at the intersection: merging ca2+ and ros signaling pathways in pollen

At the intersection: Merging Ca2+ and ROS 1

signaling pathways in pollen 2

3

Michael M. Wudick and José A. Feijó 4

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Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, 6 Maryland 20742-5815 7

and 8

Instituto Gulbenkian de Ciencia, 2780-156 Oeiras, Portugal 9

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Correspondence to [email protected] 11

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The mixed blessing of ROS: Don't you know that I'm toxic? 16

Due to plant's aerobic metabolism, reactive oxygen species (ROS) continuously 17 accrue in different tissues and organs as a by-‐product of many metabolic reactions. 18

Despite their toxic activity, plants also rely on ROS as a major form of second 19 messenger to integrate, induce and/or propagate biotic and abiotic signals and 20 signaling cascades. Being small, short lived and rapidly diffusible, makes ROS ideal 21 messenger molecules for local biochemical reactions. Hence, a delicate fine-‐tuning of 22 localized ROS production and signaling events is crucial for many physiologic 23 processes. 24

In pollen tube (PT) growth, ROS were shown to be involved in various processes, 25 including germination (Speranza et al., 2012), polarized growth (Potocký et al., 26 2007), elongation (Lassig et al., 2014), guidance and ovule targeting (Prado et al., 27 2008) as well as PT burst during fertilization (Duan et al., 2014). 28

Genetic evidence that production of ROS could be associated to members of the 29 respiratory burst oxidase homolog (Rboh) family of the plasma membrane localized 30 and PT-‐specific NADP(H) oxidases (NOX) was first shown by Potocký et al. (2007). 31 More recently, three groups provided genetic evidence that RbohH and RbohJ seem 32 to be essential for PT growth and fertilization (Boisson-‐Dernier et al., 2013; Lassig et 33

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al., 2014; Kaya et al., 2014). These proteins consist of six integral trans-‐membrane 1 segments and bear a FAD/NADP(H) binding domain in their C-‐terminal region. Like 2 other NOX, RobhH and J display two Ca2+-‐binding EF-‐hand motifs in the cytosolic N-‐3 terminus, suggesting a direct functional link between Ca2+ and ROS (Steinhorst and 4 Kudla, 2013). Circumstantial evidence for this link initially came by externally 5 applying Ca2+ to trigger an increase of ROS production in tobacco PT (Potocký et al., 6 2007). Discrepancies exist, however on the interpretation of these results. Double 7 mutants of RobhH and J were shown to have no detectable H2O2 in the cytosol, and 8 were functionally linked to the action of the receptor-‐like kinases (RLKs) ANXURs and 9 to the modulation of cytosolic free Ca2+, putatively by ROS’ control of Ca2+ channels, 10 and possibly through Rho GTPases (Boisson-‐Dernier et al., 2013). More Recently, 11 Duan et al. (2014), by making use of feronia (the RLK female homologue of ANXUR), 12 suggested that exogenous ROS, either applied to in vitro or emanating from ovules, 13 induce Ca2+ influx just prior to PT rupture. However, point mutations of the EF-‐hand 14 motifs of RobhH and J showed impaired ROS production (Kaya et al., 2014), in 15 apparent contradiction of ROS being upstream of Ca2+. Complex patterns of cytosolic 16 Ca2+ and growth-‐rate were also described when growing robhH and J double knock-‐17 out PTs in vitro (Lassig et al., 2014), which could support any of these views, or both, 18 depending on the way one interprets the sequence of events. 19

Things are further complicated by the finding that phosphorylation and Ca2+ signaling 20 act synergistically as part of a positive feedback loop that leads to ROS production in 21 plant cells, as recently demonstrated for RbohF, where phosphorylation was shown 22 necessary for Ca2+ activation of ROS production (Kimura et al., 2012). Although up to 23 date there are no genes that could account for its production in plants, it is worth 24 noting that another relevant ROS species, nitric oxide (NO), was shown to modulate 25 PT growth and orientation in a Ca2+ dependent manner (Prado et al., 2008). 26

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Putting it all together? A positive feedback model for ROS/ Ca2+signaling in pollen 28

In a minimalistic model (Figure 1), Rbohs would be activated by an initial 29 phosphorylation step, which allows them to bind Ca2+ through their EF-‐hand motifs. 30 Ca2+ binding triggers the production of ROS, which can also act on plasma membrane 31 Ca2+ channels, leading to an increase of the cytosolic Ca2+ concentration. The 32 increase in Ca2+ can also activate Ca2+-‐dependent protein kinases (CPKs) that in 33 return might amplify the phosphorylation signal. Small GTPases are likely to 34 integrate different signals in the ROS/Ca2+ signaling network, as it has been 35 demonstrated in root hairs (Cheung and Wu, 2011) and proposed in PT growth (see 36 for example Duan et al., 2014). While many experimental gaps are still present, 37 identification of the PT CPKs and Ca2+ channels (Konrad et al., 2011) involved 38 emerges as a crucial step forward. 39

CPK17 and CPK34 are candidates for Ca2+-‐dependent protein kinases that are highly 40 expressed in pollen and localize to the PT plasma membrane; their double loss-‐of-‐41

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function mutants revealed impaired PT growth and fertilization (Myers et al., 2009). 1 CPK32, CPK2 and CPK20 have also been recently implied in the regulation of PT 2 growth (Zhou et al. 2014; Gutermuth et al., 2013). The issue of specificity is still open 3 for this diverse and relevant family of kinases, and multiple targets are known for 4 each, suggesting that they target other proteins, including Rbohs. Other 5 phosphorylation enzymes, like the CBL/CIPK pairs are also possible candidates 6 (Konrad et al., 2011; Mähs et al. 2013). Indeed, it has been recently shown that the 7 direct binding of CIPK26 to the N-‐terminus of AtRbohF negatively modulates ROS 8 production when heterologously expressed in HEK cells (Kimura et al., 2013), thereby 9 establishing a molecular pathway to repress NOX activity. Further work showed that 10 CIPK26 is dependent on either CBL1 or 9 (Drerup et al. 2013). 11

More uncertainties exist about possible Ca2+ channels to be regulated by ROS. To 12 date the characterized arsenal of plant bona fide Ca2+ channels that could account 13 for the ROS-‐activated [Ca2+]cyt increase is scarce. Members of the cyclic nucleotide 14 gated channel (CNGC) family along with channels encoded by the glutamate 15 receptor-‐like (GLR) genes are the only candidates with a proven function as Ca2+ 16 channels. Loss-‐of-‐function lines for members of both families display phenotypes in 17 PT germination and reproduction (reviewed in Konrad et al. 2011; Gao et al. 2014). 18 Data is missing of any putative ROS regulation of any of these. 19

In tip-‐growing cells, small GTPases from the RAC/ROP family play important roles in 20 the integration of signaling cascades. It is thus not astonishing that they were also 21 shown to act on NOX. For instance, expression of pollen enriched NtRAC5 in tobacco 22 PT led to an increased ROS production while expression of a dominant-‐negative 23 RAC5 version led to reduced ROS levels (Potocký et al., 2012). An implication of small 24 GTPases in targeting NOX to the plasma membrane has also been proposed for ROP1 25 by acting on the assembly status of F-‐actin (Kaya et al., 2014). This status however is 26 also tightly regulated by the local Ca2+ concentration that affects the majority of 27 cytoskeleton proteins. 28

By necessity, this minimal feedback model is affected by the lack of characterization 29 of still many interacting partners. The ubiquitous action of ROS and cytosolic free 30 Ca2+ makes it very likely that what we presently know about Ca2+ binding/regulated 31 proteins and what they have to tell about the fine-‐tuning of the ROS/Ca2+ signaling is 32 only part of the whole picture. But, as in many other processes, PTs are likely to 33 stand out as one of the effective models to dissect this complexity, as two common 34 patterns for both ROS and Ca2+-‐derived signals emerge: that they are both generated 35 or focused at the tip of the PT; and that this spatial overlap in return is fundamental 36 for pollen tube growth. 37

Acknowledgements:

JF’s lab is funded by Fundação para a Ciência e Tecnologia (PTDC/BEX-‐BCM/0376/2012 and PTDC/BIA-‐PLA/4018/ 2012). MW acknowledges an FCT

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fellowship (SFRH/BPD/70739/2010). We thank Alice Cheung for careful reading of the manuscript. Many references were left out due to space constraints, we apologize the authors for that.

Cited Literature:

Boisson-‐Dernier, A., Lituiev, D., Nestorova, A., Franck, C.M., Thirugnanarajah, S., Grossniklaus, U. (2013) ANXUR Receptor-‐Like Kinases Coordinate Cell Wall Integrity with Growth at the Pollen Tube Tip Via NADPH Oxidases. PLoS Biol 11(11): e1001719.

Cheung, A.Y., Wu, H.M. (2011) THESEUS 1, FERONIA and relatives: a family of cell wall-‐sensing receptor kinases? Curr Opin Plant Biol. 14:632-‐41.

Drerup M.M., Schlücking, K., Hashimoto, K., Manishankar, P., Steinhorst, L., Kuchitsu, K., Kudla, J. (2013) The Calcineurin B-‐like calcium sensors CBL1 and CBL9 together with their interacting protein kinase CIPK26 regulate the Arabidopsis NADPH oxidase RBOHF. Mol Plant. 6:559-‐69

Duan, Q., Kita, D., Johnson, E.A., Aggarwal, M., Gates, L., Wu, H.-‐M. and Cheung, A.Y. (2014). Reactive oxygen species mediate pollen tube rupture to release sperm for fertilization in Arabidopsis. Nat. Commun. 5:3129.

Gao, Q.-‐F., Fei, C.-‐F., Dong, J.-‐Y., Gu, Li-‐Li, Wang, Y.-‐F. (2014) Arabidopsis CNGC18 Is a Ca2+-‐Permeable Channel. Mol.Plant. 7:739-‐743

Gutermuth, T., Lassig, R., Portes, M.T., Maierhofer, T., Romeis, T., Borst, J.W., Hedrich, R., Feijó, J.A., Konrad, K.R. (2013) Pollen tube growth regulation by free anions depends on the interaction between the anion channel SLAH3 and calcium-‐dependent protein kinases CPK2 and CPK20. Plant Cell. 25:4525-‐43

Kaya, H., Kakajima, R., Iwano, M., Kanaoka, M.M., Kimura, S., Takeda, S., Kawarazaki, T., Senzaki, E., Hamamura, Y., Higashiyama, T., Takayama, S., Abe, M., Kuchitsu, K. (2014). Ca2+-‐Activated Reactive Oxygen Species Production by Arabidopsis RbohH and RbohJ Is Essential for Proper Pollen Tube Tip Growth. Plant Cell. 26:1069-‐1080

Kimura, S., Kaya, H., Kawarazaki, T., Hiraoka, G., Senzaki, E., Michikawa, M. and Kuchitsu, K. (2012). Protein phosphorylation is a prerequisite for the Ca2+-‐dependent activation of Arabidopsis NADPH oxidases and may function as a trigger for the positive feedback regulation of Ca2+ and reactive oxygen species. Biochim. Biophys. Acta. 1823:398–405.

Kimura, S., Kawarazaki, T., Nibori, H., Michikawa, M., Imai, A., Kaya, H., & Kuchitsu, K. (2013). The CBL-‐interacting protein kinase CIPK26 is a novel interactor of

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Dow

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Arabidopsis NADPH oxidase AtRbohF that negatively modulates its ROS-‐producing activity in a heterologous expression system. Journal of Biochemistry, 153(2), 191–5. doi:10.1093/jb/mvs132

Konrad, K.R., Wudick, M.M. and Feijó, J.A. (2011). Calcium regulation of tip growth: new genes for old mechanisms. Curr. Opin. Plant Biol. 14:721–30.

Lassig, R., Gutermuth, T., Bey, T.D., Konrad, K.R. and Romeis, T. (2014). Pollen tube NAD(P)H oxidases act as a speed control to dampen growth rate oscillations during polarized cell growth. Plant J. 78:94–106.

Mähs A, Steinhorst L, Han JP, Shen LK, Wang Y, Kudla J. (2013) The calcineurin B-‐like Ca2+ sensors CBL1 and CBL9 function in pollen germination and pollen tube growth in Arabidopsis. Mol Plant. 6:1149-‐62

Myers, C., Romanowsky, S.M., Barron Y.D., Garg, S., Azuse, C.L., Curran, A., Davis, R.M., Hatton, J., Harmon, A.C., Harper, J.F. (2009) Calcium-‐dependent protein kinases regulate polarized tip growth in pollen tubes. Plant J. 59:528-‐39

Ogasawara, Y. et al. (2008). Synergistic activation of the Arabidopsis NADPH oxidase AtrbohD by Ca2+ and phosphorylation. J. Biol. Chem. 283:8885–92.

Potocký, M., Jones, M.A., Bezvoda, R., Smirnoff, N. and Zárský, V. (2007). Reactive oxygen species produced by NADPH oxidase are involved in pollen tube growth. New Phytol. 174:742–51.

Potocký, M., Pejchar, P., Gutkowska, M., Jiménez-‐Quesada, M.J., Potocká, A., Alché, J. de D., Kost, B. and Žárský, V. (2012). NADPH oxidase activity in pollen tubes is affected by calcium ions, signaling phospholipids and Rac/Rop GTPases. J. Plant Physiol. 169:1654–63.

Prado, A.M., Colaço, 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. 1:703–14.

Speranza, A., Crinelli, R., Scoccianti, V. and Geitmann, A. (2012). Reactive oxygen species are involved in pollen tube initiation in kiwifruit. Plant Biol. 14:64–76.

Steinhorst, L., Kudla, J. (2013) Calcium and reactive oxygen species rule the waves of signaling. Plant Physiol. 163:471-‐85

Zhou, L., Lan, W., Jiang, Y., Fang, W., Luan, S. (2014) A Calcium-‐Dependent Protein Kinase Interacts with and Activates A Calcium Channel to Regulate Pollen Tube Growth. Mol.Plant. 7:369-‐376

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at the intersection: merging ca2+ and ros signaling pathways in pollen

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