AGGTCGTGCCGTATC TCTTTC | Genotyping of fab1d-2 and RT-PCR |
sgtDs3'-1 | GGTTCCCGTCCGATT TCGACT | Genotyping of fab1c-2 |
LBb1.3 | ATTTTGCCGATTTCG GAAC | Genotyping of fab1b-2 and fab1d-2 |
AtACTIN-F | GTCGTACAACCGGTA TTGTG | Internal control for RT-PCR |
AtACTIN-R | GAGCTGGTCTTTGAG GTTTC | Internal control for RT-PCR |
表1 引物序列
Table 1 Primers used in this study
Primer name | Primer sequence (5'-3') | Purpose |
---|---|---|
F1 | GGCGAGGGATATTGA GTTCAG | Genotyping of fab1b-2 and RT-PCR |
R1 | GTCATACATGTGGGA TCACCG | Genotyping of fab1b-2 and RT-PCR |
F2 | TGGGAGAAAACAGCAA TGAAC | Genotyping of fab1c-2 and RT-PCR |
R2 | CACGACAACTTCCCCG AAGCACAA | Genotyping of fab1c-2 and RT-PCR |
F3 | AGGTTGGGATGAATGG TTTTG | Genotyping of fab1d-2 and RT-PCR |
R3 | AGGTCGTGCCGTATC TCTTTC | Genotyping of fab1d-2 and RT-PCR |
sgtDs3'-1 | GGTTCCCGTCCGATT TCGACT | Genotyping of fab1c-2 |
LBb1.3 | ATTTTGCCGATTTCG GAAC | Genotyping of fab1b-2 and fab1d-2 |
AtACTIN-F | GTCGTACAACCGGTA TTGTG | Internal control for RT-PCR |
AtACTIN-R | GAGCTGGTCTTTGAG GTTTC | Internal control for RT-PCR |
图1 FAB1B、FAB1C和FAB1D调控拟南芥根毛伸长 (A) FAB1基因结构和T-DNA插入位点; (B) RT-PCR检测FAB1B、FAB1C和FAB1D的表达; (C) 5天龄幼苗根毛长度(黄色虚线框代表定量区域) (Bar=5 mm); (D) 定量根毛长度; (E)-(M) FAB1单突变体、双突变体及YM201636 (FAB1特异性抑制剂)处理的野生型幼苗根毛表型(Bar=0.5 mm); (N) 定量(E)-(M)突变体根毛长度; (O) 根毛形态分析, 红色箭头表示根毛上的凸起和分叉(Bar=75 µm); (P) 定量分叉根毛占总根毛数量的百分比。*** P<0.001 (Student’s t-test)
Figure 1 FAB1B, FAB1C and FAB1D regulate root hair growth in Arabidopsis (A) FAB1 gene structure and T-DNA insertion sites; (B) Analysis of gene expression of FAB1B, FAB1C and FAB1D by RT-PCR; (C) Assay of root hair length in 5-day-old seedlings (the yellow dotted box represents quantitative area) (Bar=5 mm); (D) Quantification of root hair length; (E)-(M) The root hair phenotype of the FAB1 single mutants, double mutants and YM201636 (FAB1-specific inhibitor) treatment of Col-0 seedling (Bar=0.5 mm); (N) Quantification of (E)-(M) mutant root hair length; (O) Images of the typical root hair morphologies, the root hair morphologies were categorized as swollen or branched (red arrow indicates) (Bar=75 μm); (P) Quantification of frequency of branched root hairs (%). *** P<0.001 (Student’s t-test)
图2 拟南芥fab1b/c/d突变体根毛生长速率减慢 (A) 根毛伸长动力学分析(使用荧光显微镜连续50分钟记录野生型Col-0和fab1b/c/d突变体的动态生长趋势, 并每隔5分钟拍照1次) (Bar=50 µm); (B) 单位时间根毛长度; (C) 根毛生长速率(*** P<0.001, Student’s t-test); (D) 单位长度内根毛数量。
Figure 2 Slow growth of Arabidopsis fab1b/c/d mutant root hairs (A) Growth dynamics of individual Col-0 and fab1b/c/d root hairs (fluorescence microscopy was used to assess root hair elongation, showing consecutive frames of growing root hairs for a period of 50 minutes, pictures were taken every 5 minutes) (Bar=50 µm); (B) Root hair length per unit time; (C) Root hairs growth speed (*** P<0.001, Student’s t-test); (D) Average root hairs number.
图3 外源生长素能部分恢复拟南芥fab1b/c/d突变体的短根毛表型 (A)-(C) 外源添加DMSO (Mock)、10 nmol·L-1 2,4-D或0.1 μmol·L-1 NAA可部分恢复fab1b/c/d突变体根毛表型(Bar=0.5 mm); (D) 定量分析根毛长度; (E), (F) 生长素运输抑制剂TIBA (生长素输出抑制剂)和1-NOA (生长素输入抑制剂)处理后根毛表型(Bar=0.5 mm); (G) 定量分析根毛长度。* P<0.05; ** P<0.01; *** P<0.001 (Student’s t-test)
Figure 3 Exogenous auxin application partially rescued the root hair defect of Arabidopsis fab1b/c/d mutants (A)-(C) Root hairs of fab1b/c/d treated with DMSO (Mock), 10 nmol·L-1 2,4-D and 0.1 μmol·L-1 NAA, respectively, the phenotype of root hairs was rescued partially (Bar=0.5 mm); (D) Quantification of root hair length; (E), (F) Col-0 and fab1b/c/d seedlings were transferred to plates containing TIBA (auxin efflux inhibitors) and 1-NOA (auxin influx inhibitor) (Bar=0.5 mm); (G) Quantitative analysis of root hair length. * P<0.05; ** P<0.01; *** P<0.001 (Student’s t-test)
图4 FAB1影响拟南芥生长素的分布 (A) 利用DR5-GFP分析Col-0和fab1b/c/d根部的生长素分布变化(Bar=75 µm); (B) DR5-GUS在Col-0和fab1b/c/d中的表达情况(Bar=100 µm)
Figure 4 FAB1 affects auxin distribution in Arabidopsis (A) The expression analysis of DR5-GFP for auxin distribution in the root (Bar=75 µm); (B) DR5-GUS expression in Col-0 and fab1b/c/d (Bar=100 µm)
图5 拟南芥fab1b/c/d突变体根毛活性氧(ROS)含量及肌动蛋白稳定性改变 (A) 用DCF-DA (ROS染液)检测Col-0、fab1b/c/d和Col-0- YM201636根毛中的ROS含量(Bar=25 µm); (B) 定量分析DCF-DA荧光强度; (C) Col-0、fab1b/c/d和Col-0-YM201636的根毛肌动蛋白细胞骨架标记ABD2的分布(Bar=25 µm); (D) 定量分析ABD2-GFP荧光强度。*** P<0.001 (Student’s t-test)
Figure 5 Reactive oxygen species (ROS) intensity and actin stability were altered in root hairs of Arabidopsis fab1b/c/d seedlings (A) Total ROS generated by oxidation of DCF-DA in wild type, fab1b/c/d and Col-0-YM201636 root (Bar=25 µm); (B) Relative intensity of the GFP signals; (C) Distribution of actin cytoskeleton marker ABD2 in root hair of Col-0, fab1b/c/d and Col-0-YM201636 treatment (Bar=25 µm); (D) Average relative intensity of the ABD2-GFP signals. *** P<0.001 (Student’s t-test)
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摘要 |
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