Accepted Article
Received Date: 12-Aug-2014 Accepted Date: 31-Mar-2015 Article Type: Original Article
Enzyme activities of Arabidopsis inositol polyphosphate kinases AtIPK2α and AtIPK2β are involved in pollen development, pollen tube guidance and embryogenesis
Huadong Zhan1, Yujiao Zhong1, Zhongnan Yang2, and Huijun Xia1* 1
State Key Laboratory of Hybrid Rice, College of Life Sciences, Wuhan University, Wuhan,
Hubei 430072, China 2
College of Life and Environment Sciences, Shanghai Normal University, Shanghai 200234,
China
*For correspondence: Huijun Xia
Email: [email protected]; Tel and Fax: +86-27-68752112
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an 'Accepted Article', doi: 10.1111/tpj.12846 This article is protected by copyright. All rights reserved.
Accepted Article
Running title: AtIPK2 in male gametophyte and embryogenesis
Keywords: inositol polyphosphate kinase, AtIPK2α and AtIPK2β, kinase activity, pollen development, pollen tube guidance, embryogenesis, Arabidopsis thaliana
SUMMARY
Inositol polyphosphate kinase (IPK2) is a key component of inositol polyphosphate signaling. There are two highly homologous inositol polyphosphate kinases (AtIPK2α and AtIPK2β) in Arabidopsis. Previous studies that over-expressed or reduced expression of AtIPK2α and AtIPK2β had suggested roles in auxiliary shoot branching, abiotic stress responses and root growth. Here, we report that AtIPK2α and AtIPK2β act redundantly during pollen development, pollen tube guidance and embryogenesis. Single knockout mutants of atipk2α and atipk2β were indistinguishable from the wild-type, whereas atipk2αatipk2β double mutant could not be obtained. Detailed genetic and cytological investigations showed that mutation of AtIPK2α and AtIPK2β resulted in severely reduced transmission of male gametophyte due to abnormal pollen development and defective pollen tube guidance. In addition, the early embryo development of atipk2αatipk2β double mutant was also aborted. Expressing either catalytically inactive or substrate specificity-altered variants of AtIPK2β could not rescue the male gametophyte and embryogenesis defects of atipk2αatipk2β double mutant, implying that the kinase activity of AtIPK2 is required for pollen development,
This article is protected by copyright. All rights reserved.
Accepted Article
pollen tube guidance and embryogenesis. Taken together, our results provide genetic evidence for the requirement of inositol polyphosphate signaling in plant sexual reproduction.
INTRODUCTION
Male gametophyte plays a pivotal role in plant sexual reproduction. In angiosperms, successful double fertilization depends on formation of male gametophytes within the anther, which fuse with the egg cell and the central cell to give rise to the embryo and the endosperm (McCormick, 1993). Pollen tube is a highly specialized cell structure used to deliver the male gametes. After adhesion and germination on a compatible stigma, pollen tube first penetrates the stigma and style, then grows through the transmitting tissue, migrates along the septum to the funiculus, and finally enters the micropyle (Hülskamp et al., 1995; Márton and Dresselhaus, 2010). To achieve this highly precise and directional growth in pistil, pollen tubes need extensive cell communication with the female tissues and adjust their tip growth in response to guidance cues (Cheung et al., 2010; Palanivelu and Tsukamoto, 2012). In the past decade, considerable progresses have been made in understanding the female signals that guide the pollen tube (Márton et al., 2005; Okuda et al., 2009; Takeuchi and Higashiyama, 2012). Until recently, two receptor-like kinases (RLKs) preferentially expressed in the apical plasma membrane of pollen tube, LIP1 and LIP2 (Lost In Pollen Tube Guidance1 and 2) were demonstrated to be the essential components of the receptor complex in micropylar guidance (Liu et al., 2013). However, the knowledge about the intracellular signaling events in pollen tube in response to the guidance cues is still scarce.
This article is protected by copyright. All rights reserved.
Accepted Article
Amino acid signaling to mTOR mediated by inositol polyphosphate multikinase. Cell Metab. 13, 215-221 Krinke, O., Novotna, Z., Valentova, O. and Martinec, J. (2007) Inositol trisphosphate receptor in higher plants: is it real? J. Exp. Bot. 58, 361-376. Lemtiri-Chlieh F., MacRobbie E.A., Webb A.A., Manison N.F., Brownlee C., Skepper J.N., Chen J., Prestwich G.D. and Brearley C.A. (2003) Inositol hexakisphosphate mobilizes an endomembrane store of calcium in guard cells. Proc. Natl. Acad. Sci. USA. 100, 10091-10095. Li, H., Lin, Y.K., Heath, R.M., Zhu, M.X. and Yang, Z.B. (1999) Control of pollen tube tip growth by a Rop GTPase-dependent pathway that leads to tip-localized calcium influx. Plant Cell 11, 1731-1742. Li, H.J., Xue, Y., Jia, D.J., Wang, T., Hi, D.Q., Liu, J., Cui, F., Xie, Q., Ye, D. and Yang, W.C. (2011) POD1 regulates pollen tube guidance in response to micropylar female signaling and acts in early embryo patterning in Arabidopsis. Plant Cell, 23, 3288-3302. Liu, C.M. and Meinke, D.W. (1998) The titan mutants of Arabidopsis are disrupted in mitosis and cell cycle control during seed development. Plant J.16, 21-31. Liu, J.J., Zhong, S., Guo, X.Y., Hao, L.H., Wei, X.L., Huang, Q.P., Hou, Y.N., Shi, J., Wang, C.Y., Gu, H.Y. and Qu, L.J. (2013) Membrane-bound RLCKs LIP1 and LIP2 are essential male factors controlling male-female attraction in Arabidopsis. Curr. Biol. 23, 993-998. Lu, Y., Chanroj, S., Zulkifli, L., Johnson, M., Uozumi, N., Cheung, A. and Sze, H. (2011) Pollen tubes lacking a pair of K+ transporters fail to target ovules in Arabidopsis. Plant Cell 23, 81-174. Maag, D., Maxwell, M.J., Hardesty, D.A., Boucher, K.L., Choudhari, N., Hanno, A.G., Ma, J.F., Snowman, A.S., Pietropaoli, J.W., Xu, R., Storm, P.B., Saiardi, A., Snyder,
This article is protected by copyright. All rights reserved.
Accepted Article
bisphosphate to phosphatidylinositol (3,4,5)-trisphosphate (Resnick et al., 2005; Maag et al., 2011). Moreover, IPK2 can physically interact with and physiologically regulate target of rapamycin (TOR) and AMP-activated protein kinase (AMPK) in mammals (Kim et al., 2011; Bang et al., 2012). Overall, these results suggest that IPK2 is a multifunctional protein.
The inositol polyphosphate kinase in higher plants was first identified from Arabidopsis thaliana (Stevenson-Paulik et al., 2002; Xia et al., 2003). Genome survey and phylogenetic analysis reveal that there are two highly homologous inositol polyphosphate kinases (AtIPK2α and AtIPK2β) in Arabidopsis. The two AtIPK2 proteins share over 70% amino acid identity and exhibit identical 6/3 dual-specificity kinase activities, which sequentially phosphorylate inositol 1,4,5-trisphosphate to generate inositol 1,4,5,6-tetrakisphosphate and inositol 1,3,4,5,6-pentakisphosphate, like their yeast counterpart (Stevenson-Paulik et al., 2002; Xia et al., 2003). Downregulation of AtIPK2α through antisense inhibition has been shown to result in enhanced root growth and pollen germination (Xu et al., 2005), while AtIPK2β functions in auxiliary shoot branching through the auxin signaling pathway (Zhang et al., 2007). Moreover, AtIPK2β is a stress-responsive gene and its heterologous expression in tobacco lead to improved tolerance to diverse abiotic stresses (Yang et al., 2008).
The presence of two highly homologous inositol polyphosphate kinases in Arabidopsis imposes a major obstacle for defining their biological functions. In this study, through double mutant analysis, we report that AtIPK2α and AtIPK2β play a redundant and essential role in This article is protected by copyright. All rights reserved.
Accepted Article
pollen development, pollen tube guidance and embryogenesis. Moreover, we investigate the correlation between the kinase activity and function of AtIPK2 in male gametophyte and embryogenesis. Our results indicate that the kinase activity of AtIPK2 is essential for pollen development, pollen tube guidance and embryogenesis, implying the requirement of intracellular messenger inositol polyphosphates in plant sexual reproduction.
RESULTS
The male gametophyte transmission of atipk2αatipk2β double mutant is severely reduced
AtIPK2α and AtIPK2β are two closely related inositol polyphosphate kinases, sharing over 70% amino acid identity and possessing identical 6/3-kinase activities (Stevenson-Paulik et al., 2002; Xia and Yang, 2005). We obtained T-DNA insertion mutants for them. The GABIKat line (GABI_879D07, named atipk2α) contains a T-DNA insertion in the exon of AtIPK2α (Figure 1a, b). It is a null allele as AtIPK2α transcripts were not detected in this mutant (Figure 1b). The atipk2β RNA-null mutant (SALK_104995) used in this study was originally described by Zhang et al. (2007) and examined by genotyping-PCR with genespecific primers again here (Figure 1a, c). Both atipk2α and atipk2β are in the Columbia background (Col-0). No visible phenotypes were observed for atipk2α and atipk2β single mutant plants (Figure S1).
This article is protected by copyright. All rights reserved.
Accepted Article
Crosses between atipk2α and atipk2β were performed to generate double mutant. AtIPK2α and AtIPK2β are located at opposite ends of chromosome 5, which ensures a high frequency of recombination between the two loci. However, of over 200 F2 plants, no homozygous double mutant was identified. From the F2 population, we identified the lines of atipk2α/atipk2α;AtIPK2β/atipk2β (aaBb) and AtIPK2α/atipk2α; atipk2β/atipk2β (Aabb) plants. Once again no homozygous double mutant could be obtained from the self-fertilized progeny of aaBb and Aabb plants (Table 1). Moreover, when atipk2α/atipk2α;AtIPK2β/atipk2β or AtIPK2α/atipk2α;atipk2β/ atipk2β was pollinated with wild-type pollen, approximately 50% progeny contained double mutant alleles (Table 2). This suggests that atipk2αatipk2β was transmitted normally through the female parent. However, when atipk2α/atipk2α;AtIPK2β/ atipk2β or AtIPK2α/atipk2α;atipk2β/atipk2β plants were used as pollen donors, severe reduction in co-transmission of atipk2α and atipk2β was observed (Table 2). These results suggested that mutation of AtIPK2α and AtIPK2β significantly reduced the genetic transmission of male gametophyte, but did not affect the female gametophyte function.
To confirm that this abnormal segregation of atipk2αatipk2β double mutant is caused by insertions in inositol polyphosphate kinase genes, genetic complementation experiments were performed. Constructs encoding AtIPK2α/AtIPK2α-GFP or AtIPK2β/AtIPK2β-GFP under the control of their native promoters were introduced into atipk2α/atipk2α;AtIPK2β/atipk2β (aaBb) or AtIPK2α/atipk2α;atipk2β/atipk2β (Aabb) plants respectively via Agrobacterium tumefaciens-mediated transformation (Figure 1d). Homozygous This article is protected by copyright. All rights reserved.
Accepted Article
atipk2α/atipk2α;atipk2β/atipk2β (aabb) double mutants were recovered by these complementary transgenes as demonstrated by PCR genotyping (Figure 1e). Moreover, the segregation of atipk2αatipk2β double mutant alleles was restored in atipk2α/atipk2α;AtIPK2β/atipk2β and AtIPK2α/atipk2α; atipk2β/atipk2β plants carrying the homozygous AtIPK2α or AtIPK2β transgene (Table 3). These results suggested that the lethality of atipk2αatipk2β double mutant was caused by T-DNA insertions in AtIPK2 loci. It also indicated that the C-terminally fused AtIPK2α-GFP and AtIPK2β-GFP were functional.
The pollen development of atipk2αatipk2β double mutant is partially compromised
Male gametophyte transmission defect could be caused by defects in pollen viability, germination, pollen tube growth and/or fertilization. In order to further understand the transmission defect of atipk2αatipk2β, we first examined the pollen viability using Alexander’s staining (Alexander, 1969). Almost all pollen from atipk2α and atipk2β single homozygous mutants appeared full, round and red-stained, which was similar to mature pollen grains of wild-type (Figure 2a-c, t). In contrast, 11.6% pollen from atipk2α/atipk2α;AtIPK2β/atipk2β (aaBb) anthers and 13.2% pollen in AtIPK2α/ atipk2α;atipk2β/atipk2β (Aabb) anthers were completely or partially devoid of cytoplasmic contents as indicated by the reduced staining (Figure 2d, e and t). Moreover, this abnormal pollen viability staining could be restored in the homozygous atipk2αatipk2β double mutant plants carrying the AtIPK2β-GFP complementary transgene (aabb/AtIPK2β-GFP) (Figure 2f, t). Thus, the defective pollen grains observed in atipk2α/atipk2α;AtIPK2β/atipk2β and
This article is protected by copyright. All rights reserved.
Accepted Article
AtIPK2α/atipk2α; atipk2β/atipk2β plants were most likely the atipk2αatipk2β double mutant. Considering that 50% pollen grains produced by atipk2α/atipk2α;AtIPK2β/atipk2β or AtIPK2α/atipk2α;atipk2β/atipk2β plants carry the double mutant alleles, then approximately 20% atipk2αatipk2β double mutant pollen grains have compromised viability. As aborted pollen grains often have an abnormal shape, we further investigated the pollen morphology by scanning electron microscopy (SEM). Consistent with the viability staining result, pollen grains from atipk2α/atipk2α; AtIPK2β/atipk2β and AtIPK2α/atipk2α;atipk2β/atipk2β plants frequently collapsed or possessed distorted morphology (Figure 2g-l). The 4’,6-diaminophenylindole (DAPI) staining showed that almost all mature pollen grains collected from wild-type, atipk2 single mutant and complementary aabb/AtIPK2β-GFP plants had two brightly stained sperm nuclei and one faint vegetative nucleus (2s+1v) (Figure 2m-r, u). By contrast, about 10% pollen grains produced by atipk2α/atipk2α;AtIPK2β/atipk2β and AtIPK2α/ atipk2α;atipk2β/atipk2β plants were abnormally stained (Figure 2m-r, u), including one generative nucleus and one vegetative nucleus (1s+1v), only one nucleus (1v) and no discernible nucleus (Figure 2s).
The defective development of atipk2αatipk2β double mutant pollen were characterized in more detail by introgressing AtIPK2α/atipk2α;atipk2β/atipk2β into quartet1 mutant (qrt1-6, Col-0 background). In qrt1-6, four products of male meiosis fail to separate, resulting in a characteristic pollen quartet structure (Preuss et al., 1994). Pollen from qrt1-6 mutant (AABBqq) and AtIPK2α/AtIPK2α;atipk2β/ atipk2β;qrt1-6/qrt1-6 (AAbbqq) plants always produced quartets in which all four pollen grains were viable (Figure S2). By contrast, over This article is protected by copyright. All rights reserved.
Accepted Article
20% pollen quartets in AtIPK2α/atipk2α;atipk2β/atipk2β;qrt1-6/qrt1-6 (Aabbqq) plants had one or two defective pollen grains (Figure S2). Quartets contained more than two aberrant pollen grains were not observed in Aabbqq, suggesting that the function of AtIPK2 in pollen formation is likely restricted to gametophyte.
The pollen tube guidance of atipk2αatipk2β double mutant is severely impaired
As the majority of atipk2 double mutant pollen was viable, we further investigated the pollen germination and pollen tube growth of atipk2αatipk2β double mutant. Consistent with the partial defect of atipk2αatipk2β double mutant pollen, we could observe small-size and ungerminable pollen grains on germination medium when pollen form atipk2α/atipk2α;AtIPK2β/atipk2β and AtIPK2α/atipk2α;atipk2β/atipk2β plants were incubated (Figure 3a-f). In vitro analysis revealed that pollen grains from atipk2α/atipk2α;AtIPK2β/atipk2β and AtIPK2α/atipk2α;atipk2β/atipk2β plants germinate slightly slower than that of the controls. After incubation in growth medium for 6 h, about 73.2% and 72.6% pollen from aaBb and Aabb plants germinated, compared with the approximately 80% of the various controls (Figure 3s). Considering that ~20% double mutant pollen were defective, over 70% pollen germination rate for aaBb and Aabb plants indicated a competitive germination for viable atipk2αatipk2β double mutant pollen grains. The pollen tube growth was also investigated by pollinating the pre-emasculated wild-type stigmas with limited wild-type, atipk2 mutant or aabb/AtIPK2β-GFP pollen. The majority of pollen tubes from aaBb and Aabb plants (approximately 85%, n>300) were able to penetrate the stigma
This article is protected by copyright. All rights reserved.
Accepted Article
and style, and grow in the transmitting tract at a comparable rate with wild-type and single mutant controls (Figure 3g-r, t). Thus, the germination and tube growth of viable atipk2αatipk2β pollen were competent.
We further observed the path of pollen tube growth in pistils to investigate whether the pollen tube guidance of atipk2αatipk2β was compromised. To do this, wild-type stigmas were pollinated with various pollen grains, then fixed and stained with aniline blue to follow the route of pollen tubes. The wild-type, atipk2 single mutant and complementary aabb/AtIPK2βGFP pollen tubes were observed to grow on the surface of the funiculus after leaving the transmitting tissue and entered the micropyle smoothly and precisely (Figure 4a-c, f and g). However, when wild-type stigmas pollinated with pollen grains from atipk2α/atipk2α;AtIPK2β/atipk2β or AtIPK2α/ atipk2α;atipk2β/atipk2β plants, disordered patterns of pollen tube directional growth were observed (Figure 4d, e and h-j). The defective pollen tube phenotypes could be divided into two categories: (1) pollen tubes failed to shift their directional growth to the funiculus. About 27.6% (n>300) pollen tubes of atipk2α/atipk2α;AtIPK2β/atipk2β plants were found without approaching the funiculus in limited pollination (Figure 4k). (2) Pollen tubes grew randomly toward the micropyle after adhering to the funiculus, including growing on the ovule surface, or initially twisting around the micropyle and finally entering the micropyle. Approximately 12.3% (n>300) pollen tubes from atipk2α/atipk2α;AtIPK2β/atipk2β plants appeared to lose their way towards the micropyle in limited pollination (Figure 4k). These observations indicated that the pollen tube guidance of atipk2αatipk2β was disrupted. This article is protected by copyright. All rights reserved.
Accepted Article
AtIPK2α and AtIPK2β are important for embryogenesis
Considering that the male gametophyte defect in atipk2αatipk2β double mutant was not fully penetrant, homozygous atipk2α/atipk2α;atipk2β/atipk2β should be obtained from the progeny of self-fertilized atipk2α/atipk2α;AtIPK2β/atipk2β (aaBb) and AtIPK2α/atipk2α;atipk2β/atipk2β (Aabb) plants, and the segregation of aaBB:aaBb and AAbb:Aabb should be between 1:1 and 1:2. However, no atipk2αatipk2β double mutant plant was identified in PCR genotype screens, and the segregation of aaBB:aaBb and AAbb:Aabb was found to be 1:1 (Table 1). Therefore, in addition to the lethality of atipk2αatipk2β double mutant, the development of atipk2α/atipk2α; AtIPK2β/atipk2β and AtIPK2α/atipk2α;atipk2β/atipk2β embryos should be also partially aborted. To test these hypotheses, the seed set of self-pollinated atipk2α/atipk2α;AtIPK2β/atipk2β and AtIPK2α/atipk2α;atipk2β/atipk2β plants was investigated. As shown in Figure 5a, siliques from atipk2α/atipk2α;AtIPK2β/atipk2β and AtIPK2α/atipk2α;atipk2β/atipk2β plants had a variety of defective seeds, ranging from the highly degenerated seeds to discolored seeds. To verify the genotype of the aborted seeds, embryos embedded in the green and white seeds were isolated from atipk2α/atipk2α;AtIPK2β/atipk2β plants (Figure 5b). Single-embryo PCR was carried out with allele-specific primers. Among the 31 green seeds, 14 were atipk2α/atipk2α;AtIPK2β/AtIPK2β and 17 were atipk2α/atipk2α;AtIPK2β/atipk2β. While all the 15 pale embryos derived from the white seeds were found to be atipk2α/atipk2α;AtIPK2β/atipk2β. This result suggested that atipk2α/atipk2α; AtIPK2β/atipk2β could develop into either normal or abnormal seeds. Moreover, the absence of atipk2α/atipk2α;atipk2β/atipk2β in the examined white seeds indicated that the This article is protected by copyright. All rights reserved.
Accepted Article
atipk2αatipk2β double mutant embryos died at early embryonic stage, before visible seeds were formed. To characterize the process of seed abortion, developing seeds from self-pollinated atipk2α/atipk2α;AtIPK2β/atipk2β and AtIPK2α/atipk2α;atipk2β/atipk2β siliques were cleared and visualized using differential interference contrast (DIC) microscopy. When most of the sibling embryos reached the torpedo stage at 6-7 d after pollination (Figure 5c), 13 to 15% embryos were found to be retarded at the heart or earlier stages within the same silique (Figure 5d-m, q). This strongly asynchronous embryo stages supported the early embryo abortion. Consistent with this, aborted embryos could be seen as early as at the first and second round of cell division in both atipk2α/atipk2α;AtIPK2β/atipk2β and AtIPK2α/atipk2α;atipk2β/atipk2β siliques, which finally developed into the highly degenerated seeds (Figure 5d-f). Intriguingly, aberrant embryos with elongated suspensor could be observed in atipk2α/atipk2α; AtIPK2β/atipk2β plants in some cases (Figure 5k-m). The cell divisions in these elongated suspensors appeared to be normal, but the longitudinal cell expansion was dramatically increased (Figure 5k-m). This long-suspensor phenotype was only observed in atipk2α/atipk2α;AtIPK2β/atipk2β silique, suggesting that they were likely atipk2α/atipk2α;AtIPK2β/atipk2β rather than the atipk2α/atipk2α;atipk2β/ atipk2β double mutant.
In addition to the abnormalities described above, embryos that progressed to the torpedo stage but with reduced size or asymmetrical cotyledonary lobes were also observed (Figure 5n, o). Statistically, 4 to 6% embryos were found with this delayed development in atipk2α/atipk2α;AtIPK2β/atipk2β and AtIPK2α/atipk2α;atipk2β/ atipk2β siliques (Figure 5q). This article is protected by copyright. All rights reserved.
Accepted Article
These delayed embryos should be able to develop into mature seeds, as seedlings with smaller and asymmetrical cotyledons could be found in selfed offspring of atipk2α/atipk2α;AtIPK2β/atipk2β and AtIPK2α/atipk2α; atipk2β/atipk2β plants (Figure 5p, r). Molecular characterization of these cotyledon-abnormal seedlings derived from the selfpollinated atipk2α/atipk2α; AtIPK2β/atipk2β plants revealed that nearly 90% (27 of 31) of them were with the genotype of atipk2α/atipk2α;AtIPK2β/atipk2β.
Taken together, these results demonstrate that AtIPK2α and AtIPK2β are important for embryo development.
Kinase activity of AtIPK2 is required for the male gametophyte function and embryogenesis
As described above, AtIPK2α and AtIPK2β are essential for male gametophyte and embryogenesis. However, whether the function of AtIPK2 in these processes is associated with their kinase activity is completely unknown. To confirm this idea, experimental approaches specifically disrupting or altering the kinase activity of AtIPK2 were employed.
The consensus PxxxDxKxG motif is responsible for IP binding and highly conserved in all known IPK2s (Dubois et al., 2000; Stevenson-Paulik et al., 2002). Mutations in this region abolished the inositol polyphosphate kinase activity without affecting the protein stability. Recently, two lysine sites were reported to be required for 3-kinase activity of AtIPK2 based
This article is protected by copyright. All rights reserved.
Accepted Article
on crystal structure information and biochemical analysis (Endo-Streeter et al., 2012). Mutation of these two lysine sites significantly reduced 3-kinase activity of AtIPK2, while the 6-kinase activity was normal. Therefore, we constructed kinase-dead (AtIPK2β-D100A and AtIPK2β-D100A;K102A) and substrate specificity altered (AtIPK2β-K119W; K123W) variants for AtIPK2β (designated as AtIPK2β*) (Figure 6a). These AtIPK2β* variants, together with wild-type control AtIPK2β, were introduced into AtIPK2α/atipk2α;atipk2β/atipk2β (Aabb) plants under control of either the native promoter or the pollen-specific promoter Lat52 to examine their complementation of atipk2αatipk2β double mutant (Figure 6b). In series of the T2 transgenic plants driven by the native promoter, atipk2αatipk2β double mutant was only identified by the control AtIPK2β complementary transgene. By contrast, the AtIPK2β* variants with either lost kinase activity or inhibited 3-kinase activity could not complement the lethality of atipk2αatipk2β double mutant (Table S2), suggesting that the catalytic activity of AtIPK2 was responsible, at least for the embryonic lethality of atipk2 double mutant.
The lethality of atipk2αatipk2β double mutant was caused by combination of defects in male gametophyte and embryogenesis. We further investigated the effect of AtIPK2 kinase activity in male gametophyte using pollen-specific Lat52 promoter. Lat52 promoter was specifically expressed in pollen and pollen tube. So even if the pollen function was fully rescued, the homozygous mutant would still be unobtainable due to defective embryogenesis. Through genotyping-PCR screening, we identified AtIPK2α/atipk2α;atipk2β/atipk2β plants with homozygous normal AtIPK2β and variant AtIPK2β* complementary transgenes This article is protected by copyright. All rights reserved.
Accepted Article
(Aabb/Lat52-AtIPK2β; Lat52-AtIPK2β and Aabb/Lat52-AtIPK2β*;Lat52-AtIPK2β*). RTPCR analysis confirmed the expression of AtIPK2β/AtIPK2β* in inflorescence tissues of transgenic Arabidopsis (Figure 6c). Microscopic examination showed that the male gametophyte abnormalities of atipk2αatipk2β were rescued only by normal AtIPK2β. The ratio of abnormal pollen and defected pollen tube guidance were reduced to 2.3% and 3.7% respectively in Aabb/Lat52-AtIPK2β;Lat52-AtIPK2β plants (Figure 6d, e). By contrast, AtIPK2β* variants could not restore the male gametophyte defects (Figure 6d, e). Taken together, these results indicated that the kinase activity of AtIPK2 was required for the male gametophyte function and embryogenesis.
AtIPK2α and AtIPK2β have overlapping expression in male gametophyte
Previous GUS staining analyses revealed the expression of AtIPK2α and AtIPK2β in male gametophyte (Xia et al., 2003; Xu et al., 2005). As AtIPK2α and AtIPK2β are identified to be essential for pollen development and pollen tube guidance in this work, we further investigated the expression of the AtIPK2α and AtIPK2β in male gametophyte using GFP fusion protein under the control of their native promoters (Figure 1d). As expected, fluorescent signals of AtIPK2α-GFP and AtIPK2β-GFP were detected in mature pollen (Figure 7a-c). When pollen grains transformed with AtIPK2α-GFP or AtIPK2β-GFP were further examined under confocal laser scanning microscopy, GFP signals were observed in both the cytoplasm and nucleus of the vegetative cell (Figure 7d-i), while a relatively weak GFP fluorescence was present in the sperm cells (Figure 7d-i). In growing pollen tube, both
This article is protected by copyright. All rights reserved.
Accepted Article
AtIPK2α-GFP and AtIPK2β-GFP appeared to unevenly accumulate at the tip of the pollen tube (Figure 7j-o). This tip localization of AtIPK2α and AtIPK2β in pollen tube was consistent with their role of signaling function in pollen tube guidance.
DISCUSSION Prior work by overexpressing AtIPK2β in Arabidopsis and tobacco revealed its roles in auxiliary shoot branching and abiotic stress responses (Zhang et al., 2007; Yang et al., 2008). However, the endogenous function for AtIPK2 is still unknown. Moreover, the presence of two closely related inositol polyphosphate kinases (AtIPK2α and AtIPK2β) has raised the issue of functional redundancy for them (Zhang et al., 2007; Yang et al., 2008; Tsui and York, 2010). In this study, we demonstrate that AtIPK2α and AtIPK2β act redundantly to regulate male gametophyte and embryogenesis. Previously, Xu et al, (2005) reported that specific antisense inhibition of AtIPK2a led to enhanced pollen germination and pollen tube growth through in vitro analysis. However, the in vivo germination and tube growth of AtIPK2α-antisense pollen were not investigated. In this work, through T-DNA knockout mutants, we analyzed the in-pistil germination and tube growth of atipk2α and atipk2β single mutant pollen. Our results indicated that the pollen and pollen tube of atipk2α and atipk2β single mutant could germinate and grow normally in pistil. Moreover, our genetic results showed that the single atipk2 mutant could be normally transmitted (Table 2). Given that the atipk2a knockdown improves the pollen tube growth, this will not affect the atipk2a
This article is protected by copyright. All rights reserved.
Accepted Article
gametophyte transmission. Thus, it seems that AtIPK2a or AtIPK2β single mutation might not be of functional significance in pollen germination and pollen tube growth in vivo.
Genetic analysis demonstrated that the transmission of atipk2αatipk2β double mutant male gametophyte was severely impaired (Table 2). Approximate 20% double mutant pollen grains were observed with compromised cytoplasmic content or abnormal nuclear constitution (Figure 2). The mitosis of more that 10% pollen grains produced by atipk2α/atipk2α;AtIPK2β/atipk2β and AtIPK2α/atipk2α;atipk2β/atipk2β plants were abnormal. It is likely that the abnormal mitosis in the double mutant pollen leads to the defective pollen formation. However, the majority of atipk2αatipk2β pollens are similar with wild-type. Both in vitro and in vivo assays indicated that the germination and tube growth of viable atipk2αatipk2β double mutant pollen were competent (Figure 3). However, the directional growth of these double mutant pollen tubes towards the ovule were severely disrupted (Figure 4). These suggest that AtIPK2α and AtIPK2β specifically affect the responses of pollen tube to gametophytic maternal (ovule) guiding cues, while have no obvious effect on responses to sporophytic maternal (stigma, style and transmitting tract) signals. It should be noted that some atipk2αatipk2β pollen tubes were able to enter the ovule, but no double mutant plant could be obtained. Silique dissection proved that the embryogenesis of atipk2αatipk2β double mutant was aborted at early stage (Figure 5). The defects of atipk2αatipk2β double mutant in pollen development, pollen tube guidance and embryogenesis suggests that inositol polyphosphate signaling is essential in plant reproduction. This article is protected by copyright. All rights reserved.
Accepted Article
The function of IPK2 in modulating inositol polyphosphate signaling is conserved in all eukaryotes. Nevertheless, recent studies suggest that IPK2 also plays a kinase activityindependent role. In mammals, IPK2/IPMK could interact with AMP-activated protein kinase (AMPK) and target of rapamycin (TOR) to monitor the cell energy status and coordinate the responses to nutrient availability (Kim et al., 2011; Bang et al., 2012). In addition, catalytically inactive yeast IPK2 could restore the transcriptional regulation of argininedependent genes (Bosch and Saiardi, 2012). In these cases, kinase activity of IPK2 was not required to establish the physical association. Thus, both the kniase activity and non-kinase activity are required for the function of IPK2. However, whether the kinase activity of IPK2 is involved in specific physiological function or developmental process in plant is completely unknown. In this study, we investigated the relationship between the kinase activity and function of AtIPK2 in male gametophyte and embryogenesis (Figure 6). We found that only the normal AtIPK2β could restore the defective male gametophyte and embryogenesis of atipk2αatipk2β double mutant, while AtIPK2β variants with either lost kinase activity or inhibited 3-kinase activity could not complement the atipk2αatipk2β double mutant (Figure 6). Our results provide strong genetic evidence that the kinase activity of AtIPK2 is critical for male gametophyte and embryogenesis.
Kinase activity-dependent manner of AtIPK2 implies the requirement of intracellular messenger inositol polyphosphate in male gametophyte, especially in pollen tube guidance. In Arabidopsis, pollen tube guidance by the ovule is divided into two phases: funicular guidance and micropylar guidance. It is believed that female gametophyte could produce This article is protected by copyright. All rights reserved.
Accepted Article
guidance cues to direct the growth of pollen tube (Márton et al., 2005; Okuda et al., 2009; Takeuchi and Higashiyama, 2012). To date, only limited number of male specific components in pollen tube guidance has been identified, including POD1, K+ transporters (CHX21/CHX23), receptor-like kinases (LIP1/LIP2) and mitogen-activated protein kinases (MPK3/MPK6) (Li et al., 2011; Lu et al., 2011; Liu et al., 2013; Guan et al., 2014). Considering the role of inositol polyphosphate in signaling cascade, AtIPK2 should be a male factor directly involved in pollen tube guidance, compared with the indirect mechanism proposed for ER localized POD1 and CHX21/CHX23 (Li et al., 2011; Lu et al., 2011). Moreover, unlike the specific role for POD1, LIP1/LIP2 and MPK3/6 in either funicular guidance or micropylar guidance, AtIPK2 affects both the processes, which suggests that inositol polyphosphates might be universal signals underlying funicular guidance and micropylar guidance. Furthermore, it seems that IP5 and/or IP6 might play a more important role than IP3/IP4 in pollen tube guidance, as inhibition of the 3-kinase of AtIPK2β could not complement the male gametophyte defects of double mutant. Consistently, IP6 was reported to be more potent than IP3 in eliciting Ca2+ release (Lemtiri-Chlieh et al., 2003). In addition, Perera et al. (2008) reported that tremendous reduction of IP3 level in Arabidopsis by expressing the mammalian type 1 inositol polyphosphate 5-phosphatase caused no obvious alteration in plant morphology, indicating that IP3 alone might not be sufficient to modulate plant growth and development. Emerging evidence suggests that Ca2+ dynamics at the pollen tube tip are involved in cell-cell communication in plant reproduction (Iwano et al., 2012). Ca2+ influx facilitated by glutamate receptor-like channels (GLRs) and Ca2+ homeostasis mediated by ER luminal protein POD1 have been proposed to be required for pollen tube
This article is protected by copyright. All rights reserved.
Accepted Article
directional growth and guidance (Michard et al., 2011; Li et al., 2011). Moreover, Ca2+ has been shown to serve as a downstream messenger in nitric oxide (NO) mediated pollen tube redirection (Prado et al., 2008). For these reasons, we presume that Ca2+ signal mediated by inositol polyphosphate might also be involved in regulation of pollen tube guidance. It should be noted that no receptor for inositol polyphosphate recognition has been identified from plant (Krinke et al., 2007). Hence, elucidation of the downstream signaling events modulated by inositol polyphosphate is needed.
AtIPK2 is a central, but not the only regulator of inositol polyphosphate signaling. Other kinases like inositol 1,3,4,5,6-pentakisphosphate 2-kinase (AtIPK1) and inositol 1,3,4trisphosphate 5/6-kinase (AtITPK) are also required (Wilson et al., 1997; Shi et al., 2003; Stevenson-Paulik et al., 2005; Sweetman et al., 2006). Stevenson-Paulik et al. (2005) demonstrated that coincident disruption of AtIPK2β and AtIPK1 nearly ablates seed IP6 (phytate) biosynthesis. Hence, in addition to the specific roles for these kinases, they also likely work together to maintain the integrity and plasticity of IP signaling network. For this reason, we cannot preclude that AtIPK2 may act with AtIPK1 or AtITPK to regulate plant growth and development. This would explain the partial developmental defect for atipk2αatipk2β double mutant pollen and the lack of post-embryonic phenotype in atipk2α/atipk2α; AtIPK2β/atipk2β and AtIPK2α/atipk2α;atipk2β/atipk2β plants (Figure S3).
This article is protected by copyright. All rights reserved.
Accepted Article
In summary, our results in this study demonstrate that AtIPK2α and AtIPK2β play an essential role in pollen development, pollen tube guidance and embryogenesis in a kinase activity-dependent manner. Our data provides insights into the underlying molecular mechanism of pollen tube guidance.
EXPERIMENTAL PROCEDURES
Plant materials and growth conditions
Arabidopsis thaliana ecotype Columbia (Col-0) was used as the wild-type control in this study. The T-DNA insertion line atipk2α (GABI_879D07) was obtained from the Nottingham Arabidopsis Stock Centre. The atipk2β T-DNA mutant (SALK_104995) was originally described by Zhang et al. (2007). The T-DNA insertions were verified using primers designed by the T-DNA Primer Design website. The detailed sequences of these primers were listed in Table S1.
Seeds were surface sterilized with 75% ethanol for 5 min, then washed three times with sterile water and plated on solid Murashige and Skoog (MS) media. For growth in both plates and soil, seeds were stratified for 2 days at 4°C and grown in chamber at 23°C under16-hlight/8-h-dark conditions.
RNA Extraction and RT-PCR analysis Total RNA was isolated using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. For RT-PCR analysis, first strand cDNAs were synthesized from DNaseI-treated This article is protected by copyright. All rights reserved.
Accepted Article
total RNA using RevertAidTM cDNA Synthesis Kit (Fermentas). The Actin gene was amplified as internal controls. Gene specific primers used for RT-PCR are listed in Table S1.
Genetic analysis
For all crosses, flowers at floral stage 12c (Smyth et al., 1990) was emasculated, and pollen grains from wild-type or mutant plants were dispersed onto the recipient stigma. For selfcross analysis, plants with various genotypes were allowed to self-pollinate and progeny seeds were collected. The progeny plants were genotyped and scored with PCR. For studies of gametophyte transmission of atipk2α and/or atipk2β alleles, reciprocal crosses were performed between wild-type and mutant materials. The progenies were genotyped for the presence of atipk2α and atipk2β T-DNA insertions by PCR.
Complementation of atipk2αatipk2β double mutants For complementation, AtIPK2a and AtIPK2β genomic fragments were amplified using KODFX polymerase (Takara Biotechnology) with gene specific primers (Table S1). After verification by sequencing, the fragments were cloned into pCAMBIA1302. The resulting vectors (pAtIPK2α-AtIPK2α and pAtIPK2β-AtIPK2β) were then introduced into Agrobacterium tumefaciens GV3101 and transformed into atipk2α/atipk2α;AtIPK2β/atipk2β and AtIPK2α/atipk2α;atipk2β/atipk2β plants respectively by the floral dip method (Clough
This article is protected by copyright. All rights reserved.
Accepted Article
and Bent, 1998). Transgenic seeds were selected on half-strength MS medium supplied with 30 mg/L hygromycin.
Subcellular localization
The coding region of AtIPK2α and AtIPK2β were cloned into the pCAMBIA1302 vector to generate AtIPK2α-GFP and AtIPK2β-GFP fusion protein expression cassettes under the control of their native promoters. The resulting constructs (pAtIPK2α-AtIPK2α-GFP and pAtIPK2β-AtIPK2β-GFP) were then introduced into atipk2α/atipk2α;AtIPK2β/atipk2β and AtIPK2α/atipk2α;atipk2β/atipk2β plants, respectively. The fluorescent signals were observed under a confocal microscope (Olympus FV1000).
Generation of kinase-dead and substrate specificity-altered constructs
Point mutations that ablate (D100A and D100A;D102A) or alter (K119W;K123W) the kinase activity of AtIPK2β were created using the site-directed PCR mutagenesis. These mutated fragments were ligated into pCAMBIA1302 under the control of either the native AtIPK2β promoter or the pollen specific promoter Lat52. All constructs were verified by sequencing. The primers used for site-directed mutagenesis are listed in Table S1.
This article is protected by copyright. All rights reserved.
Accepted Article
Microscopy and phenotypic characterization
Pollen viability was assessed using Alexander’s staining (Alexander, 1969) and viewed under the light microscopy. For morphological observation of pollen grains, mature pollen were mounted on double-sided tape and coated with gold. The samples were then observed using a scanning electron microscope (SEM). For DAPI (4’,6-diamidino-2-phenylindole) staining, pollen grains were released into a staining solution [0.1 M sodium phosphate (pH7), 1 mM EDTA, 0.1% Triton X-100, 300 ng/mL DAPI] and observed under UV illumination. In vitro pollen germination analysis was performed as described by Li et al. (1999). Pollen grains were brushed evenly onto the surface of a gar plates and immediately transferred to a moisture box at 23°C for indicated time (Boavida and McCormick, 2007). For in vivo pollen germination and pollen tube growth assay, pre-emasculated wild-type flowers were pollinated with a limited number of wild-type or mutant pollen grains. The pollen tubes in the pistils were stained with aniline blue and viewed under a fluorescence microscope. To visualize the pollen tube growth in pistils, aniline blue staining was performed as described by Huck et al. (2003). Siliques were fixed overnight in FAA solution (1.5% formaldehyde, 2% acetic acid and 30% ethanol), and washed in an alcohol series (70, 50, 30 and 10%) for 10 min for each step. The sample was further softened with 10% chloral hydrate at 60°C for 10 min, subsequently rinsed twice with sodium phosphate buffer (100 mM, pH 7) and then in 5 M NaOH at 60°C, for 5 minutes. Pollen tubes were stained with 0.1% aniline blue for 4 h under dark and observed with a microscope under UV irradiation condition. Embryos were obtained by removing the seed coat, and the isolated embryos were then rinsed at least three times with distilled water. For single-embryo PCR, embryo DNA isolation was performed as described This article is protected by copyright. All rights reserved.
Accepted Article
by Park et al. (2008). Isolated embryos were homogenized in 40 μL 250 mM NaOH followed by heating to 100°C for 30 s. To the sample, 20 μL buffer (500 mM Tris-HCl, pH 8.0, 0.25% [v/v] NP-40) followed by 40 μL of 250 mM HCl were added, and the sample was mixed and incubated at 100°C for 2 min. Samples were cooled to ambient temperature and centrifuged at ambient temperature for 10 min at 16,000 g. The supernatant was used directly for PCR analysis using allele-specific primers. For embryo observation, developing seeds were clarified in Hoyer’s solution (Liu and Meinke, 1998) for 24 h and observed with Olympus microscope equipped with differential interference contrast (DIC) system.
ACKNOWLEDGEMENTS We sincerely thank Prof. De Ye (China Agricultural University) for providing quartet1 mutant. This work was supported by the National Natural Science Foundation of China (Grant no. 31271319 and 31170270).
SUPPORTING INFORMATION
Figure S1. Phenotypic characterization of atipk2α and atipk2β single mutants
Figure S2. Development of atipk2αatipk2β double mutant pollen in quartet1-6 background
Figure S3. Phenotypic analysis of atipk2α/atipk2α;AtIPK2β/atipk2β and AtIPK2α/ atipk2α;atipk2β/atipk2β self-crossed progeny
This article is protected by copyright. All rights reserved.
Accepted Article
Table S1. List of synthetic oligonucleotide sequences used in this study
Table S2. Analysis of the complementation of atipk2αatipk2β double mutant by AtIPK2β* variants under control of the native promoter.
REFERENCE Alexander, M.P. (1969) Differential staining of aborted and non-aborted pollen. Stain Technol. 44, 117-122. Bang, S., Kim, S., Dailey, M.J., Chen, Y., Moran, T.H., Snyder, S.H., and Kim, S.F. (2012) AMP-activated protein kinase is physiologically regulated by inositol polyphosphate multikinase. Proc. Natl. Acad. Sci. USA. 109, 616-620. Bercy, J., Dubois, E., and Messenguy, F. (1987) Regulation of arginine metabolism in Saccharomyces cerevisiae: expression of the three ARGR regulatory genes and cellular localization of their products. Gene. 55, 277-285. Boavida, L.C. and McCormick, S. (2007) Temperature as a determinant factor for increased and reproducible in vitro pollen germination in Arabidopsis thaliana. Plant J. 52, 570-582. Bosch, D. and Saiardi, A. (2012) Arginine transcriptional response does not require inositol phosphate synthesis. J. Biol. Chem. 287, 38347-38355. Boss, W.F. and Im, Y.J. (2012) Phosphoinositide Signaling. Annu. Rev. Plant Biol. 63, 409429. Burnette, R.N., Gunesekera, B.M. and Gillaspy, G.E. (2003) An Arabidopsis inositol 5phosphatase gain-of-function alters abscisic acid signaling. Plant Physiol. 132, 1011-1019.
This article is protected by copyright. All rights reserved.
Accepted Article
Cheung, A.Y., Boavida, L.C., Aggarwal, M., Wu, H.M. and Feijo, J.A. (2010) The pollen tube journey in the pistil and imaging the in vivo process by two-photon microscopy. J. Exp. Bot. 61, 1907-1915. Clough, S.J. and Bent, A.F. (1998) Floral dip: a simplified method for Agrobacteriummediated transformation of Arabidopsis thaliana. Plant J. 16, 735-743. Dubois, E., Dewaste, V., Erneux, C. and Messenguy, F. (2000) Inositol polyphosphate kinaseactivity of Arg82/ArgRIII is not required for the regulation of the arginine metabolism in yeast. FEBS Lett. 486, 300-304. El Alami, M., Messenguy, F., Scherens, B. and Dubois, E. (2003) Arg82p is a bifunctional protein whose inositol polyphosphate kinase activity is essential for nitrogen and PHO gene expression but not for Mcm1p chaperoning in yeast. Mol. Microbiol. 49, 457-468. Endo-Streeter, S., Tsui, M.K., Odom, A.R., Block, J. and York, J.D. (2012) Structural studies and protein engineering of inositol phosphate multikinase. J. Biol. Chem. 287, 35360-35369. Guan, Y.F., Lu, J.P., Xu, J., McClure, B. and Zhang, S.Q. (2014) Two mitogen- activated protein kinases, MPK3 and MPK6, are required for funicular guidance of pollen tubes in Arabidopsis. Plant Physiol. 165, 528-533. Huck, N., Moore, J.M., Federer, M. and Grossniklaus, U. (2003) The Arabidopsis mutant feronia disrupts the female gametophytic control of pollen tube reception. Development 130, 2149-2159. Hülskamp, M., Schneitz, K. and Pruitt, R. (1995) Genetic evidence for a long-range activity that directs pollen tube guidance in Arabidopsis. Plant Cell 7, 57-64. Kim, S., Kim, S.F., Maag, D., Maxwell, M.J., Resnick, A.C., Juluri, K.R., Chakraborty, A., Koldobskiy, M.A., Cha, S.H., Barrow, R, Snowman, A.M. and Snyder, SH. (2011)
This article is protected by copyright. All rights reserved.
Accepted Article
Amino acid signaling to mTOR mediated by inositol polyphosphate multikinase. Cell Metab. 13, 215-221 Krinke, O., Novotna, Z., Valentova, O. and Martinec, J. (2007) Inositol trisphosphate receptor in higher plants: is it real? J. Exp. Bot. 58, 361-376. Lemtiri-Chlieh F., MacRobbie E.A., Webb A.A., Manison N.F., Brownlee C., Skepper J.N., Chen J., Prestwich G.D. and Brearley C.A. (2003) Inositol hexakisphosphate mobilizes an endomembrane store of calcium in guard cells. Proc. Natl. Acad. Sci. USA. 100, 10091-10095. Li, H., Lin, Y.K., Heath, R.M., Zhu, M.X. and Yang, Z.B. (1999) Control of pollen tube tip growth by a Rop GTPase-dependent pathway that leads to tip-localized calcium influx. Plant Cell 11, 1731-1742. Li, H.J., Xue, Y., Jia, D.J., Wang, T., Hi, D.Q., Liu, J., Cui, F., Xie, Q., Ye, D. and Yang, W.C. (2011) POD1 regulates pollen tube guidance in response to micropylar female signaling and acts in early embryo patterning in Arabidopsis. Plant Cell, 23, 3288-3302. Liu, C.M. and Meinke, D.W. (1998) The titan mutants of Arabidopsis are disrupted in mitosis and cell cycle control during seed development. Plant J.16, 21-31. Liu, J.J., Zhong, S., Guo, X.Y., Hao, L.H., Wei, X.L., Huang, Q.P., Hou, Y.N., Shi, J., Wang, C.Y., Gu, H.Y. and Qu, L.J. (2013) Membrane-bound RLCKs LIP1 and LIP2 are essential male factors controlling male-female attraction in Arabidopsis. Curr. Biol. 23, 993-998. Lu, Y., Chanroj, S., Zulkifli, L., Johnson, M., Uozumi, N., Cheung, A. and Sze, H. (2011) Pollen tubes lacking a pair of K+ transporters fail to target ovules in Arabidopsis. Plant Cell 23, 81-174. Maag, D., Maxwell, M.J., Hardesty, D.A., Boucher, K.L., Choudhari, N., Hanno, A.G., Ma, J.F., Snowman, A.S., Pietropaoli, J.W., Xu, R., Storm, P.B., Saiardi, A., Snyder,
This article is protected by copyright. All rights reserved.
Accepted Article
S.H. and Resnick, A.C. (2011) Inositol polyphosphate multikinase is a physiologic PI3kinase that activates Akt/PKB. Proc. Natl. Acad. Sci. USA. 108, 1391-1396. Márton, M.L., Cordts, S., Broadhvest, J. and Dresselhaus, T. (2005) Micropylar pollen tube guidance by egg apparatus 1 of maize. Science 307, 573-576. Márton, M.L. and Dresselhaus, T. (2010) Female gametophyte-controlled pollen tube guidance. Biochem. Soc. Trans. 38, 627-630. McCormick, S. (1993) Male Gametophyte Development. Plant Cell 5, 1265-1275. Michard, E., Lima, P.T., Borges, F., Silva, A.C., Portes, M.T., Carvalho, J.E., Gilliham, M., Liu, L.H., Obermeyer, G. and Feijo, J.A. (2011) Glutamate receptor like genes form Ca2+ channels in pollen tubes and are regulated by pistil D-serine. Science 332, 434-437. Odom, A.R., Stahlberg, A., Wente, S.R. and York, J.D. (2000) A role for nuclear inositol 1,4,5-trisphosphate kinase in transcriptional control. Science 287, 2026-2029. Okuda, S., Tsutsui, H., Shiina, K., Sprunck, S., Takeuchi, H., Yui, R., Kasahara, R.D., Hamamura, Y., Mizukami, A., Susaki, D., Kawano, N., Sakakibara, T., Namiki, S., Itoh, K., Otsuka, K., Matsuzaki, M., Nozaki, H., Kuroiwa, T., Nakano, A., Kanaoka, M.M., Dresselhaus, T., Sasaki, N. and Higashiyama, T. (2009) Defensin-like polypeptide LUREs are pollen tube attractants secreted from synergid cells. Nature 458, 357-361. Palanivelu, R. and Tsukamoto, T. (2012) Pathfinding in angiosperm reproduction: pollen tube guidance by pistils ensures successful double fertilization. WIREs Dev Biol. 1, 96-113. Park, S., Rancour, D.M. and Bednarek, S.Y. (2008) In planta analysis of the cell cycledependent localization of AtCDC48A and its critical roles in cell division, expansion, and differentiation. Plant Physiol. 148, 246-258.
This article is protected by copyright. All rights reserved.
Accepted Article
Perera, I.Y., Hung, C.Y., Moore, C.D., Stevenson-Paulik, J. and Boss, W.F. (2008) Transgenic Arabidopsis plants expressing the type I inositol 5-phosphatase exhibit increased drought tolerance and altered abscisic acid signaling. Plant Cell 20, 2876-2893. Preuss, D., Rhee, S.Y. and Davis, R.W. (1994) Tetrad analysis possible in Arabidopsis with mutation of the QUARTET (QRT) genes. Science 264, 1458-1460. Prado, A.M., Colaco, R., Moreno, N., Silva, A.C. and Feijó, J.A. (2008) Targeting of pollen tubes to ovules is dependent on nitric oxide (NO) signaling. Mol. Plant. 1, 703-714. Resnick, A.C., Snowman, A.M., Kang, B.N., Hurt, K.J., Snyder, S.H. and Saiardi, A. (2005) Inositol polyphosphate multikinase is a nuclear PI3 kinase with transcriptional regulatory activity. Proc. Natl. Acad. Sci. USA. 102, 12783-12788. Seeds, A.M., Frederick, J.P., Tsui, M.M. and York, J.D. (2007) Roles for inositol polyphosphate kinases in the regulation of nuclear processes and developmental biology. Adv. Enzyme Regul. 47, 10-25. Shears, S.B. (2004) How versatile are inositol phosphate kinases? Biochem. J. 377, 265-280. Shi, J.R., Wang, H.Y., Wu, Y.S., Hazebroek, J., Meeley, R.B. and Ertl, D.S. (2003) The maize low-phytic acid mutant lpa2 is caused by mutation in an inositol phosphate kinase gene. Plant Physiol. 131, 507-515. Staxen, I.I., Pical, C., Montgomery, L.T., Gray, J.E., Hetherington, M. and McAinsh, M.R. (1999) Abscisic acid induces oscillations in guard-cell cytosolic free calcium that involve phosphoinositide-specific phospholipase C. Proc. Natl. Acad. Sci. USA 96, 17791784. Steger, D.J., Haswell, E.S., Miller, A.L., Wente, S.R. and OòShea, E.K. (2003) Regulation of chromatin remodeling by inositol polyphosphates. Science 299, 114-116.
This article is protected by copyright. All rights reserved.
Accepted Article
Stevenson-Paulik, J., Bastidas, R.J., Chiou, S.T., Frye, R.A. and York, J.D. (2005) Generation of phytate-free seeds in Arabidopsis through disruption of inositol polyphosphate kinases. Proc. Natl. Acad. Sci. USA 102, 12612-12617. Stevenson-Paulik, J., Odom, A.R. and York J.D. (2002) Molecular and biochemical characterization of two plant inositol polyphosphate 6-/3-/5-kinases. J. Biol. Chem. 277: 42711-42718. Smyth, D.R., Bowman, J.L. and Meyerowitz, E.M. (1990) Early flower development in Arabidopsis. Plant Cell 2, 755-767. Sweetman, D., Johnson, S., Caddick, S.E., Hanke, D.E. and Brearley, C.A. (2006) Characterization of an Arabidopsis inositol 1,3,4,5,6-pentakisphosphate 2-kinase (AtIPK1). Biochem. J. 394, 95-103. Takeuchi, H. and Higashiyama, T. (2012) A species-specific cluster of defensin-like genes encodes diffusible pollen tube attractants in Arabidopsis. PLoS Biol. 10, e1001449. Tsui,
M.M.
and
York,
J.D.
(2010)
Roles of inositol
phosphates and inositol
pyrophosphates in development, cell signalingand nuclear processes. Adv Enzyme Regul. 50, 324-337. Wilson, M.P. and Majerus, P.W. (1997) Characterization of a cDNA encoding Arabidopsis thaliana inositol 1,3,4-trisphosphate 5/6-kinase, Biochem. Biophys. Res. Commun. 232, 678-681. Xia, H.J., Brearley, C., Elge, S., Kaplan, B., Fromm, H. and Mueller-Roeber, B. (2003) Arabidopsis inositol polyphosphate 6-/3-kinase is a nuclear protein that complements a yeast mutant lacking a functional ArgR-Mcm1 transcription complex. Plant Cell 15, 449463. Xia, H.J. and Yang, G. (2005) Inositol 1,4,5-trisphosphate 3-kinases: functions and regulations. Cell Res. 15, 83-91.
This article is protected by copyright. All rights reserved.
Accepted Article
Xu, J., Brearley, C.A., Lin, W.H., Wang, Y., Ye, R., Mueller-Roeber, B., Xu, Z.H. and Xue, H.W. (2005) A role of Arabidopsis inositol polyphosphate kinase AtIPK2α, in pollen germination and root growth. Plant Physiol. 137, 94-103. Yang, L., Tang, R.J., Zhu, J.Q., Liu, H., Mueller-Roeber, B., Xia, H.J. and Zhang, H.X. (2007) Enhancement of stress tolerance in transgenic tobacco plants constitutively expressing AtIPK2β, an inositol polyphosphate 6-/3-kinase from Arabidopsis thaliana. Plant Mol. Biol. 66, 329-343. York, J.D., Odom, A.R., Murphy, R., Ives, E.B. and Wente, S.R. (1999) A phospholipase C-dependent inositol polyphosphate kinase pathway required for efficient messenger RNA export. Science 285, 96-100. Zhang, Z.B., Yang, G., Arana, F., Chen, Z., Li, Y. and Xia, H.J. (2007) Arabidopsis inositol polyphosphate 6-/3-kinase (AtIpk2β) is involved in auxiliary shoot branching via auxin signaling. Plant Physiol. 144, 942-951.
TABLES Table 1. Segregation analysis of atipk2α and atipk2β mutants Parent genotype AtIPK2α/atipk2α;AtIPK2β/AtIPK2β
AABB
AaBB
aaBB
(AaBB)
35
67
34
AtIPK2α/AtIPK2α;AtIPK2β/atipk2β
AABB
AABb
AAbb
(AABb)
28
57
30
aaBB
aaBb
aabb
291
284
0
AAbb
Aabb
aabb
326
335
0
atipk2α/atipk2α;AtIPK2β/atipk2β (aaBb) AtIPK2α/atipk2α;atipk2β/atipk2β (Aabb) a
Genotype of progeny
Expected
Observed
1:2:1
1:1.91:0.97
1:2:1
1:2.04:1.07
1:2:1
1:0.98:0a
1:2:1
1:1.03:0a
Significantly different from the expected 1:2:1 segregation ratio (P
Received Date: 12-Aug-2014 Accepted Date: 31-Mar-2015 Article Type: Original Article
Enzyme activities of Arabidopsis inositol polyphosphate kinases AtIPK2α and AtIPK2β are involved in pollen development, pollen tube guidance and embryogenesis
Huadong Zhan1, Yujiao Zhong1, Zhongnan Yang2, and Huijun Xia1* 1
State Key Laboratory of Hybrid Rice, College of Life Sciences, Wuhan University, Wuhan,
Hubei 430072, China 2
College of Life and Environment Sciences, Shanghai Normal University, Shanghai 200234,
China
*For correspondence: Huijun Xia
Email: [email protected]; Tel and Fax: +86-27-68752112
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an 'Accepted Article', doi: 10.1111/tpj.12846 This article is protected by copyright. All rights reserved.
Accepted Article
Running title: AtIPK2 in male gametophyte and embryogenesis
Keywords: inositol polyphosphate kinase, AtIPK2α and AtIPK2β, kinase activity, pollen development, pollen tube guidance, embryogenesis, Arabidopsis thaliana
SUMMARY
Inositol polyphosphate kinase (IPK2) is a key component of inositol polyphosphate signaling. There are two highly homologous inositol polyphosphate kinases (AtIPK2α and AtIPK2β) in Arabidopsis. Previous studies that over-expressed or reduced expression of AtIPK2α and AtIPK2β had suggested roles in auxiliary shoot branching, abiotic stress responses and root growth. Here, we report that AtIPK2α and AtIPK2β act redundantly during pollen development, pollen tube guidance and embryogenesis. Single knockout mutants of atipk2α and atipk2β were indistinguishable from the wild-type, whereas atipk2αatipk2β double mutant could not be obtained. Detailed genetic and cytological investigations showed that mutation of AtIPK2α and AtIPK2β resulted in severely reduced transmission of male gametophyte due to abnormal pollen development and defective pollen tube guidance. In addition, the early embryo development of atipk2αatipk2β double mutant was also aborted. Expressing either catalytically inactive or substrate specificity-altered variants of AtIPK2β could not rescue the male gametophyte and embryogenesis defects of atipk2αatipk2β double mutant, implying that the kinase activity of AtIPK2 is required for pollen development,
This article is protected by copyright. All rights reserved.
Accepted Article
pollen tube guidance and embryogenesis. Taken together, our results provide genetic evidence for the requirement of inositol polyphosphate signaling in plant sexual reproduction.
INTRODUCTION
Male gametophyte plays a pivotal role in plant sexual reproduction. In angiosperms, successful double fertilization depends on formation of male gametophytes within the anther, which fuse with the egg cell and the central cell to give rise to the embryo and the endosperm (McCormick, 1993). Pollen tube is a highly specialized cell structure used to deliver the male gametes. After adhesion and germination on a compatible stigma, pollen tube first penetrates the stigma and style, then grows through the transmitting tissue, migrates along the septum to the funiculus, and finally enters the micropyle (Hülskamp et al., 1995; Márton and Dresselhaus, 2010). To achieve this highly precise and directional growth in pistil, pollen tubes need extensive cell communication with the female tissues and adjust their tip growth in response to guidance cues (Cheung et al., 2010; Palanivelu and Tsukamoto, 2012). In the past decade, considerable progresses have been made in understanding the female signals that guide the pollen tube (Márton et al., 2005; Okuda et al., 2009; Takeuchi and Higashiyama, 2012). Until recently, two receptor-like kinases (RLKs) preferentially expressed in the apical plasma membrane of pollen tube, LIP1 and LIP2 (Lost In Pollen Tube Guidance1 and 2) were demonstrated to be the essential components of the receptor complex in micropylar guidance (Liu et al., 2013). However, the knowledge about the intracellular signaling events in pollen tube in response to the guidance cues is still scarce.
This article is protected by copyright. All rights reserved.
Accepted Article
Amino acid signaling to mTOR mediated by inositol polyphosphate multikinase. Cell Metab. 13, 215-221 Krinke, O., Novotna, Z., Valentova, O. and Martinec, J. (2007) Inositol trisphosphate receptor in higher plants: is it real? J. Exp. Bot. 58, 361-376. Lemtiri-Chlieh F., MacRobbie E.A., Webb A.A., Manison N.F., Brownlee C., Skepper J.N., Chen J., Prestwich G.D. and Brearley C.A. (2003) Inositol hexakisphosphate mobilizes an endomembrane store of calcium in guard cells. Proc. Natl. Acad. Sci. USA. 100, 10091-10095. Li, H., Lin, Y.K., Heath, R.M., Zhu, M.X. and Yang, Z.B. (1999) Control of pollen tube tip growth by a Rop GTPase-dependent pathway that leads to tip-localized calcium influx. Plant Cell 11, 1731-1742. Li, H.J., Xue, Y., Jia, D.J., Wang, T., Hi, D.Q., Liu, J., Cui, F., Xie, Q., Ye, D. and Yang, W.C. (2011) POD1 regulates pollen tube guidance in response to micropylar female signaling and acts in early embryo patterning in Arabidopsis. Plant Cell, 23, 3288-3302. Liu, C.M. and Meinke, D.W. (1998) The titan mutants of Arabidopsis are disrupted in mitosis and cell cycle control during seed development. Plant J.16, 21-31. Liu, J.J., Zhong, S., Guo, X.Y., Hao, L.H., Wei, X.L., Huang, Q.P., Hou, Y.N., Shi, J., Wang, C.Y., Gu, H.Y. and Qu, L.J. (2013) Membrane-bound RLCKs LIP1 and LIP2 are essential male factors controlling male-female attraction in Arabidopsis. Curr. Biol. 23, 993-998. Lu, Y., Chanroj, S., Zulkifli, L., Johnson, M., Uozumi, N., Cheung, A. and Sze, H. (2011) Pollen tubes lacking a pair of K+ transporters fail to target ovules in Arabidopsis. Plant Cell 23, 81-174. Maag, D., Maxwell, M.J., Hardesty, D.A., Boucher, K.L., Choudhari, N., Hanno, A.G., Ma, J.F., Snowman, A.S., Pietropaoli, J.W., Xu, R., Storm, P.B., Saiardi, A., Snyder,
This article is protected by copyright. All rights reserved.
Accepted Article
bisphosphate to phosphatidylinositol (3,4,5)-trisphosphate (Resnick et al., 2005; Maag et al., 2011). Moreover, IPK2 can physically interact with and physiologically regulate target of rapamycin (TOR) and AMP-activated protein kinase (AMPK) in mammals (Kim et al., 2011; Bang et al., 2012). Overall, these results suggest that IPK2 is a multifunctional protein.
The inositol polyphosphate kinase in higher plants was first identified from Arabidopsis thaliana (Stevenson-Paulik et al., 2002; Xia et al., 2003). Genome survey and phylogenetic analysis reveal that there are two highly homologous inositol polyphosphate kinases (AtIPK2α and AtIPK2β) in Arabidopsis. The two AtIPK2 proteins share over 70% amino acid identity and exhibit identical 6/3 dual-specificity kinase activities, which sequentially phosphorylate inositol 1,4,5-trisphosphate to generate inositol 1,4,5,6-tetrakisphosphate and inositol 1,3,4,5,6-pentakisphosphate, like their yeast counterpart (Stevenson-Paulik et al., 2002; Xia et al., 2003). Downregulation of AtIPK2α through antisense inhibition has been shown to result in enhanced root growth and pollen germination (Xu et al., 2005), while AtIPK2β functions in auxiliary shoot branching through the auxin signaling pathway (Zhang et al., 2007). Moreover, AtIPK2β is a stress-responsive gene and its heterologous expression in tobacco lead to improved tolerance to diverse abiotic stresses (Yang et al., 2008).
The presence of two highly homologous inositol polyphosphate kinases in Arabidopsis imposes a major obstacle for defining their biological functions. In this study, through double mutant analysis, we report that AtIPK2α and AtIPK2β play a redundant and essential role in This article is protected by copyright. All rights reserved.
Accepted Article
pollen development, pollen tube guidance and embryogenesis. Moreover, we investigate the correlation between the kinase activity and function of AtIPK2 in male gametophyte and embryogenesis. Our results indicate that the kinase activity of AtIPK2 is essential for pollen development, pollen tube guidance and embryogenesis, implying the requirement of intracellular messenger inositol polyphosphates in plant sexual reproduction.
RESULTS
The male gametophyte transmission of atipk2αatipk2β double mutant is severely reduced
AtIPK2α and AtIPK2β are two closely related inositol polyphosphate kinases, sharing over 70% amino acid identity and possessing identical 6/3-kinase activities (Stevenson-Paulik et al., 2002; Xia and Yang, 2005). We obtained T-DNA insertion mutants for them. The GABIKat line (GABI_879D07, named atipk2α) contains a T-DNA insertion in the exon of AtIPK2α (Figure 1a, b). It is a null allele as AtIPK2α transcripts were not detected in this mutant (Figure 1b). The atipk2β RNA-null mutant (SALK_104995) used in this study was originally described by Zhang et al. (2007) and examined by genotyping-PCR with genespecific primers again here (Figure 1a, c). Both atipk2α and atipk2β are in the Columbia background (Col-0). No visible phenotypes were observed for atipk2α and atipk2β single mutant plants (Figure S1).
This article is protected by copyright. All rights reserved.
Accepted Article
Crosses between atipk2α and atipk2β were performed to generate double mutant. AtIPK2α and AtIPK2β are located at opposite ends of chromosome 5, which ensures a high frequency of recombination between the two loci. However, of over 200 F2 plants, no homozygous double mutant was identified. From the F2 population, we identified the lines of atipk2α/atipk2α;AtIPK2β/atipk2β (aaBb) and AtIPK2α/atipk2α; atipk2β/atipk2β (Aabb) plants. Once again no homozygous double mutant could be obtained from the self-fertilized progeny of aaBb and Aabb plants (Table 1). Moreover, when atipk2α/atipk2α;AtIPK2β/atipk2β or AtIPK2α/atipk2α;atipk2β/ atipk2β was pollinated with wild-type pollen, approximately 50% progeny contained double mutant alleles (Table 2). This suggests that atipk2αatipk2β was transmitted normally through the female parent. However, when atipk2α/atipk2α;AtIPK2β/ atipk2β or AtIPK2α/atipk2α;atipk2β/atipk2β plants were used as pollen donors, severe reduction in co-transmission of atipk2α and atipk2β was observed (Table 2). These results suggested that mutation of AtIPK2α and AtIPK2β significantly reduced the genetic transmission of male gametophyte, but did not affect the female gametophyte function.
To confirm that this abnormal segregation of atipk2αatipk2β double mutant is caused by insertions in inositol polyphosphate kinase genes, genetic complementation experiments were performed. Constructs encoding AtIPK2α/AtIPK2α-GFP or AtIPK2β/AtIPK2β-GFP under the control of their native promoters were introduced into atipk2α/atipk2α;AtIPK2β/atipk2β (aaBb) or AtIPK2α/atipk2α;atipk2β/atipk2β (Aabb) plants respectively via Agrobacterium tumefaciens-mediated transformation (Figure 1d). Homozygous This article is protected by copyright. All rights reserved.
Accepted Article
atipk2α/atipk2α;atipk2β/atipk2β (aabb) double mutants were recovered by these complementary transgenes as demonstrated by PCR genotyping (Figure 1e). Moreover, the segregation of atipk2αatipk2β double mutant alleles was restored in atipk2α/atipk2α;AtIPK2β/atipk2β and AtIPK2α/atipk2α; atipk2β/atipk2β plants carrying the homozygous AtIPK2α or AtIPK2β transgene (Table 3). These results suggested that the lethality of atipk2αatipk2β double mutant was caused by T-DNA insertions in AtIPK2 loci. It also indicated that the C-terminally fused AtIPK2α-GFP and AtIPK2β-GFP were functional.
The pollen development of atipk2αatipk2β double mutant is partially compromised
Male gametophyte transmission defect could be caused by defects in pollen viability, germination, pollen tube growth and/or fertilization. In order to further understand the transmission defect of atipk2αatipk2β, we first examined the pollen viability using Alexander’s staining (Alexander, 1969). Almost all pollen from atipk2α and atipk2β single homozygous mutants appeared full, round and red-stained, which was similar to mature pollen grains of wild-type (Figure 2a-c, t). In contrast, 11.6% pollen from atipk2α/atipk2α;AtIPK2β/atipk2β (aaBb) anthers and 13.2% pollen in AtIPK2α/ atipk2α;atipk2β/atipk2β (Aabb) anthers were completely or partially devoid of cytoplasmic contents as indicated by the reduced staining (Figure 2d, e and t). Moreover, this abnormal pollen viability staining could be restored in the homozygous atipk2αatipk2β double mutant plants carrying the AtIPK2β-GFP complementary transgene (aabb/AtIPK2β-GFP) (Figure 2f, t). Thus, the defective pollen grains observed in atipk2α/atipk2α;AtIPK2β/atipk2β and
This article is protected by copyright. All rights reserved.
Accepted Article
AtIPK2α/atipk2α; atipk2β/atipk2β plants were most likely the atipk2αatipk2β double mutant. Considering that 50% pollen grains produced by atipk2α/atipk2α;AtIPK2β/atipk2β or AtIPK2α/atipk2α;atipk2β/atipk2β plants carry the double mutant alleles, then approximately 20% atipk2αatipk2β double mutant pollen grains have compromised viability. As aborted pollen grains often have an abnormal shape, we further investigated the pollen morphology by scanning electron microscopy (SEM). Consistent with the viability staining result, pollen grains from atipk2α/atipk2α; AtIPK2β/atipk2β and AtIPK2α/atipk2α;atipk2β/atipk2β plants frequently collapsed or possessed distorted morphology (Figure 2g-l). The 4’,6-diaminophenylindole (DAPI) staining showed that almost all mature pollen grains collected from wild-type, atipk2 single mutant and complementary aabb/AtIPK2β-GFP plants had two brightly stained sperm nuclei and one faint vegetative nucleus (2s+1v) (Figure 2m-r, u). By contrast, about 10% pollen grains produced by atipk2α/atipk2α;AtIPK2β/atipk2β and AtIPK2α/ atipk2α;atipk2β/atipk2β plants were abnormally stained (Figure 2m-r, u), including one generative nucleus and one vegetative nucleus (1s+1v), only one nucleus (1v) and no discernible nucleus (Figure 2s).
The defective development of atipk2αatipk2β double mutant pollen were characterized in more detail by introgressing AtIPK2α/atipk2α;atipk2β/atipk2β into quartet1 mutant (qrt1-6, Col-0 background). In qrt1-6, four products of male meiosis fail to separate, resulting in a characteristic pollen quartet structure (Preuss et al., 1994). Pollen from qrt1-6 mutant (AABBqq) and AtIPK2α/AtIPK2α;atipk2β/ atipk2β;qrt1-6/qrt1-6 (AAbbqq) plants always produced quartets in which all four pollen grains were viable (Figure S2). By contrast, over This article is protected by copyright. All rights reserved.
Accepted Article
20% pollen quartets in AtIPK2α/atipk2α;atipk2β/atipk2β;qrt1-6/qrt1-6 (Aabbqq) plants had one or two defective pollen grains (Figure S2). Quartets contained more than two aberrant pollen grains were not observed in Aabbqq, suggesting that the function of AtIPK2 in pollen formation is likely restricted to gametophyte.
The pollen tube guidance of atipk2αatipk2β double mutant is severely impaired
As the majority of atipk2 double mutant pollen was viable, we further investigated the pollen germination and pollen tube growth of atipk2αatipk2β double mutant. Consistent with the partial defect of atipk2αatipk2β double mutant pollen, we could observe small-size and ungerminable pollen grains on germination medium when pollen form atipk2α/atipk2α;AtIPK2β/atipk2β and AtIPK2α/atipk2α;atipk2β/atipk2β plants were incubated (Figure 3a-f). In vitro analysis revealed that pollen grains from atipk2α/atipk2α;AtIPK2β/atipk2β and AtIPK2α/atipk2α;atipk2β/atipk2β plants germinate slightly slower than that of the controls. After incubation in growth medium for 6 h, about 73.2% and 72.6% pollen from aaBb and Aabb plants germinated, compared with the approximately 80% of the various controls (Figure 3s). Considering that ~20% double mutant pollen were defective, over 70% pollen germination rate for aaBb and Aabb plants indicated a competitive germination for viable atipk2αatipk2β double mutant pollen grains. The pollen tube growth was also investigated by pollinating the pre-emasculated wild-type stigmas with limited wild-type, atipk2 mutant or aabb/AtIPK2β-GFP pollen. The majority of pollen tubes from aaBb and Aabb plants (approximately 85%, n>300) were able to penetrate the stigma
This article is protected by copyright. All rights reserved.
Accepted Article
and style, and grow in the transmitting tract at a comparable rate with wild-type and single mutant controls (Figure 3g-r, t). Thus, the germination and tube growth of viable atipk2αatipk2β pollen were competent.
We further observed the path of pollen tube growth in pistils to investigate whether the pollen tube guidance of atipk2αatipk2β was compromised. To do this, wild-type stigmas were pollinated with various pollen grains, then fixed and stained with aniline blue to follow the route of pollen tubes. The wild-type, atipk2 single mutant and complementary aabb/AtIPK2βGFP pollen tubes were observed to grow on the surface of the funiculus after leaving the transmitting tissue and entered the micropyle smoothly and precisely (Figure 4a-c, f and g). However, when wild-type stigmas pollinated with pollen grains from atipk2α/atipk2α;AtIPK2β/atipk2β or AtIPK2α/ atipk2α;atipk2β/atipk2β plants, disordered patterns of pollen tube directional growth were observed (Figure 4d, e and h-j). The defective pollen tube phenotypes could be divided into two categories: (1) pollen tubes failed to shift their directional growth to the funiculus. About 27.6% (n>300) pollen tubes of atipk2α/atipk2α;AtIPK2β/atipk2β plants were found without approaching the funiculus in limited pollination (Figure 4k). (2) Pollen tubes grew randomly toward the micropyle after adhering to the funiculus, including growing on the ovule surface, or initially twisting around the micropyle and finally entering the micropyle. Approximately 12.3% (n>300) pollen tubes from atipk2α/atipk2α;AtIPK2β/atipk2β plants appeared to lose their way towards the micropyle in limited pollination (Figure 4k). These observations indicated that the pollen tube guidance of atipk2αatipk2β was disrupted. This article is protected by copyright. All rights reserved.
Accepted Article
AtIPK2α and AtIPK2β are important for embryogenesis
Considering that the male gametophyte defect in atipk2αatipk2β double mutant was not fully penetrant, homozygous atipk2α/atipk2α;atipk2β/atipk2β should be obtained from the progeny of self-fertilized atipk2α/atipk2α;AtIPK2β/atipk2β (aaBb) and AtIPK2α/atipk2α;atipk2β/atipk2β (Aabb) plants, and the segregation of aaBB:aaBb and AAbb:Aabb should be between 1:1 and 1:2. However, no atipk2αatipk2β double mutant plant was identified in PCR genotype screens, and the segregation of aaBB:aaBb and AAbb:Aabb was found to be 1:1 (Table 1). Therefore, in addition to the lethality of atipk2αatipk2β double mutant, the development of atipk2α/atipk2α; AtIPK2β/atipk2β and AtIPK2α/atipk2α;atipk2β/atipk2β embryos should be also partially aborted. To test these hypotheses, the seed set of self-pollinated atipk2α/atipk2α;AtIPK2β/atipk2β and AtIPK2α/atipk2α;atipk2β/atipk2β plants was investigated. As shown in Figure 5a, siliques from atipk2α/atipk2α;AtIPK2β/atipk2β and AtIPK2α/atipk2α;atipk2β/atipk2β plants had a variety of defective seeds, ranging from the highly degenerated seeds to discolored seeds. To verify the genotype of the aborted seeds, embryos embedded in the green and white seeds were isolated from atipk2α/atipk2α;AtIPK2β/atipk2β plants (Figure 5b). Single-embryo PCR was carried out with allele-specific primers. Among the 31 green seeds, 14 were atipk2α/atipk2α;AtIPK2β/AtIPK2β and 17 were atipk2α/atipk2α;AtIPK2β/atipk2β. While all the 15 pale embryos derived from the white seeds were found to be atipk2α/atipk2α;AtIPK2β/atipk2β. This result suggested that atipk2α/atipk2α; AtIPK2β/atipk2β could develop into either normal or abnormal seeds. Moreover, the absence of atipk2α/atipk2α;atipk2β/atipk2β in the examined white seeds indicated that the This article is protected by copyright. All rights reserved.
Accepted Article
atipk2αatipk2β double mutant embryos died at early embryonic stage, before visible seeds were formed. To characterize the process of seed abortion, developing seeds from self-pollinated atipk2α/atipk2α;AtIPK2β/atipk2β and AtIPK2α/atipk2α;atipk2β/atipk2β siliques were cleared and visualized using differential interference contrast (DIC) microscopy. When most of the sibling embryos reached the torpedo stage at 6-7 d after pollination (Figure 5c), 13 to 15% embryos were found to be retarded at the heart or earlier stages within the same silique (Figure 5d-m, q). This strongly asynchronous embryo stages supported the early embryo abortion. Consistent with this, aborted embryos could be seen as early as at the first and second round of cell division in both atipk2α/atipk2α;AtIPK2β/atipk2β and AtIPK2α/atipk2α;atipk2β/atipk2β siliques, which finally developed into the highly degenerated seeds (Figure 5d-f). Intriguingly, aberrant embryos with elongated suspensor could be observed in atipk2α/atipk2α; AtIPK2β/atipk2β plants in some cases (Figure 5k-m). The cell divisions in these elongated suspensors appeared to be normal, but the longitudinal cell expansion was dramatically increased (Figure 5k-m). This long-suspensor phenotype was only observed in atipk2α/atipk2α;AtIPK2β/atipk2β silique, suggesting that they were likely atipk2α/atipk2α;AtIPK2β/atipk2β rather than the atipk2α/atipk2α;atipk2β/ atipk2β double mutant.
In addition to the abnormalities described above, embryos that progressed to the torpedo stage but with reduced size or asymmetrical cotyledonary lobes were also observed (Figure 5n, o). Statistically, 4 to 6% embryos were found with this delayed development in atipk2α/atipk2α;AtIPK2β/atipk2β and AtIPK2α/atipk2α;atipk2β/ atipk2β siliques (Figure 5q). This article is protected by copyright. All rights reserved.
Accepted Article
These delayed embryos should be able to develop into mature seeds, as seedlings with smaller and asymmetrical cotyledons could be found in selfed offspring of atipk2α/atipk2α;AtIPK2β/atipk2β and AtIPK2α/atipk2α; atipk2β/atipk2β plants (Figure 5p, r). Molecular characterization of these cotyledon-abnormal seedlings derived from the selfpollinated atipk2α/atipk2α; AtIPK2β/atipk2β plants revealed that nearly 90% (27 of 31) of them were with the genotype of atipk2α/atipk2α;AtIPK2β/atipk2β.
Taken together, these results demonstrate that AtIPK2α and AtIPK2β are important for embryo development.
Kinase activity of AtIPK2 is required for the male gametophyte function and embryogenesis
As described above, AtIPK2α and AtIPK2β are essential for male gametophyte and embryogenesis. However, whether the function of AtIPK2 in these processes is associated with their kinase activity is completely unknown. To confirm this idea, experimental approaches specifically disrupting or altering the kinase activity of AtIPK2 were employed.
The consensus PxxxDxKxG motif is responsible for IP binding and highly conserved in all known IPK2s (Dubois et al., 2000; Stevenson-Paulik et al., 2002). Mutations in this region abolished the inositol polyphosphate kinase activity without affecting the protein stability. Recently, two lysine sites were reported to be required for 3-kinase activity of AtIPK2 based
This article is protected by copyright. All rights reserved.
Accepted Article
on crystal structure information and biochemical analysis (Endo-Streeter et al., 2012). Mutation of these two lysine sites significantly reduced 3-kinase activity of AtIPK2, while the 6-kinase activity was normal. Therefore, we constructed kinase-dead (AtIPK2β-D100A and AtIPK2β-D100A;K102A) and substrate specificity altered (AtIPK2β-K119W; K123W) variants for AtIPK2β (designated as AtIPK2β*) (Figure 6a). These AtIPK2β* variants, together with wild-type control AtIPK2β, were introduced into AtIPK2α/atipk2α;atipk2β/atipk2β (Aabb) plants under control of either the native promoter or the pollen-specific promoter Lat52 to examine their complementation of atipk2αatipk2β double mutant (Figure 6b). In series of the T2 transgenic plants driven by the native promoter, atipk2αatipk2β double mutant was only identified by the control AtIPK2β complementary transgene. By contrast, the AtIPK2β* variants with either lost kinase activity or inhibited 3-kinase activity could not complement the lethality of atipk2αatipk2β double mutant (Table S2), suggesting that the catalytic activity of AtIPK2 was responsible, at least for the embryonic lethality of atipk2 double mutant.
The lethality of atipk2αatipk2β double mutant was caused by combination of defects in male gametophyte and embryogenesis. We further investigated the effect of AtIPK2 kinase activity in male gametophyte using pollen-specific Lat52 promoter. Lat52 promoter was specifically expressed in pollen and pollen tube. So even if the pollen function was fully rescued, the homozygous mutant would still be unobtainable due to defective embryogenesis. Through genotyping-PCR screening, we identified AtIPK2α/atipk2α;atipk2β/atipk2β plants with homozygous normal AtIPK2β and variant AtIPK2β* complementary transgenes This article is protected by copyright. All rights reserved.
Accepted Article
(Aabb/Lat52-AtIPK2β; Lat52-AtIPK2β and Aabb/Lat52-AtIPK2β*;Lat52-AtIPK2β*). RTPCR analysis confirmed the expression of AtIPK2β/AtIPK2β* in inflorescence tissues of transgenic Arabidopsis (Figure 6c). Microscopic examination showed that the male gametophyte abnormalities of atipk2αatipk2β were rescued only by normal AtIPK2β. The ratio of abnormal pollen and defected pollen tube guidance were reduced to 2.3% and 3.7% respectively in Aabb/Lat52-AtIPK2β;Lat52-AtIPK2β plants (Figure 6d, e). By contrast, AtIPK2β* variants could not restore the male gametophyte defects (Figure 6d, e). Taken together, these results indicated that the kinase activity of AtIPK2 was required for the male gametophyte function and embryogenesis.
AtIPK2α and AtIPK2β have overlapping expression in male gametophyte
Previous GUS staining analyses revealed the expression of AtIPK2α and AtIPK2β in male gametophyte (Xia et al., 2003; Xu et al., 2005). As AtIPK2α and AtIPK2β are identified to be essential for pollen development and pollen tube guidance in this work, we further investigated the expression of the AtIPK2α and AtIPK2β in male gametophyte using GFP fusion protein under the control of their native promoters (Figure 1d). As expected, fluorescent signals of AtIPK2α-GFP and AtIPK2β-GFP were detected in mature pollen (Figure 7a-c). When pollen grains transformed with AtIPK2α-GFP or AtIPK2β-GFP were further examined under confocal laser scanning microscopy, GFP signals were observed in both the cytoplasm and nucleus of the vegetative cell (Figure 7d-i), while a relatively weak GFP fluorescence was present in the sperm cells (Figure 7d-i). In growing pollen tube, both
This article is protected by copyright. All rights reserved.
Accepted Article
AtIPK2α-GFP and AtIPK2β-GFP appeared to unevenly accumulate at the tip of the pollen tube (Figure 7j-o). This tip localization of AtIPK2α and AtIPK2β in pollen tube was consistent with their role of signaling function in pollen tube guidance.
DISCUSSION Prior work by overexpressing AtIPK2β in Arabidopsis and tobacco revealed its roles in auxiliary shoot branching and abiotic stress responses (Zhang et al., 2007; Yang et al., 2008). However, the endogenous function for AtIPK2 is still unknown. Moreover, the presence of two closely related inositol polyphosphate kinases (AtIPK2α and AtIPK2β) has raised the issue of functional redundancy for them (Zhang et al., 2007; Yang et al., 2008; Tsui and York, 2010). In this study, we demonstrate that AtIPK2α and AtIPK2β act redundantly to regulate male gametophyte and embryogenesis. Previously, Xu et al, (2005) reported that specific antisense inhibition of AtIPK2a led to enhanced pollen germination and pollen tube growth through in vitro analysis. However, the in vivo germination and tube growth of AtIPK2α-antisense pollen were not investigated. In this work, through T-DNA knockout mutants, we analyzed the in-pistil germination and tube growth of atipk2α and atipk2β single mutant pollen. Our results indicated that the pollen and pollen tube of atipk2α and atipk2β single mutant could germinate and grow normally in pistil. Moreover, our genetic results showed that the single atipk2 mutant could be normally transmitted (Table 2). Given that the atipk2a knockdown improves the pollen tube growth, this will not affect the atipk2a
This article is protected by copyright. All rights reserved.
Accepted Article
gametophyte transmission. Thus, it seems that AtIPK2a or AtIPK2β single mutation might not be of functional significance in pollen germination and pollen tube growth in vivo.
Genetic analysis demonstrated that the transmission of atipk2αatipk2β double mutant male gametophyte was severely impaired (Table 2). Approximate 20% double mutant pollen grains were observed with compromised cytoplasmic content or abnormal nuclear constitution (Figure 2). The mitosis of more that 10% pollen grains produced by atipk2α/atipk2α;AtIPK2β/atipk2β and AtIPK2α/atipk2α;atipk2β/atipk2β plants were abnormal. It is likely that the abnormal mitosis in the double mutant pollen leads to the defective pollen formation. However, the majority of atipk2αatipk2β pollens are similar with wild-type. Both in vitro and in vivo assays indicated that the germination and tube growth of viable atipk2αatipk2β double mutant pollen were competent (Figure 3). However, the directional growth of these double mutant pollen tubes towards the ovule were severely disrupted (Figure 4). These suggest that AtIPK2α and AtIPK2β specifically affect the responses of pollen tube to gametophytic maternal (ovule) guiding cues, while have no obvious effect on responses to sporophytic maternal (stigma, style and transmitting tract) signals. It should be noted that some atipk2αatipk2β pollen tubes were able to enter the ovule, but no double mutant plant could be obtained. Silique dissection proved that the embryogenesis of atipk2αatipk2β double mutant was aborted at early stage (Figure 5). The defects of atipk2αatipk2β double mutant in pollen development, pollen tube guidance and embryogenesis suggests that inositol polyphosphate signaling is essential in plant reproduction. This article is protected by copyright. All rights reserved.
Accepted Article
The function of IPK2 in modulating inositol polyphosphate signaling is conserved in all eukaryotes. Nevertheless, recent studies suggest that IPK2 also plays a kinase activityindependent role. In mammals, IPK2/IPMK could interact with AMP-activated protein kinase (AMPK) and target of rapamycin (TOR) to monitor the cell energy status and coordinate the responses to nutrient availability (Kim et al., 2011; Bang et al., 2012). In addition, catalytically inactive yeast IPK2 could restore the transcriptional regulation of argininedependent genes (Bosch and Saiardi, 2012). In these cases, kinase activity of IPK2 was not required to establish the physical association. Thus, both the kniase activity and non-kinase activity are required for the function of IPK2. However, whether the kinase activity of IPK2 is involved in specific physiological function or developmental process in plant is completely unknown. In this study, we investigated the relationship between the kinase activity and function of AtIPK2 in male gametophyte and embryogenesis (Figure 6). We found that only the normal AtIPK2β could restore the defective male gametophyte and embryogenesis of atipk2αatipk2β double mutant, while AtIPK2β variants with either lost kinase activity or inhibited 3-kinase activity could not complement the atipk2αatipk2β double mutant (Figure 6). Our results provide strong genetic evidence that the kinase activity of AtIPK2 is critical for male gametophyte and embryogenesis.
Kinase activity-dependent manner of AtIPK2 implies the requirement of intracellular messenger inositol polyphosphate in male gametophyte, especially in pollen tube guidance. In Arabidopsis, pollen tube guidance by the ovule is divided into two phases: funicular guidance and micropylar guidance. It is believed that female gametophyte could produce This article is protected by copyright. All rights reserved.
Accepted Article
guidance cues to direct the growth of pollen tube (Márton et al., 2005; Okuda et al., 2009; Takeuchi and Higashiyama, 2012). To date, only limited number of male specific components in pollen tube guidance has been identified, including POD1, K+ transporters (CHX21/CHX23), receptor-like kinases (LIP1/LIP2) and mitogen-activated protein kinases (MPK3/MPK6) (Li et al., 2011; Lu et al., 2011; Liu et al., 2013; Guan et al., 2014). Considering the role of inositol polyphosphate in signaling cascade, AtIPK2 should be a male factor directly involved in pollen tube guidance, compared with the indirect mechanism proposed for ER localized POD1 and CHX21/CHX23 (Li et al., 2011; Lu et al., 2011). Moreover, unlike the specific role for POD1, LIP1/LIP2 and MPK3/6 in either funicular guidance or micropylar guidance, AtIPK2 affects both the processes, which suggests that inositol polyphosphates might be universal signals underlying funicular guidance and micropylar guidance. Furthermore, it seems that IP5 and/or IP6 might play a more important role than IP3/IP4 in pollen tube guidance, as inhibition of the 3-kinase of AtIPK2β could not complement the male gametophyte defects of double mutant. Consistently, IP6 was reported to be more potent than IP3 in eliciting Ca2+ release (Lemtiri-Chlieh et al., 2003). In addition, Perera et al. (2008) reported that tremendous reduction of IP3 level in Arabidopsis by expressing the mammalian type 1 inositol polyphosphate 5-phosphatase caused no obvious alteration in plant morphology, indicating that IP3 alone might not be sufficient to modulate plant growth and development. Emerging evidence suggests that Ca2+ dynamics at the pollen tube tip are involved in cell-cell communication in plant reproduction (Iwano et al., 2012). Ca2+ influx facilitated by glutamate receptor-like channels (GLRs) and Ca2+ homeostasis mediated by ER luminal protein POD1 have been proposed to be required for pollen tube
This article is protected by copyright. All rights reserved.
Accepted Article
directional growth and guidance (Michard et al., 2011; Li et al., 2011). Moreover, Ca2+ has been shown to serve as a downstream messenger in nitric oxide (NO) mediated pollen tube redirection (Prado et al., 2008). For these reasons, we presume that Ca2+ signal mediated by inositol polyphosphate might also be involved in regulation of pollen tube guidance. It should be noted that no receptor for inositol polyphosphate recognition has been identified from plant (Krinke et al., 2007). Hence, elucidation of the downstream signaling events modulated by inositol polyphosphate is needed.
AtIPK2 is a central, but not the only regulator of inositol polyphosphate signaling. Other kinases like inositol 1,3,4,5,6-pentakisphosphate 2-kinase (AtIPK1) and inositol 1,3,4trisphosphate 5/6-kinase (AtITPK) are also required (Wilson et al., 1997; Shi et al., 2003; Stevenson-Paulik et al., 2005; Sweetman et al., 2006). Stevenson-Paulik et al. (2005) demonstrated that coincident disruption of AtIPK2β and AtIPK1 nearly ablates seed IP6 (phytate) biosynthesis. Hence, in addition to the specific roles for these kinases, they also likely work together to maintain the integrity and plasticity of IP signaling network. For this reason, we cannot preclude that AtIPK2 may act with AtIPK1 or AtITPK to regulate plant growth and development. This would explain the partial developmental defect for atipk2αatipk2β double mutant pollen and the lack of post-embryonic phenotype in atipk2α/atipk2α; AtIPK2β/atipk2β and AtIPK2α/atipk2α;atipk2β/atipk2β plants (Figure S3).
This article is protected by copyright. All rights reserved.
Accepted Article
In summary, our results in this study demonstrate that AtIPK2α and AtIPK2β play an essential role in pollen development, pollen tube guidance and embryogenesis in a kinase activity-dependent manner. Our data provides insights into the underlying molecular mechanism of pollen tube guidance.
EXPERIMENTAL PROCEDURES
Plant materials and growth conditions
Arabidopsis thaliana ecotype Columbia (Col-0) was used as the wild-type control in this study. The T-DNA insertion line atipk2α (GABI_879D07) was obtained from the Nottingham Arabidopsis Stock Centre. The atipk2β T-DNA mutant (SALK_104995) was originally described by Zhang et al. (2007). The T-DNA insertions were verified using primers designed by the T-DNA Primer Design website. The detailed sequences of these primers were listed in Table S1.
Seeds were surface sterilized with 75% ethanol for 5 min, then washed three times with sterile water and plated on solid Murashige and Skoog (MS) media. For growth in both plates and soil, seeds were stratified for 2 days at 4°C and grown in chamber at 23°C under16-hlight/8-h-dark conditions.
RNA Extraction and RT-PCR analysis Total RNA was isolated using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. For RT-PCR analysis, first strand cDNAs were synthesized from DNaseI-treated This article is protected by copyright. All rights reserved.
Accepted Article
total RNA using RevertAidTM cDNA Synthesis Kit (Fermentas). The Actin gene was amplified as internal controls. Gene specific primers used for RT-PCR are listed in Table S1.
Genetic analysis
For all crosses, flowers at floral stage 12c (Smyth et al., 1990) was emasculated, and pollen grains from wild-type or mutant plants were dispersed onto the recipient stigma. For selfcross analysis, plants with various genotypes were allowed to self-pollinate and progeny seeds were collected. The progeny plants were genotyped and scored with PCR. For studies of gametophyte transmission of atipk2α and/or atipk2β alleles, reciprocal crosses were performed between wild-type and mutant materials. The progenies were genotyped for the presence of atipk2α and atipk2β T-DNA insertions by PCR.
Complementation of atipk2αatipk2β double mutants For complementation, AtIPK2a and AtIPK2β genomic fragments were amplified using KODFX polymerase (Takara Biotechnology) with gene specific primers (Table S1). After verification by sequencing, the fragments were cloned into pCAMBIA1302. The resulting vectors (pAtIPK2α-AtIPK2α and pAtIPK2β-AtIPK2β) were then introduced into Agrobacterium tumefaciens GV3101 and transformed into atipk2α/atipk2α;AtIPK2β/atipk2β and AtIPK2α/atipk2α;atipk2β/atipk2β plants respectively by the floral dip method (Clough
This article is protected by copyright. All rights reserved.
Accepted Article
and Bent, 1998). Transgenic seeds were selected on half-strength MS medium supplied with 30 mg/L hygromycin.
Subcellular localization
The coding region of AtIPK2α and AtIPK2β were cloned into the pCAMBIA1302 vector to generate AtIPK2α-GFP and AtIPK2β-GFP fusion protein expression cassettes under the control of their native promoters. The resulting constructs (pAtIPK2α-AtIPK2α-GFP and pAtIPK2β-AtIPK2β-GFP) were then introduced into atipk2α/atipk2α;AtIPK2β/atipk2β and AtIPK2α/atipk2α;atipk2β/atipk2β plants, respectively. The fluorescent signals were observed under a confocal microscope (Olympus FV1000).
Generation of kinase-dead and substrate specificity-altered constructs
Point mutations that ablate (D100A and D100A;D102A) or alter (K119W;K123W) the kinase activity of AtIPK2β were created using the site-directed PCR mutagenesis. These mutated fragments were ligated into pCAMBIA1302 under the control of either the native AtIPK2β promoter or the pollen specific promoter Lat52. All constructs were verified by sequencing. The primers used for site-directed mutagenesis are listed in Table S1.
This article is protected by copyright. All rights reserved.
Accepted Article
Microscopy and phenotypic characterization
Pollen viability was assessed using Alexander’s staining (Alexander, 1969) and viewed under the light microscopy. For morphological observation of pollen grains, mature pollen were mounted on double-sided tape and coated with gold. The samples were then observed using a scanning electron microscope (SEM). For DAPI (4’,6-diamidino-2-phenylindole) staining, pollen grains were released into a staining solution [0.1 M sodium phosphate (pH7), 1 mM EDTA, 0.1% Triton X-100, 300 ng/mL DAPI] and observed under UV illumination. In vitro pollen germination analysis was performed as described by Li et al. (1999). Pollen grains were brushed evenly onto the surface of a gar plates and immediately transferred to a moisture box at 23°C for indicated time (Boavida and McCormick, 2007). For in vivo pollen germination and pollen tube growth assay, pre-emasculated wild-type flowers were pollinated with a limited number of wild-type or mutant pollen grains. The pollen tubes in the pistils were stained with aniline blue and viewed under a fluorescence microscope. To visualize the pollen tube growth in pistils, aniline blue staining was performed as described by Huck et al. (2003). Siliques were fixed overnight in FAA solution (1.5% formaldehyde, 2% acetic acid and 30% ethanol), and washed in an alcohol series (70, 50, 30 and 10%) for 10 min for each step. The sample was further softened with 10% chloral hydrate at 60°C for 10 min, subsequently rinsed twice with sodium phosphate buffer (100 mM, pH 7) and then in 5 M NaOH at 60°C, for 5 minutes. Pollen tubes were stained with 0.1% aniline blue for 4 h under dark and observed with a microscope under UV irradiation condition. Embryos were obtained by removing the seed coat, and the isolated embryos were then rinsed at least three times with distilled water. For single-embryo PCR, embryo DNA isolation was performed as described This article is protected by copyright. All rights reserved.
Accepted Article
by Park et al. (2008). Isolated embryos were homogenized in 40 μL 250 mM NaOH followed by heating to 100°C for 30 s. To the sample, 20 μL buffer (500 mM Tris-HCl, pH 8.0, 0.25% [v/v] NP-40) followed by 40 μL of 250 mM HCl were added, and the sample was mixed and incubated at 100°C for 2 min. Samples were cooled to ambient temperature and centrifuged at ambient temperature for 10 min at 16,000 g. The supernatant was used directly for PCR analysis using allele-specific primers. For embryo observation, developing seeds were clarified in Hoyer’s solution (Liu and Meinke, 1998) for 24 h and observed with Olympus microscope equipped with differential interference contrast (DIC) system.
ACKNOWLEDGEMENTS We sincerely thank Prof. De Ye (China Agricultural University) for providing quartet1 mutant. This work was supported by the National Natural Science Foundation of China (Grant no. 31271319 and 31170270).
SUPPORTING INFORMATION
Figure S1. Phenotypic characterization of atipk2α and atipk2β single mutants
Figure S2. Development of atipk2αatipk2β double mutant pollen in quartet1-6 background
Figure S3. Phenotypic analysis of atipk2α/atipk2α;AtIPK2β/atipk2β and AtIPK2α/ atipk2α;atipk2β/atipk2β self-crossed progeny
This article is protected by copyright. All rights reserved.
Accepted Article
Table S1. List of synthetic oligonucleotide sequences used in this study
Table S2. Analysis of the complementation of atipk2αatipk2β double mutant by AtIPK2β* variants under control of the native promoter.
REFERENCE Alexander, M.P. (1969) Differential staining of aborted and non-aborted pollen. Stain Technol. 44, 117-122. Bang, S., Kim, S., Dailey, M.J., Chen, Y., Moran, T.H., Snyder, S.H., and Kim, S.F. (2012) AMP-activated protein kinase is physiologically regulated by inositol polyphosphate multikinase. Proc. Natl. Acad. Sci. USA. 109, 616-620. Bercy, J., Dubois, E., and Messenguy, F. (1987) Regulation of arginine metabolism in Saccharomyces cerevisiae: expression of the three ARGR regulatory genes and cellular localization of their products. Gene. 55, 277-285. Boavida, L.C. and McCormick, S. (2007) Temperature as a determinant factor for increased and reproducible in vitro pollen germination in Arabidopsis thaliana. Plant J. 52, 570-582. Bosch, D. and Saiardi, A. (2012) Arginine transcriptional response does not require inositol phosphate synthesis. J. Biol. Chem. 287, 38347-38355. Boss, W.F. and Im, Y.J. (2012) Phosphoinositide Signaling. Annu. Rev. Plant Biol. 63, 409429. Burnette, R.N., Gunesekera, B.M. and Gillaspy, G.E. (2003) An Arabidopsis inositol 5phosphatase gain-of-function alters abscisic acid signaling. Plant Physiol. 132, 1011-1019.
This article is protected by copyright. All rights reserved.
Accepted Article
Cheung, A.Y., Boavida, L.C., Aggarwal, M., Wu, H.M. and Feijo, J.A. (2010) The pollen tube journey in the pistil and imaging the in vivo process by two-photon microscopy. J. Exp. Bot. 61, 1907-1915. Clough, S.J. and Bent, A.F. (1998) Floral dip: a simplified method for Agrobacteriummediated transformation of Arabidopsis thaliana. Plant J. 16, 735-743. Dubois, E., Dewaste, V., Erneux, C. and Messenguy, F. (2000) Inositol polyphosphate kinaseactivity of Arg82/ArgRIII is not required for the regulation of the arginine metabolism in yeast. FEBS Lett. 486, 300-304. El Alami, M., Messenguy, F., Scherens, B. and Dubois, E. (2003) Arg82p is a bifunctional protein whose inositol polyphosphate kinase activity is essential for nitrogen and PHO gene expression but not for Mcm1p chaperoning in yeast. Mol. Microbiol. 49, 457-468. Endo-Streeter, S., Tsui, M.K., Odom, A.R., Block, J. and York, J.D. (2012) Structural studies and protein engineering of inositol phosphate multikinase. J. Biol. Chem. 287, 35360-35369. Guan, Y.F., Lu, J.P., Xu, J., McClure, B. and Zhang, S.Q. (2014) Two mitogen- activated protein kinases, MPK3 and MPK6, are required for funicular guidance of pollen tubes in Arabidopsis. Plant Physiol. 165, 528-533. Huck, N., Moore, J.M., Federer, M. and Grossniklaus, U. (2003) The Arabidopsis mutant feronia disrupts the female gametophytic control of pollen tube reception. Development 130, 2149-2159. Hülskamp, M., Schneitz, K. and Pruitt, R. (1995) Genetic evidence for a long-range activity that directs pollen tube guidance in Arabidopsis. Plant Cell 7, 57-64. Kim, S., Kim, S.F., Maag, D., Maxwell, M.J., Resnick, A.C., Juluri, K.R., Chakraborty, A., Koldobskiy, M.A., Cha, S.H., Barrow, R, Snowman, A.M. and Snyder, SH. (2011)
This article is protected by copyright. All rights reserved.
Accepted Article
Amino acid signaling to mTOR mediated by inositol polyphosphate multikinase. Cell Metab. 13, 215-221 Krinke, O., Novotna, Z., Valentova, O. and Martinec, J. (2007) Inositol trisphosphate receptor in higher plants: is it real? J. Exp. Bot. 58, 361-376. Lemtiri-Chlieh F., MacRobbie E.A., Webb A.A., Manison N.F., Brownlee C., Skepper J.N., Chen J., Prestwich G.D. and Brearley C.A. (2003) Inositol hexakisphosphate mobilizes an endomembrane store of calcium in guard cells. Proc. Natl. Acad. Sci. USA. 100, 10091-10095. Li, H., Lin, Y.K., Heath, R.M., Zhu, M.X. and Yang, Z.B. (1999) Control of pollen tube tip growth by a Rop GTPase-dependent pathway that leads to tip-localized calcium influx. Plant Cell 11, 1731-1742. Li, H.J., Xue, Y., Jia, D.J., Wang, T., Hi, D.Q., Liu, J., Cui, F., Xie, Q., Ye, D. and Yang, W.C. (2011) POD1 regulates pollen tube guidance in response to micropylar female signaling and acts in early embryo patterning in Arabidopsis. Plant Cell, 23, 3288-3302. Liu, C.M. and Meinke, D.W. (1998) The titan mutants of Arabidopsis are disrupted in mitosis and cell cycle control during seed development. Plant J.16, 21-31. Liu, J.J., Zhong, S., Guo, X.Y., Hao, L.H., Wei, X.L., Huang, Q.P., Hou, Y.N., Shi, J., Wang, C.Y., Gu, H.Y. and Qu, L.J. (2013) Membrane-bound RLCKs LIP1 and LIP2 are essential male factors controlling male-female attraction in Arabidopsis. Curr. Biol. 23, 993-998. Lu, Y., Chanroj, S., Zulkifli, L., Johnson, M., Uozumi, N., Cheung, A. and Sze, H. (2011) Pollen tubes lacking a pair of K+ transporters fail to target ovules in Arabidopsis. Plant Cell 23, 81-174. Maag, D., Maxwell, M.J., Hardesty, D.A., Boucher, K.L., Choudhari, N., Hanno, A.G., Ma, J.F., Snowman, A.S., Pietropaoli, J.W., Xu, R., Storm, P.B., Saiardi, A., Snyder,
This article is protected by copyright. All rights reserved.
Accepted Article
S.H. and Resnick, A.C. (2011) Inositol polyphosphate multikinase is a physiologic PI3kinase that activates Akt/PKB. Proc. Natl. Acad. Sci. USA. 108, 1391-1396. Márton, M.L., Cordts, S., Broadhvest, J. and Dresselhaus, T. (2005) Micropylar pollen tube guidance by egg apparatus 1 of maize. Science 307, 573-576. Márton, M.L. and Dresselhaus, T. (2010) Female gametophyte-controlled pollen tube guidance. Biochem. Soc. Trans. 38, 627-630. McCormick, S. (1993) Male Gametophyte Development. Plant Cell 5, 1265-1275. Michard, E., Lima, P.T., Borges, F., Silva, A.C., Portes, M.T., Carvalho, J.E., Gilliham, M., Liu, L.H., Obermeyer, G. and Feijo, J.A. (2011) Glutamate receptor like genes form Ca2+ channels in pollen tubes and are regulated by pistil D-serine. Science 332, 434-437. Odom, A.R., Stahlberg, A., Wente, S.R. and York, J.D. (2000) A role for nuclear inositol 1,4,5-trisphosphate kinase in transcriptional control. Science 287, 2026-2029. Okuda, S., Tsutsui, H., Shiina, K., Sprunck, S., Takeuchi, H., Yui, R., Kasahara, R.D., Hamamura, Y., Mizukami, A., Susaki, D., Kawano, N., Sakakibara, T., Namiki, S., Itoh, K., Otsuka, K., Matsuzaki, M., Nozaki, H., Kuroiwa, T., Nakano, A., Kanaoka, M.M., Dresselhaus, T., Sasaki, N. and Higashiyama, T. (2009) Defensin-like polypeptide LUREs are pollen tube attractants secreted from synergid cells. Nature 458, 357-361. Palanivelu, R. and Tsukamoto, T. (2012) Pathfinding in angiosperm reproduction: pollen tube guidance by pistils ensures successful double fertilization. WIREs Dev Biol. 1, 96-113. Park, S., Rancour, D.M. and Bednarek, S.Y. (2008) In planta analysis of the cell cycledependent localization of AtCDC48A and its critical roles in cell division, expansion, and differentiation. Plant Physiol. 148, 246-258.
This article is protected by copyright. All rights reserved.
Accepted Article
Perera, I.Y., Hung, C.Y., Moore, C.D., Stevenson-Paulik, J. and Boss, W.F. (2008) Transgenic Arabidopsis plants expressing the type I inositol 5-phosphatase exhibit increased drought tolerance and altered abscisic acid signaling. Plant Cell 20, 2876-2893. Preuss, D., Rhee, S.Y. and Davis, R.W. (1994) Tetrad analysis possible in Arabidopsis with mutation of the QUARTET (QRT) genes. Science 264, 1458-1460. Prado, A.M., Colaco, R., Moreno, N., Silva, A.C. and Feijó, J.A. (2008) Targeting of pollen tubes to ovules is dependent on nitric oxide (NO) signaling. Mol. Plant. 1, 703-714. Resnick, A.C., Snowman, A.M., Kang, B.N., Hurt, K.J., Snyder, S.H. and Saiardi, A. (2005) Inositol polyphosphate multikinase is a nuclear PI3 kinase with transcriptional regulatory activity. Proc. Natl. Acad. Sci. USA. 102, 12783-12788. Seeds, A.M., Frederick, J.P., Tsui, M.M. and York, J.D. (2007) Roles for inositol polyphosphate kinases in the regulation of nuclear processes and developmental biology. Adv. Enzyme Regul. 47, 10-25. Shears, S.B. (2004) How versatile are inositol phosphate kinases? Biochem. J. 377, 265-280. Shi, J.R., Wang, H.Y., Wu, Y.S., Hazebroek, J., Meeley, R.B. and Ertl, D.S. (2003) The maize low-phytic acid mutant lpa2 is caused by mutation in an inositol phosphate kinase gene. Plant Physiol. 131, 507-515. Staxen, I.I., Pical, C., Montgomery, L.T., Gray, J.E., Hetherington, M. and McAinsh, M.R. (1999) Abscisic acid induces oscillations in guard-cell cytosolic free calcium that involve phosphoinositide-specific phospholipase C. Proc. Natl. Acad. Sci. USA 96, 17791784. Steger, D.J., Haswell, E.S., Miller, A.L., Wente, S.R. and OòShea, E.K. (2003) Regulation of chromatin remodeling by inositol polyphosphates. Science 299, 114-116.
This article is protected by copyright. All rights reserved.
Accepted Article
Stevenson-Paulik, J., Bastidas, R.J., Chiou, S.T., Frye, R.A. and York, J.D. (2005) Generation of phytate-free seeds in Arabidopsis through disruption of inositol polyphosphate kinases. Proc. Natl. Acad. Sci. USA 102, 12612-12617. Stevenson-Paulik, J., Odom, A.R. and York J.D. (2002) Molecular and biochemical characterization of two plant inositol polyphosphate 6-/3-/5-kinases. J. Biol. Chem. 277: 42711-42718. Smyth, D.R., Bowman, J.L. and Meyerowitz, E.M. (1990) Early flower development in Arabidopsis. Plant Cell 2, 755-767. Sweetman, D., Johnson, S., Caddick, S.E., Hanke, D.E. and Brearley, C.A. (2006) Characterization of an Arabidopsis inositol 1,3,4,5,6-pentakisphosphate 2-kinase (AtIPK1). Biochem. J. 394, 95-103. Takeuchi, H. and Higashiyama, T. (2012) A species-specific cluster of defensin-like genes encodes diffusible pollen tube attractants in Arabidopsis. PLoS Biol. 10, e1001449. Tsui,
M.M.
and
York,
J.D.
(2010)
Roles of inositol
phosphates and inositol
pyrophosphates in development, cell signalingand nuclear processes. Adv Enzyme Regul. 50, 324-337. Wilson, M.P. and Majerus, P.W. (1997) Characterization of a cDNA encoding Arabidopsis thaliana inositol 1,3,4-trisphosphate 5/6-kinase, Biochem. Biophys. Res. Commun. 232, 678-681. Xia, H.J., Brearley, C., Elge, S., Kaplan, B., Fromm, H. and Mueller-Roeber, B. (2003) Arabidopsis inositol polyphosphate 6-/3-kinase is a nuclear protein that complements a yeast mutant lacking a functional ArgR-Mcm1 transcription complex. Plant Cell 15, 449463. Xia, H.J. and Yang, G. (2005) Inositol 1,4,5-trisphosphate 3-kinases: functions and regulations. Cell Res. 15, 83-91.
This article is protected by copyright. All rights reserved.
Accepted Article
Xu, J., Brearley, C.A., Lin, W.H., Wang, Y., Ye, R., Mueller-Roeber, B., Xu, Z.H. and Xue, H.W. (2005) A role of Arabidopsis inositol polyphosphate kinase AtIPK2α, in pollen germination and root growth. Plant Physiol. 137, 94-103. Yang, L., Tang, R.J., Zhu, J.Q., Liu, H., Mueller-Roeber, B., Xia, H.J. and Zhang, H.X. (2007) Enhancement of stress tolerance in transgenic tobacco plants constitutively expressing AtIPK2β, an inositol polyphosphate 6-/3-kinase from Arabidopsis thaliana. Plant Mol. Biol. 66, 329-343. York, J.D., Odom, A.R., Murphy, R., Ives, E.B. and Wente, S.R. (1999) A phospholipase C-dependent inositol polyphosphate kinase pathway required for efficient messenger RNA export. Science 285, 96-100. Zhang, Z.B., Yang, G., Arana, F., Chen, Z., Li, Y. and Xia, H.J. (2007) Arabidopsis inositol polyphosphate 6-/3-kinase (AtIpk2β) is involved in auxiliary shoot branching via auxin signaling. Plant Physiol. 144, 942-951.
TABLES Table 1. Segregation analysis of atipk2α and atipk2β mutants Parent genotype AtIPK2α/atipk2α;AtIPK2β/AtIPK2β
AABB
AaBB
aaBB
(AaBB)
35
67
34
AtIPK2α/AtIPK2α;AtIPK2β/atipk2β
AABB
AABb
AAbb
(AABb)
28
57
30
aaBB
aaBb
aabb
291
284
0
AAbb
Aabb
aabb
326
335
0
atipk2α/atipk2α;AtIPK2β/atipk2β (aaBb) AtIPK2α/atipk2α;atipk2β/atipk2β (Aabb) a
Genotype of progeny
Expected
Observed
1:2:1
1:1.91:0.97
1:2:1
1:2.04:1.07
1:2:1
1:0.98:0a
1:2:1
1:1.03:0a
Significantly different from the expected 1:2:1 segregation ratio (P
