Arabidopsis AtVPS15 is essential for pollen development and germination through modulating phosphatidylinositol 3-phosphate formation
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Arabidopsis thaliana phosphatidylinositol 3-kinase (AtVPS34) functions in the development and germination of pollen by catalyzing the biosynthesis of phosphatidylinositol 3-phosphate (PI3P). In yeast, Vps15p is required for the membrane targeting and activity of Vps34. The expression of Arabidopsis thaliana VPS15 (AtVPS15), an ortholog of yeast Vps15, is mainly detected in pollen grains and pollen tubes. To determine its role in pollen development and pollen tube growth, we attempted to isolate the T-DNA insertion mutants of AtVPS15; however, homozygous lines of atvps15 were not obtained from the progeny of atvps15/+ heterozygotes. Genetic analysis revealed that the abnormal segregation is due to the failure of transmission of the atvps15 allele through pollen. Most pollen grains from the atvps15/+ genotype are viable, with normal exine structure and nuclei, but some mature pollen grains are characterized with unusual large vacuoles that are not observed in pollen grains from the wild AtVPS15 genotype. The germination ratio of pollen from the atvps15/+ genotype is about half when compared to that from the wild AtVPS15 genotype. When supplied with PI3P, in vitro pollen germination of the atvps15/+ genotype is greatly improved. Presumably, AtVPS15 functions in pollen development and germination by regulating PI3P biosynthesis in Arabidopsis.
KeywordsAtVPS15 Pollen germination Vacuole Phosphatidylinositol 3-phosphate Arabidopsis
In higher plants, pollen grains hydrate, germinate, and protrude pollen tubes on the compatible stigma which then grow into the style towards the ovules. The two sperm cells move in the growing pollen tube to enter the female gametophyte for completion of the process of double fertilization. Failure of pollen germination or pollen tube formation will abort fertilization and further seed development. Signaling molecules and genes required for pollen germination and pollen tube growth have been identified in plants (Hepler et al. 2001; Johnson and Preuss 2002; McCormick 2004; Ge et al. 2010). Phospholipids, comprising about 1% of membrane lipids, play critical roles in regulating plant development and environment response (Xue et al. 2009). Phosphatidylinositol 3-phosphate (PI3P) is one type of phospholipid involved in receptor signaling and membrane/vesicle trafficking (Petiot et al. 2003; Roth 2004). In plant cells, PI3P is synthesized in the trans-Golgi/endosome network and transported to the vacuole through the prevacuolar compartment (Kim et al. 2001). As an essential component of plant growth, PI3P signaling functions in diverse physiological processes such as pollen development, germination, and pollen tube growth (Lee et al. 2008a; Xue et al. 2009; Zhang and McCormick 2010).
Yeast Vps34 is a phosphatidylinositol 3-kinase (PI3K) catalyzing phosphoinositide to PI3P (Herman and Emr 1990). Arabidopsis AtVPS34, a class III PI3K, is an ortholog of yeast Vps34 and is involved in the PI3P synthesis. Knockdown of AtVPS34 results in an embryo lethal phenotype in transgenic Arabidopsis (Welters et al. 1994). In root cells, the mutant of pi3K/AtVPS34 shows reduced endocytosis and reactive oxygen species production under salt stress (Leshem et al. 2007). Pollen grains of the T-DNA insertion mutants of AtVPS34 are sterile and are characterized by abnormal large vacuoles and a reduced number of nuclei (Lee et al. 2008b). In yeast, Vps34 forms a class III PI3K complex with Vps15p and Vps30 (ATG6) (Kihara et al. 2001). Yeast Vps15p is a Ser/Thr protein kinase containing HEAT repeats within its central region and a series of WD-40 domains within its C-terminus (Stack et al. 1993). Vps15p is required for the membrane targeting and activity of Vps34 (Yan and Backer 2007). The ArabidopsisAtVPS15 (At4g29380) was identified as the putative ortholog of yeast Vps15 (Mueller-Roeber and Pical 2002); however, its function in Arabidopsis development is unknown. In this study, we found that AtVPS15 was mainly detected in anthers. The pollen of the AtVPS15 mutants is defective in vacuolar morphology and pollen germination, but an exogenous supply of PI3P could restore pollen germination of mutants. Apparently, AtVPS15 regulates the formation of PI3P and plays an essential role during the development and germination of pollen grains.
AtVPS15 appears to be an ortholog of yeast Vps15p and human hVps15
Transcription of AtVPS15 and subcellular localization of AtVPS15
The atvps15 mutation reduces male gametophyte transmission
Segregation analysis of selfed progeny of atvps15/+ mutants
Analysis of the genetic transmission of atvps15 alleles
Genotype (Female × Male)
atvps15-1/+ × Col-0
WT × atvps15-1/+
atvps15-2/+ × WT
WT × atvps15-2/+
Pollen grains carrying the atvps15 mutation are defective for pollen germination
The atvps15 mutation affects vacuolar organization of pollen
Population of mature pollen with abnormal vacuoles of atvps15/+
13.84 ± 0.48% (15/123)*
22.41 ± 2.51% (17/94)*
Exogenous PI3P supply restores pollen germination of the atvps15 mutants
Phosphoinositides function in membrane trafficking and polarized growth of pollen tube (Monteiro et al. 2005; Samaj et al. 2006; Zonia and Munnik 2008; Ischebeck et al. 2008; Sousa et al. 2008). In yeast, Vps15p plays its role by forming a class III PI3K complex with Vps34 and Vps30. Vps34 has lipid kinase activity and functions in PI3P synthesis (Herman and Emr 1990). In Arabidopsis, the atvps34 mutation impairs the production of PI3P and results in failure of pollen germination (Lee et al. 2008b). In yeast, Vps15 is required for membrane targeting and function of Vps34 (Stack et al. 1993). Our results revealed that AtVPS15 is important for pollen development and germination, and further that an exogenous supply of PI3P improves the pollen germination of the atvps15 mutants. Due to the close phenotype between the atvps15 mutants and the atvps34 mutants, AtVPS15 potentially interacts with AtVPS34 and regulates AtVPS34 activity and PI3P synthesis in vivo, as in yeast (Stack et al. 1993). Additionally, AtAtg6 (AtVPS30) plays roles in pollen germination of Arabidopsis. The mutation of AtAtg6 leads to failure of pollen germination (Fujiki et al. 2007; Qin et al. 2007; Harrison-Lowe and Olsen 2008). PI3K complex is involved in autophagy and vacuolar protein sorting (VPS) in yeast (Yorimitsu and Klionsky 2005). However, whether the Arabidopsis Atg-regulated autophagy plays a role in pollen germination remains unknown (Fujiki et al. 2007; Qin et al. 2007; Harrison-Lowe and Olsen 2008).
In Arabidopsis, PI3K can phosphorylate the phosphatidylinositol at the D-3 position to generate the PI3P, and then the PIKfyve/Fab1 family will phosphorylate the PI3P at the D-5 position to produce the phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) (Cantley 2002). Arabidopsis FAB1A and FAB1B are orthologs of the PIKfyve/Fab1 family proteins. The Arabidopsisfab1a/fab1b mutants are defective in pollen development and germination, which may be caused by the decrease of the PI(3,5)P2 level in pollen (Whitley et al. 2009). ATVPS15 may affect the PI(3,5)P2 synthesis in pollen by regulating ATVPS34 activity and the PI3P level. The similar vacuolar phenotype of atvps15, atvps34 and fab1a/fab1b mutants provides an additional line of evidence. Studies in non-plant cells revealed that PI(3,5)P2 mediates the fission of vacuoles (or its equivalent organelles) by increasing the surface-to-volume ratio of intracellular organelles (Weisman 2003; Sbrissa and Shisheva 2005). Although the atvps15, atvps34, and fab1a/fab1b mutants are similar in pollen germination, they differ in other stages during the pollen development. The atvps34 and fab1a/fab1b mutants associate with reduced pollen viability, but some pollen grains of fab1a/fab1b mutant are collapsed; however, atvps15 pollen had normal viability and morphology. Additionally, the atvps15 mutants associate with the largest fraction of pollen with normal vacuoles, while the fab1a/fab1b mutant has the least fraction of pollen with normal vacuoles. The different levels of PI(3,5)P2 in these mutants may lead to different phenotypes in pollen development. How ATVPS15, ATVPS34, and FAB1A/FAB1B contribute to the PI(3,5)P2 accumulation in pollen is likely conditioned by their position in the PI(3,5)P2 metabolism pathway. FAB1A/FAB1B directly catalyzes the production of PI(3,5)P2 and the fab1a/fab1b mutations cause significant changes in PI(3,5)P2 level. ATVPS15 may only cause minor change in PI(3,5)P2 level since the ATVPS15 controls the PI3P accumulation by regulating the ATVPS34 activity. Alternatively, AtVPS15, AtVPS34, and FAB1A/FAB1B may be involved in other pathways that regulate pollen development and germination.
Materials and methods
Two T-DNA insertion lines of Arabidopsisthaliana (Col-0 ecotype) of AtVPS15 (GABI_015F04 and SALK_004719) were obtained from NASC and ABRC, respectively. The genotypes of T-DNA insertion line plants were determined by a PCR-based method. The following primers were used: LB1 (5′-ATATTGACCATCATACTCATTG-3′), LP1 (5′- CTCCGTCTGATCACTCAAAGC -3′), and RP1 (5′-CAATGAACGCAAGCACAGTAC-3′) for GABI_015F04 and LB2 (5′-ATTTTGCCGATTTCGGAAC-3′), LP2 (5′-GAATGATTTTGCAGCTTCTGG-3′), and RP2 (5′-CAAGAGAATCGATGCGAAAAG-3′) for SALK_004719. The wild-type Arabidopsis and T-DNA insertion plants were grown at 22°C in a 16/8 h light/dark cycle.
Pollen analysis and microscopy
For pollen staining, mature pollen grains or the anthers were used. The mature anthers were soaked in Alexander stain for 1–2 days to detect pollen viability according to Alexander (1969) under a BX51 microscope (Olympus, www.olympus.com). To observe the exines of pollen grains, we used 10 μg/ml (w/v) propidium iodide (Sigma, www.sigmaaldrich.com) to stain mature pollen grains followed by observation under Zeiss CLSM 510 meta (www.zeiss.com), according to Tian et al. (2006). The nuclei of pollen grains were stained with 1 μg/ml (w/v) DAPI (Sigma, www.sigmaaldrich.com) for 30 min and observed by epifluorescence microscopy (Olympus BX51 microscope). For the sections of anthers and pollen grains, the materials were embedded in Spurr resin and after-fixed with a solution of 4% (w/v) glutaraldehyde and 1% (w/v) OsO4 in phosphate buffer (pH 7.2). The semi-thick sections were stained with 0.5% (w/v) toluidine blue-O (Sigma, www.sigmaaldrich.com) and observed with an Olympus BX51 microscope.
Pollen germination in vitro
To analyze pollen germination in vitro, the pollen grains at anthesis of wild-type Arabidopsis and T-DNA insertion lines were collected and cultured on medium containing 1 mM CaCl2, 1 mM Ca(NO3)2, 1 mM MgSO4, 0.01% (w/v) H3BO3, and 18% (w/v) sucrose solidified with 0.5% (w/v) agar (pH 7.0). To analyze the role of PIP3 in pollen germination, PIP3 (PtdIns(3)P di-C16, P-3016) and its carrier (Carrier 3, P-9C3) (Echelon Biosciences Inc, www.echelon-inc.com) were added into the pollen germination medium according to the manufacturer’s recommendation. In medium, the ratio of PIP3 and carrier is 1:1 to form the PIP3–carrier complex at a final concentration of 6.25 × 10−3 mg/ml. The pollen grains were cultured immediately at 28°C, 100% relative humidity, and cool light at 30 mmol/m2s. The germinated pollen grains were counted and images were captured at 4 h after germination.
Quantitative Real-time PCR (qRT-PCR)
The Arabidopsis seedlings of 3-week-old (for seedling, root and leaf) and 5-week-old (for stem, flower, anther, pistil and silique) were collected for AtVPS15 expression analysis. Total RNA was isolated with the Trizol isolation reagent (Invitrogen, www.invitrogen.com) for reverse transcription. qRT-PCR was conducted to determine AtVPS15 transcript levels with the primer pairs VPS15RT-sense (5′-GGAAGTTACCAAGCAATAAGG-3′) and VPS15RT-antisense (5′- AGAGCGGATACCAGGAAG -3′). Tubulin was used as a quantitative control and amplified using the primers, TUB2-RT-sense (5′-ATCCGTGAAGAGTACCCAGAT-3′) and TUB2-RT-antisense (5′-AAGAACCATGCACTCATCAGC-3′). The line arrangement of the PCR products was established by comparing samples that were run for different numbers of cycles. All experiments were repeated three times.
Transient expression YFP fusion protein in tobacco pollen tubes
Transient expression of YFP-AtVPS15 fusion protein in pollen tubes of tobacco was used to study the intracellular localization of AtVPS15. The full-length cDNA of AtVPS15 was amplified by PCR using GATEWAY-compatible primers VPS15-sense (5′-CACCATGGGAAACAAAATCGCTCGTACGACAC-3′) and VPS15-antisense (5′-TTACTTCCAGACCTTTATGGCTCCATCT-3′) and cloned into the pENTY/D-TOPO vector (Gateway, Invitrogen, www.invitrogen.com) for sequencing, and then recombined into pAL20 to create Lat52::YFP-AtVPS15 construct. The Lat52::YFP construct was used as the control. Each construct was introduced into tobacco (Nicotiana tabacum) pollen using a pneumatic particle gun (PDS-1000/He; Bio-Rad, www.bio-rad.com), as described by Zhang and McCormick (2007). After 4–8 h cultivation on medium, the pollen grains were visualized under a fluorescence microscope (Olympus BX51).
RNA in situ hybridization
RNA in situ hybridization was performed as previously described by Li et al. (2010). The specific AtVPS15 probe is a 570-bp fragment amplified in the coding region of AtVPS15 with the primers VPS15-IF (5′-TCACAAGGGAGGTAGTTTATCG-3′) and VPS15-IR (5′-GCTTAGGCACTGGGACCGAACT-3′). The samples were observed using an Olympus BX51 microscope.
For the observation of pollen morphology, mature pollen grains coated with gold palladium were observed under a JSM-6610LV scanning electron microscope (JEOL, Japan) and photographed. Transmission electron microscopy was performed as described by Wang et al. (2009), with modification. The mature anthers were fixed in 4% (v/v) glutaraldehyde and 1% (w/v) osmic acid (Sigma). After washing, the specimens were dehydrated in a graded ethanol series and embedded in Epon 812 resin (SPI-CHEM, USA). Ultrathin sections were stained with acetate uranium and lead citrate. The specimens were observed using a JEM-1200EX transmission electron microscope (JEOL, Japan).
We thank Dr. Yan Zhang (Shandong Agricultural University) for her helpful discussions and providing pAL20 vector, and Dr. Sharman D. O’Neill (University of California, Davis, USA) for her critical reading for the manuscript. We thank Mr. Yan-Kui Guo (Shandong Agricultural University) for his kind help with electron microscopy. We are also grateful to the Arabidopsis Biological Resource Center and the European Arabidopsis Stock Centre for kindly providing the Arabidopsis mutant seeds. This study is funded by the Major Research Plan from the Ministry of Science and Technology of China (No. 2007CB947600).
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