Loss-of-function and gain-of-function mutations in FAB1A/B impair endomembrane homeostasis, conferring pleiotropic developmental abnormalities in Arabidopsis

In eukaryotic cells, PtdIns 3, 5-kinase, Fab1/PIKfyve produces PtdIns (3,5) P 2 from PtdIns 3-P, and functions in vacuole/lysosome homeostasis. Herein, we show that expression of Arabidopsis FAB1A/B in S. pombe fab1 knockout cells fully complements the vacuole morphology phenotype. Subcellular localizations of FAB1A and FAB1B fused with green fluorescent protein revealed that FAB1A/B-GFPs localize to the endosomes in root epidermal cells of Arabidopsis. Furthermore, reduction in the expression levels of FAB1A/B by RNA interference impairs vacuolar acidification and endocytosis. These results indicate that Arabidopsis FAB1A/B functions as PtdIns 3, 5-kinase in plants and in fission yeast. Conditional knockdown mutant shows various phenotypes including root growth inhibition, hyposensitivity to exogenous auxin, and disturbance of root gravitropism. These phenotypes are observed also in the overproducing mutants of FAB1A and FAB1B . The overproducing mutants reveal additional morphological phenotypes including dwarfism, male-gametophyte sterility, and abnormal floral organs. Taken together, this evidence indicates that imbalanced expression of FAB1A/B impairs endomembrane homeostasis including endocytosis, vacuole formation, and vacuolar acidification, which causes pleiotropic developmental phenotypes mostly related to the auxin signaling in Arabidopsis. kinase-deficient Schizosaccharomyces pombe Fab1p homologues are phosphatidylinositol 3-phosphate 5-kinases.

In yeast, the fab1 mutant shows an enlarged vacuolar phenotype. It is defective in terms of vacuolar acidification, osmoregulation, and inheritance of vacuoles, and exhibits a growth defect at high temperatures (Gary et al., 1998;Odorizzi et al., 1998).
Functionally, ScFab1p is necessary both for retrograde vesicle transport from vacuoles to ER/Golgi and for the sorting of cell membrane-integrated proteins in MVBs (Gary et al., 1998;Odorizzi et al., 1998;Shaw et al., 2003). Fission yeast Schizosaccharomyces pombe also has a Fab1p homologue (designated as SpFab1p), which restores the PtdIns (3,5)P 2 deficiency in Scfab1 disruptant cells (McEwen et al., 1999). The S. pombe fab1 mutant also has an enlarged vacuolar structure (Morishita et al., 2002). The mammalian orthologue of Fab1p is designated as PIKfyve. Like Fab1p in yeast, PIKfyve is also necessary for endomembrane homeostasis. Overexpression of a dominant kinase-inactive mutant exhibits an enlarged lysosome phenotype 6 (Ikonomov et al., 2001). Consequently, a shared feature of Fab1p/PIKfyve mutants in yeasts and animals is the formation of swollen vacuolar/lysosomal structures, suggesting the presence of a conserved function for Fab1p/PIKfyve in the regulation of endomembrane homeostasis (Efe et al., 2005).
In Arabidopsis, four genes encoding putative Fab1p/PIKfyve proteins, FAB1A In this study, we attempt to address the role of Fab1p/PIKfyve proteins by analyzing the phenotypes of inducible artificial microRNA (amiRNA) mutants and constitutive or inducible gain-of-function mutants of FAB1A and FAB1B. Our results demonstrated that Fab1p/PIKfyve protein is important for endomembrane homeostasis including endocytosis, vacuole formation, and vacuolar acidification. Moreover, defective FAB1 function causes pleiotropic developmental abnormalites in Arabidopsis.   Arabidopsis FAB1A and FAB1B function as PtdIns 3P-5 kinase in S. pombe.

FAB1A/B-GFPs localize to endosomes in Arabidopsis root cells
To determine the subcellular localizations of FAB1A/B proteins, we generated transgenic Arabidopsis plants expressing GFP-fused FAB1A or FAB1B (FAB1A-GFP, FAB1B-GFP) under control of their native promoters. Then we observed the subcellular localizations of these proteins in root cells.
In epidermal cells in the root differentiation zone, the fluorescence of FAB1A-GFP localized to punctate structures throughout the cytosol ( Fig. 2A). Most of these punctate structures overlapped with FM4-64 labeled endosomal compartments, implying that FAB1A-GFP localized to the endosomes of root epidermal cells (Fig. 2, B and C). In addition, FAB1B-GFP exhibited identical localization patterns to those of FAB1A-GFP (Fig. 2, E-G). These data revealed that FAB1A and FAB1B localize to the endosomes in Arabidopsis root epidermal cells.

Knockdown lines of FAB1A/B exhibit a root growth inhibition phenotype
In the previous study, the functions of FAB1A and FAB1B genes were analyzed using To examine the physiological function of FAB1A/B in vegetative tissues, we generated conditional knockdown mutant plants using the artificial micro RNA (amiRNA) technique (Alvarez et al., 2006;Niu et al., 2006;Schwab et al., 2006;Warthmann et al., 2008). AmiRNAs, which are normally absent in plants, are artificially designed 12 mer single-stranded RNAs. The amiRNAs specifically downregulate not only a single target, but also multiple protein coding genes having similar sequences (Schwab et al., 2006;Ossowski et al., 2008). We computed optimal amiRNA sequences for only attenuating FAB1A and were also shortened considerably compared with the uninduced condition (Fig. S1).
No growth inhibitions of root and root hair were observed in the control plant, which exogenously expressed GFP in the presence of estradiol (Figs. 3D, S1).

Reduction in FAB1A/B expression causes severe defects in vacuole acidification and endocytosis
Typical features of Fab1p/PIKfyve mutants in yeasts and animals are defective in vacuole acidification, fluid phase endocytosis, and formation of normal vacular/lysosomal structures (Gary et al., 1998;Odorizzi et al., 1998, McEwen et al., 1999Ikonomov et al., 2001). To elucidate whether the cellular phenotypes of knockdown and overexpressing mutants of FAB1A/B resemble those in yeast and animal cells, we assessed vacuole acidification and endocytosis using an acidification marker (acridine orange) and a fluorescent endocytosis marker (FM4-64).
Results show that conditional knockdowns of FAB1A and FAB1B ( were fully labeled with the dye (Fig. S3E).
These results show that the cellular phenotypes of Arabidopsis fab1 mutants are similar to those in other eukaryotic cells.

Auxin signaling phenotypes of knockdown and overexpression mutants of FAB1A/B
Reportedly, both fab1a/fab1a and fab1b/fab1b double homozygous mutant lines revealed a leaf curling phenotype in rosette leaves (Whitley et al., 2009). This leaf curling phenotype is known as a typical phenotype of the auxin-resistant mutants in Arabidopsis (Hobbie and Estelle, 1995). Therefore, we investigated whether the FAB1A/B knockdown and overexpression mutants can be expected to alter the sensitivity to exogenous auxin. First, we investigated auxin-dependent lateral root formation in these mutants, which is a typical auxin-responsive phenotype in Arabidopsis. The four-day-old seedlings grown on 1/2 MS agar plate were transferred on 1/2 MS plates with or without 0.1 μM 2,4-D. After day five, the numbers of lateral roots of the mutants were counted.
In the absence of estradiol, the lateral roots of the amiRNA plant (line 42 in  (Fig. 7D).
Next, we tested whether the root gravitropic response was changed when the expressions of FAB1A and FAB1B were increased or decreased conditionally. In the amiRNA lines (lines 41 and 42), treatment with 10 μM estradiol interfered strongly with the gravitropic response; particularly, the gravitropic response in line 42 was severely impaired, suggesting that root gravitropism is inhibited dose-dependently (Fig. 8, A-D).
Similarly, conditional overexpression lines of FAB1A and FAB1B revealed the root gravitropic phenotype in the presence of estradiol (Fig. 8, E-H). From these data, we concluded that auxin signaling is inhibited in both the loss-of-function and gain-of-function mutations in FAB1A/B.

Abnormal flower morphology of FAB1A and FAB1B overexpression mutants
The constitutive overexpression lines of FAB1A and FAB1B showed abnormal phenotypes in flower and leaf organ morphology. The phenotypes were categorized into six typical types based on morphology including an ovule-like structure that appeared inside of a curled sepal (Fig. 9A), sepals with a carpel-like structure (Fig.   9B), ovule-like structures in a curled leaf (Fig. 9C), a stamen fused to the sepal (Fig.   9D), papilla that emerged on the tip of the stamen (Fig. 9E), and three flowers branching from a single place (Fig. 9F). The abnormal morphology in flowers of cFAB1B-OX lines resembled that in the cFAB1A-OX lines (data not shown). These floral phenotypes suggest that floral organ identity genes might be misregulated dose-dependently in the FAB1A/B gain-of-function mutants.

Discussion
In an earlier study, the functions of FAB1A and FAB1B were analyzed using the T-DNA insertional mutation lines of both genes in Arabidopsis (Whitley et al., 2009) These defects are known as common phenotypes of fab1 mutation in yeast and animal cells (Gary et al., 1998;Odorizzi et al., 1998;McEwen et al., 1999;Ikonomov et al., 2001). Therefore, we conclude that the primary function of FAB1A and FAB1B is the endosome-resident PtdIns3P-5 kinase, FAB1/PIKfyve, in plants, just as it is in other eukaryotic organisms.

Why do loss-of-function and gain-of-function mutants reveal the same phenotypes on auxin signaling?
Because the double homozygous fab1a/fab1b knockout plants failed to generate any seeds because of a fatality of the pollen grains having mutations in both genes, it was impossible to analyze the biological function of FAB1A/B in the developmental process of the vegetative tissues in Arabidopsis despite the fact that only subtle leaf curling phenotype was observed in both fab1a and fab1b single homozygous plants, (Whitley et al., 2009).
To avoid that difficulty, we generated transgenic plants that were able to reduce the expressions of FAB1A/B conditionally by addition of a trace amount of estradiol.
We analyzed the phenotypes the conditional loss-of-function mutation in FAB1A/B. Atg18 (a FAB1 effector), and a scaffold protein, Vac14 (Duex et al., 2006;Sbrissa et al., 2007;Efe et al., 2007;Michell et al., 2009). All genes encoding Fab1 complex proteins, except Vac7, have also been found in the Arabidopsis genome. The enzymatic activity of Arabidopsis FAB1 is also likely to be regulated with these FAB1-regulatory proteins in a complex form. Therefore, a possible explanation of why the FAB1A/B loss-of-function and gain-of-function mutants show a similar phenotype is that an imbalance in the expression of FAB1A/B might inhibit proper complex formation of FAB1 with its regulatory proteins, thereby disrupting precise control of PtdIns (3,5)P 2 production in response to various environmental stresses.
Indeed, controlled expression of Arabidopsis FAB1A/B proteins in S. pombe ste12 mutant that was attained by adding various concentrations of thiamine altered the vacuolar shape of the mutant dose-dependently (data not shown). That result suggests that precise expression control of FAB1 gene is important for its function.

Plant growth conditions
Arabidopsis thaliana ecotype Columbia was used for all experiments described herein. Plants were grown under white light with 16-hour-light and 8-hour-dark cycles at 22°C.

Plasmid construction
Arabidopsis cDNAs were synthesized using AMV reverse transcriptase (Takara) from total RNA isolated from Arabidopsis seedlings using an RNeasy mini kit (Qiagen   Table S1.

Agrobacterium transformation and generating transgenic plants
The binary constructs were introduced into Agrobacterium tumefaciens strain GV3101 by electroporation; then Arabidopsis wild-type plants (Col-0) were transformed by the floral dipping method (Clough and Bent, 1998). Screening of transgenic plants was performed on 1/2 MS plates containing 50 μ g·mL -1 hygromycin.
The names and numbers of generating transgenic plants are also listed in Table S1.
Complementation assay in S. pombe www.plantphysiol.org on August 31, 2017 -Published by Downloaded from Copyright © 2010 American Society of Plant Biologists. All rights reserved.

FM4-64 staining
Yeast cells were harvested and then labeled with 2 μM of the endocytosis marker,

Acridine orange treatment
Acridine orange (Sigma-Aldrich Corp.) was added to 5-day-old seedlings of conditional loss-of-function and gain-of-function mutants to a final concentration of 50 μ M. After incubation at room temperature in the dark for 100 min, the seedlings were washed twice with water. They were then observed using confocal microscopy.

Confocal microscopy
GFP fluorescence signals and differential interference contrast (DIC) images were obtained using a laser scanning microscope (Eclipse E600; Nikon Instruments Co.) equipped with the C1si ready confocal system (Nikon). The collected images were processed using image analysis software (EZ-C1; Nikon).

Semi-quantitative RT-PCR
Total RNA was extracted from the transgenic lines. Then reverse transcription was performed using reverse transcriptase XL (Takara) using 2 μ g of total RNA as a template with an oligo-d(T) 20 and random primer mixture for 1 hr at 42°C. Of the reaction mixture, 1 μL was taken for a subsequent PCR reaction. The Arabidopsis