A Role of Arabidopsis Phosphoinositide Kinase, FAB1, in Root Hair Growth

doi:10.11983/CBB19242

Abstract

Abstract: Lipid kinase FORMATION of APLOID and BINUCLEATE CELLS 1 (FAB1/PIKfyve) is a key enzyme that generates PI(3,5)P₂. PI(3,5)P₂ plays an important role in regulating membrane trafficking in yeast and animal cells, but its function in plants remains poorly understood. Here we study the functional role of PI(3,5)P₂ via analysis of phenotypes of Arabidopsis FAB1 T-DNA knockout lines. The Arabidopsis FAB1 family contains four orthologous genes: FAB1A, FAB1B, FAB1C, and FAB1D. The fab1a/b double mutant showed a complete male gametophyte lethal phenotype. fab1b/c/d mutant was successfully isolated. Genetic analyses showed that the loss of FAB1B, FAB1C and FAB1D function disrupts root hairs elongation. Further pharmacological analysis showed that the FAB1-specific inhibitor YM201636 inhibited the root hairs growth. In addition, the transcription level of DR5-GFP, an indicator of auxin expression and distribution, was downregulated in single mutation of FAB1B, FAB1C and FAB1D. Moreover, triple mutant phenotypes (short root hairs) were partially rescued by exogenous application of auxin analog 2,4-D and NAA. However, the mutant’s sensitivity to 1-NOA (auxin influx inhibitor) and TIBA (auxin efflux inhibitor) in root hair elongation assay is identical to that of the wild-type. Furthermore, loss of FAB1B, FAB1C and FAB1D function reduces the level of reactive oxygen species (ROS) and affects actin expression in root hairs. Taken together, the FAB1B/C/D affects Arabidopsis root hair elongation by regulating auxin distribution, levels of ROS, and actin expression.

Key words: FAB1, root hair, auxin, ROS, actin, Arabidopsis thaliana

Yuting Yao,Jiaqi Ma,Xiaoli Feng,Jianwei Pan,Chao Wang. A Role of Arabidopsis Phosphoinositide Kinase, FAB1, in Root Hair Growth[J]. Chinese Bulletin of Botany, 2020, 55(2): 126-136.

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URL: https://www.chinbullbotany.com/EN/10.11983/CBB19242

https://www.chinbullbotany.com/EN/Y2020/V55/I2/126

Figures/Tables 6

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

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)

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.

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)

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)

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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