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Title: Defensin-Like ZmES4 Mediates Pollen Tube Burst in Maize via Opening of the Potassium Channel KZM1}
Article:
Abstract:
In contrast to animals and lower plant species, sperm cells of flowering plants are non-motile and are transported to the female gametes via the pollen tube, i. e. the male gametophyte. Upon arrival at the female gametophyte two sperm cells are discharged into the receptive synergid cell to execute double fertilization. The first players involved in inter-gametophyte signaling to attract pollen tubes and to arrest their growth have been recently identified. In contrast the physiological mechanisms leading to pollen tube burst and thus sperm discharge remained elusive. Here, we describe the role of polymorphic defensin-like cysteine-rich proteins ZmES1-4( Zea mays embryo sac) from maize, leading to pollen tube growth arrest, burst, and explosive sperm release. ZmES1-4 genes are exclusively expressed in the cells of the female gametophyte. ZmES4-GFP fusion proteins accumulate in vesicles at the secretory zone of mature synergid cells and are released during the fertilization process. Using RNAi knock-down and synthetic ZmES4 proteins, we found that ZmES4 induces pollen tube burst in a species-preferential manner. Pollen tube plasma membrane depolarization, which occurs immediately after ZmES4 application, as well as channel blocker experiments point to a role of K+-influx in the pollen tube rupture mechanism. Finally, we discovered the intrinsic rectifying K+ channel KZM1 as a direct target of ZmES4. Following ZmES4 application, KZM1 opens at physiological membrane potentials and closes after wash-out. In conclusion, we suggest that vesicles containing ZmES4 are released from the synergid cells upon male-female gametophyte signaling. Subsequent interaction between ZmES4 and KZM1 results in channel opening and K+ influx. We further suggest that K+ influx leads to water uptake and culminates in osmotic tube burst. The species-preferential activity of polymorphic ZmES4 indicates that the mechanism described represents a pre-zygotic hybridization barrier and may be a component of reproductive isolation in plants.
Introduction:
Flowering plants( angiosperms) emerged some 180–140 MYA[1] and have since inhabitated most ecological environments, which are often far away from humid conditions. Hence, new reproductive mechanisms were acquired to keep cells from drying out and to realize species-specific interactions between male and female reproductive structures, partly over long distances. Adaptive selection led to the reduction of the haploid male gametophyte to a three-cellular pollen grain and pollen tube, respectively, which is able to be transported over long distances and to grow deeply inside female reproductive tissues. As a further consequence the sperm cells lost their motility. The reduced female gametophyte( embryo sac), which is haploid in most plant species, is deeply embedded and protected in the maternal tissues of the ovule and ovary and harbors the female gametes( egg and central cell) as well as some accessory cells( synergid and antipodal cells; Figure 1A). Of these, the synergid cells are involved in pollen tube signaling, sperm delivery, and transport[2]. Extensive cell-cell communication events are likely to take place between both gametophytes, among male and female gametophyte cells, respectively, as well as the surrounding sporophytic tissue[3]. The central events preceding fertilization involve signaling towards the pollen tube to arrest growth and to induce discharge of the two sperm cells, a process first described by the famous German-Polish botanist Eduard Strasburger in 1884[4]. It took more than 120 years to identify the first molecular players involved in these processes. Cross-talk between both gametophytes to arrest pollen tube growth has recently been shown to depend on the FER receptor-like kinase( RLK)[5] and the GPI-anchored protein LRE[6] localized at the synergid plasma membrane. Nitric oxide and reactive oxygen species seem to play additional roles in this process[7],[8], and the activity of the Ca2+ pump ACA9[9] localized at the pollen tube plasma membrane was shown to be required for pollen tube rupture. We were investigating whether small secreted cysteine-rich proteins( CRPs), specifically expressed in the embryo sac cells, play a role in these processes. Plant genomes encode large classes of CRPs accounting for more than 800 genes in Arabidopsis and approximately 600 genes in rice. Among them, defensins/defensin-like proteins( DEFs/DEFLs), lipid transfer proteins( LTPs), rapid alkanization factor( RALF) proteins, and thionins resemble the largest classes[10]. The majority of these genes are expressed in reproductive tissues and a number of genes have been shown to be specifically expressed in the cells of the embryo sac[11],[12],[13],[14],[15],[16]. Here, we report the functional analysis of a small gene-family of four members encoding DEFL proteins in the maize inbred line A188. All four genes are specifically expressed in the embryo sac and have been shown previously to be down-regulated immediately upon fertilization[12].
Discussion:
After the first description of fertilization in flowering plants more than 120 y ago[4], we are now beginning to understand the underlying molecular and physiological mechanisms involved in pollen tube growth arrest and burst leading to sperm discharge and gamete fusion. Here, we described the activity of the synergid cell secreted DEFL protein ZmES4, required for species-preferential pollen tube burst in maize and its activity on the pollen tube expressed potassium channel KZM1. ZmES4 application triggered membrane depolarization and opening of the pollen tube expressed potassium channel KZM1 at physiological conditions before tube burst, which occurred on average within less than a minute. ZmES4 action is very fast, suggesting that a rapid decrease in the cytosolic water potential represents the main physiological cause of pollen tube burst. Moreover, ZmES4 activity was significantly delayed in the presence of Cs+, a K+ channel specific blocker, supporting the finding that changes in potassium fluxes play a central role for pollen tube burst in maize. Indeed, osmotic processes represent important forces driving plant growth, movements, and development. Besides sugars, potassium is the major osmotically active solute to maintain plant cell turgor and drives irreversible cell expansion and reversible changes in cell volume[27]. Although the potassium concentration in the filiform apparatus, the entry point of the pollen tube inside the embryo sac, is not known, it was shown that synergid cells contain considerably high potassium concentrations[28]. Once released by the degenerating receptive synergid cell, elevated apoplastic potassium concentrations together with a given hyperpolarized pollen tube membrane potential might provide the necessary driving force allowing rapid potassium influx upon K+ channel opening. This hypothesis is further supported by the observation that high cytosolic potassium concentrations in synergid cells are concomitantly lost with pollen tube discharge[28]. In summary we showed that ZmES4-GFP fusion proteins accumulate before fertilization in vesicles at the secretory zone of mature synergid cells, which have been considered to represent the glandular cells of the female gametophyte[29],[30],[31]. Upon pollen tube arrival, vesicles contents seem to be released and disappear within 24 h. Using an RNAi knock-down approach, we found that pollen tubes were guided towards the female gametes but fail to release their contents and occasionally show overgrowth inside the egg apparatus. Using chemically synthesized ZmES4 protein, we further showed that it induced pollen tube rupture in vitro in a species-preferential manner. Moreover, ZmES4 converts the potassium channel KZM1 from a voltage-dependent inward rectifier in a voltage independent, non-rectifying channel. KZM1 is assumed to represent a major housekeeping channel in many tissues and accounts for K+ homeostasis. KZM1 has also been considered as a mediator of K+ uptake into the phloem as well as guard cells in the leaf epidermis[26]. Pollen tubes have been shown to be especially sensitive to osmotic changes and it is therefore not surprising that a large number of ion channels are involved in osmotic adjustment and K+ homeostasis and are expressed during pollen development and tube growth( see also Figure S6)[32]. KZM1 is strongly expressed in pollen tubes, but it cannot be excluded that ZmES proteins also act at other pollen tube expressed targets including Ca2+ channels—a property reminiscent to animal toxins capable of modulating various ion channels simultaneously[18],[33]. However, all described activities are capable to lead to a rapid increase in osmotic pressure in the pollen tube, which ultimately results in pollen tube rupture at its weakest point, the very tip lacking callose containing cell wall material. As a consequence, sperm cells and pollen tube tip factors required for fertilization processes are released explosively into the receptive synergid, a process described previously already at the cellular level in Torenia fournieri[34]. As described above, this process is species-preferential, as ZmES4 strongly modulated KZM1 activity but obviously showed low activity on ion-channels of related T. dactyloides pollen tubes and no significant effect on tubes of other plants tested. We discovered an up to now unknown additional species-specific reproductive hybridization barrier that could represent a further component of reproductive isolation and, thus, speciation in plants[29],[35],[36]. Moreover, this finding opens the possibility to introduce ZmES related CRPs of the DEFL subclass in embryo sac cells or potassium channels in pollen tubes of various grass species to overcome species-specific crossing barriers. The surface loop that is responsible for species-specific antifungal activity of the plant DEF RsAFP2 has been mapped as C5-C6-loop[21],[37], the same loop that protrudes from the ZmES1 structural model( arrow in Figure 4A). This region differs in ZmES1 and ZmES2-4 indicating that ZmES CRPs may indeed target different proteins. Additionally, this region indicates a first sub-domain that could be engineered in order to overcome its species-specific properties. Considering that the egg apparatus secretes a large cocktail of diverse small CRPs[11],[12],[13],[14],[15],[16], perhaps more ion channels and/or receptors are affected simultaneously culminating in explosive tube discharge. This hypothesis is supported by the finding that RNAi silencing of the whole ZmES family did not lead to the expected 50% reduction of the fertilization rate. However, it also cannot be ruled out that the knock-down effect was indeed 100% efficient. Thus a systematic approach is now required to analyze the functions and activities of the various embryo sac expressed DEFL genes on all pollen tube expressed ion channels and other receptors. Interestingly, our findings show that pollen tube growth inhibition and discharge is not only mechanistically related to defense mechanisms against fungal attack, but moreover similar molecular players are involved. While many DEFs are required to protect both plant and animal reproductive tissues from pathogens[29],[38],[39], ZmES proteins obviously evolved a novel function, although still possessing low antifungal activity. As indicated above, a large number of DEFL genes are also expressed in the Arabidopsis and Torenia fournieri female gametophyte[11],[15],[20] indicating that the reported physiological mechanism of tube burst might well be conserved in angiosperms and that more DEFL genes obtained other functions. Hoverer, due to the polymorphic nature of DEFL genes, orthologous genes cannot be predicted and have to be identified experimentally. For example, related DEFL genes LURE1 and LURE2 from Torenia fournieri have recently been shown to encode the female gametophyte secreted attractants of the pollen tube[20] providing further support for the assumption that DEFL genes have evolved specific functions during plant reproduction. Interestingly, a similar evolutionary phenomenon has recently been proposed for the FER RLK: a homozygous fer mutant in Arabidopsis leads to fungal resistance[40], although the function of this RLK in the ovule seems to be restricted to pollen tube growth arrest[5]. Thus, after the identification of the first players involved in pollen tube growth arrest and burst, it will now be exciting to find out how the pathways are connected and how they evolved: for example, do FER or LRE[6] signaling pathways lead to secretion of ZmES1-4 containing vesicles similar to the release of cortical granules of animal species[41]? Do ZmES proteins also act on maize homologs of Arabidopsis ACA9 Ca2+ pumps[9] whose activity is also required for pollen tube burst? And finally, can we switch back the evolutionary clock and engineer “reproductive” DEFLs into “defense” DEFLs and vice versa? Systematic approaches to study female gametophyte expressed DEFL gene families will provide exciting answers during the next few years.
Sperm cells of animals and lower plants are mobile and can swim to the oocyte or egg cell . In contrast , flowering plants generate immobile sperm encased in a pollen coat to protect them from drying out and are transported via the pollen tube cell towards the egg apparatus to achieve double fertilization . Upon arrival the pollen tube tip bursts to deliver two sperm cells , one fusing with the egg cell to generate the embryo and the other fusing with the central cell to generate the endosperm . Here , we report the mechanisms leading to pollen tube burst and sperm discharge in maize . We found that before fertilization the defensin-like protein ZmES1-4 is stored in the secretory zone of the egg apparatus cells and that pollen tubes cannot discharge sperm in ZmES1-4 knock-down plants . Application of chemically synthesized ZmES4 leads to pollen tube burst within seconds in maize , but not in other plant species , suggesting this mechanism may be species specific . Finally , we identified the pollen tube-expressed potassium channel KZM1 as a target of ZmES4 , which opens after ZmES4 treatment and probably leads to K+ influx and sperm release after osmotic burst .