Abstract
During gamete delivery in Arabidopsis thaliana, intercellular communication between the attracted pollen tube and the receptive synergid cell leads to subcellular events in both cells culminating in the rupture of the tip-growing pollen tube and release of the sperm cells to achieve double fertilization. Live imaging of pollen tube reception revealed dynamic subcellular changes that occur in the female synergid cells. Pollen tube arrival triggers the trafficking of NORTIA (NTA) MLO protein from Golgi-associated compartments and the accumulation of endosomes at or near the synergid filiform apparatus, a membrane-rich region that acts as the site of communication between the pollen tube and synergids. Domain swaps and site-directed mutagenesis suggest that NTA’s C-terminal cytoplasmic tail with its calmodulin-binding domain regulates NTA movement and function in pollen tube reception. Signal-mediated trafficking of NTA to the filiform apparatus upon pollen tube arrival may facilitate intercellular communication that leads to pollen tube rupture.
Introduction
Intercellular communication is central to the proper development and maintenance of all multicellular organisms. During this communication, signals from one cell are perceived by receptors in another cell and translated into various subcellular responses. These include signal transduction cascades leading to transcription of other genes, calcium signaling, and trafficking of proteins to different organelles or regions of the cell. A well-studied example of signal-induced protein trafficking in plant development is the redistribution of the PIN polar auxin transporters to different sides of the cell during important developmental events such as embryo patterning, leaf initiation and lateral root initiation (Petrasek et al., 2006; Naramoto, 2017; Salanenka et al., 2018). In plants, most intercellular communication occurs between cells that are genetically identical and connected by adjoining cell walls. One exception is pollination, in which pollen (the male gametophyte) is released from an anther, transported to a receptive stigma, and produces a tip-growing pollen tube that grows through the female tissues of the pistil and delivers the two sperm cells to the female gametophyte (also known as the embryo sac, Fig 1A). The pollen tube’s journey through the pistil requires cell-to-cell interactions with the female that allows water and nutrient uptake and enables the detection of cues important for guidance toward the female gametes (Johnson et al., 2019).
In the model plant Arabidopsis thaliana, complex signaling events ranging from pollen landing on the stigma to fusion of gametes occur over several hours. Most of our knowledge about the signaling pathways involved along the pollen tube’s journey through the female is limited to the final stages of pollination and involve a highly specialized pair of female gametophyte cells known as synergids. During female gametophyte development, meiosis followed by three rounds of mitosis produce the egg cell and central cell along with 2 synergid cells flanking the egg cell and 3 antipodal cells on the chalazal end of the embryo sac (Drews and Yadegari, 2002), Fig 1A). The synergid cells are accessory cells that control the behavior of the pollen tube during the final stages of pollination. Before pollen tube arrival, they secrete cysteine-rich LURE peptides that act as short-range pollen tube attractants that are recognized by receptor-like kinases in the tip of the pollen tube to regulate the direction of pollen tube growth and guide the pollen tube to the micropyle of the ovule (Okuda et al., 2009; Takeuchi and Higashiyama, 2016; Wang et al., 2016). After pollen tube arrival, the synergids communicate with the pollen tube to induce changes that result in pollen tube rupture and delivery of the sperm cells (Kessler and Grossniklaus, 2011; Johnson et al., 2019). Thus, synergids are critical for ensuring that double fertilization can occur to produce seeds.
In Arabidopsis, live imaging has been used to examine the behavior of both the pollen tube and the synergids during the process of pollen tube reception. A pollen tube follows the gradient of LURE attractants, enters the micropyle of the ovule, and pauses its growth for 30 min to 1 h just outside the receptive synergid (Iwano et al., 2012; Denninger et al., 2014; Ngo et al., 2014). During this pause in pollen tube growth, communication occurs between the pollen tube and the synergids that leads to subcellular changes and ultimately to the death of both the pollen tube and the receptive synergid. Cytoplasmic calcium ([Ca2+]cyto) oscillations occur in both the tip of the pollen tube and in the 2 synergid cells during this communication phase. [Ca2+]cyto levels continue to increase in both cell types until the pollen tube starts to grow again and bursts to release the sperm cells, a catastrophic event for both the pollen tube and the receptive synergid, which also degenerates (Iwano et al., 2012; Denninger et al., 2014; Ngo et al., 2014). Mutations in genes that regulate communication between the synergids and pollen tube during pollen tube reception result in a pollen tube overgrowth phenotype in which the pollen tubes are attracted normally to the ovules, but do not get the signal to burst and release the sperm cells. Presumably, synergid-induced changes in the cell wall of the pollen tube tip do not occur in these mutants, therefore the pollen tube continues to grow and coil inside the embryo sac. Synergid-expressed genes that participate in pollen tube reception include the FERONIA (FER) receptor-like kinase, the GPI-anchored protein LORELEI (LRE), and the Mildew Resistance Locus-O (MLO) protein NORTIA (NTA, also known as AtMLO7) (Escobar-Restrepo et al., 2007; Capron et al., 2008; Kessler et al., 2010; Ngo et al., 2014; Li et al., 2015; Liu et al., 2016). Mutations in all of these genes lead to the pollen tube overgrowth phenotype due to disruption of the pollen tube-synergid communication pathway.
FER and LRE are necessary for the calcium oscillations that occur in synergids in response to pollen tube arrival (Ngo et al., 2014). In contrast, nta-1 mutants have [Ca2+]cyto oscillations at lower amplitudes, indicating that NTA may participate in modulating Ca2+ fluxes in the synergids during communication with the pollen tube and likely acts downstream of FER and LRE (Ngo et al., 2014). Like all members of the MLO gene family, NTA has seven membrane-spanning domains and a predicted calmodulin-binding domain (CaMBD) in its C-terminal intracellular tail (Devoto et al., 2003; Kusch et al., 2016). Calmodulin (CaM) is a small protein that binds Ca2+ and is involved in signal transduction for many cellular processes (Yang and Poovaiah, 2003). We previously showed that the C-terminal domain of NTA is necessary and sufficient for MLO function in pollen tube reception (Jones et al., 2017), but the significance of the CaMBD in pollen tube reception remains an open question.
The subcellular localization of these important pollen tube reception proteins is not always predictive of their function in communicating with the pollen tube. As expected for early response proteins, both FER and LRE are expressed in synergid cells where they localize in or near a specialized region called the filiform apparatus, a membrane rich area located at the micropyle end of the synergids (Huck et al., 2003; Rotman et al., 2003; Escobar-Restrepo et al., 2007; Capron et al., 2008; Li et al., 2015; Lindner et al., 2015; Liu et al., 2016). The filiform apparatus is thought to be important for the secretion of attractant peptides and is the first site of interaction between the pollen tube and synergid cell prior to pollen tube reception (Mansfield et al., 1991; Huang and Russell, 1992; Leshem et al., 2013). In contrast, before pollen tube arrival, NTA is sequestered in a Golgi-associated compartment within the synergid cell and excluded from the filiform apparatus region (Jones et al., 2017). At the end of pollen tube reception, NTA protein is only detected in the region of the filiform apparatus, indicating that this protein changes its subcellular localization during pollen tube reception (Kessler et al., 2010). This suggests that pollen tube-triggered regulation of the synergid secretory system may be a crucial subcellular response to pollen tube arrival and that NTA function may be related to its subcellular distribution; however, the precise timing and significance of NTA’s redistribution are still unclear.
In this study, we take advantage of a live-imaging system to further characterize synergid cellular dynamics during pollen tube reception and to determine the timing and significance of the polar redistribution of NTA to the filiform apparatus. To investigate the link between Ca2+ and MLO function in pollen tube reception, we assayed the influence of the CaMBD on NTA’s function and subcellular distribution through C-terminal truncations and a point mutation disrupting the CaMBD. We show that the polar redistribution of NTA is triggered by the approach of a pollen tube, is important for pollen tube reception, and is regulated by the CaMBD. While most subcellular compartments remain distributed throughout the synergid cells during pollen tube reception, recycling endosomes respond to pollen tube arrival by accumulating towards the filiform apparatus. Moreover, we show that targeting NTA to the filiform apparatus before pollen tube attraction does not induce synergid cell death.
Results
Time-lapse imaging of NORTIA redistribution using a semi-in vivo assay
Pollen tube reception is a complex process that requires the synergid cells to recognize the approaching pollen tube and to send signals back to the pollen tube that result in release of the sperm cells at the correct time and place. We previously showed that the NTA-GFP fusion protein localizes to a Golgi-associated compartment of the synergids before pollen tube attraction (Jones et al., 2017). When imaged after pollen tube reception, NTA-GFP is concentrated at the micropylar end of the synergid (in or near the filiform apparatus) (Kessler et al., 2010). NTA-GFP doesn’t accumulate near the filiform apparatus in fer ovules with pollen tube overgrowth, suggesting that FER-mediated signaling that occurs during pollen tube reception triggers NTA-GFP redistribution that in turn contributes to the interaction of the synergid with the pollen tube (Kessler et al., 2010). An alternative hypothesis is that pollen tube rupture triggers NTA-GFP redistribution and is a symptom of pollen tube reception rather than an important contributor to the signaling pathway. To distinguish between these two possibilities, we used a semi-in vivo pollination system combined with spinning disk confocal microscopy to determine the timing of NTA-GFP redistribution during the pollen tube reception process. In the semi-in vivo system, pollen tubes grow out of a cut style and are attracted to ovules arranged on pollen germination media (Palanivelu and Preuss, 2006). This system has previously been used to quantify and track pollen tube attraction to ovules and to image ([Ca2+]cyto reporters during pollen tube reception (Hamamura et al., 2011; Hamamura et al., 2012; Denninger et al., 2014; Hamamura et al., 2014; Ngo et al., 2014). In order to follow subcellular changes in NTA-GFP protein localization before, during, and after pollen tube arrival, we used pollen from plants expressing the pollen-specific AUTOINHIBITED Ca2+-ATPASE9pro::DsRed (ACA9pro::DsRed) reporter and ovules expressing NTApro::NTA-GFP in the semi-in vivo system. Approximately 4 h after pollination, pollen tubes emerged from the style onto the media and were attracted to ovules (Fig 1B). Images in the red and green channels were collected every 5 min from when a pollen tube approached an ovule until after the pollen tube ruptured inside the ovule. In our system, most of the ovules displayed successful pollen tube attraction and reception, while others did not attract a pollen tube during the time course of the imaging experiments (Fig 1B and S1). A second group were imaged under the same conditions and serve as a negative control for environmentally-induced changes in NTA-GFP localization. 83% of the ovules that attracted a pollen tube that successfully burst to deliver the sperm cells (n=93) displayed NTA-GFP redistributed to the micropylar end of the synergid cell (Fig 1C-E). Ovules without NTA-GFP redistribution displayed abnormal pollen tube behavior in which pollen tubes were attracted but stopped growing and never ruptured to release the sperm cells. Neighboring ovules that did not attract a pollen tube (n=103) but were imaged under the same semi-in vivo conditions did not have redistribution of NTA-GFP (Fig 1E), nor did ovules that were incubated on pollen germination media without a pollinated pistil (n=133, Fig 1E). These data suggest that pollen tube arrival is necessary for NTA-GFP redistribution and that the imaging conditions do not trigger redistribution (Fig 1E).
Our semi-in vivo system also allowed us to determine the timing of NTA-GFP redistribution in relation to the position of the pollen tube as it approached the synergids. We defined the 0 min timepoint as the time where the pollen tube just reached the micropylar opening of the ovule (Fig 1C, ovules with yellow stars, Movies S1 and S2). In all cases, NTA-GFP movement also started from this time point. During the following 30–50 min, pollen tubes grew through the micropyle region of ovule and arrived at the filiform apparatus of the receptive synergid cell. During this time, three quarters to half of the NTA-GFP signal moved to the micropylar end of synergid cells, indicating that the approach of the pollen tube triggers NTA-GFP movement. As reported in (Ngo et al., 2014) and (Denninger et al., 2014), the arriving pollen tubes paused their growth outside the filiform apparatus for 30–50 min, presumably for cell-to-cell communication. During this period, NTA-GFP signal continued to move toward the filiform apparatus. The whole movement took around 70–80 min, and after the redistribution completed, pollen tubes resumed growth and ruptured to deliver the sperm cells and complete double fertilization (Fig 1C and Movies S1 and S2). Even though only one of the synergids receives the pollen tube, NTA-GFP was actively redistributed to the filiform apparatus in both synergid cells in response to pollen tube arrival, similar to the activation of [Ca2+]cyto oscillations in both synergids during pollen tube reception reported in (Ngo et al., 2014).
The Golgi apparatus does not concentrate at the filiform apparatus during pollen tube reception
We previously determined that NTA is sequestered in a Golgi-associated compartment in synergid cells that have not attracted a pollen tube (Jones et al., 2017). Our live-imaging data suggests that NTA-GFP is selectively moved out of the Golgi and trafficked to the region of the filiform apparatus in response to pollen tube arrival; however, it is possible that the observed NTA-GFP movement is a result of massive reorganization of subcellular compartments. To distinguish between these possibilities, we investigated the behavior of Golgi in synergid cells during pollen tube reception. We used the semi-in vivo imaging system described above with a synergid-expressed Golgi marker (Man49-mCherry) co-expressed with NTA-GFP (Jones et al., 2018). In all replicates, the Golgi marker was distributed throughout the synergids, excluded from the filiform apparatus, and co-localized with NTA-GFP as reported previously (Fig 2A). When a pollen tube approached the synergids, NTA-GFP redistributed to the filiform apparatus region of the synergids as observed previously (Fig 1), but the Golgi-mCherry marker remained consistently distributed throughout the synergid cells and did not concentrate near the filiform apparatus (Fig 2B). In order to examine the behavior of the Golgi during later stages of pollen tube reception, we used the synergid-expressed Golgi marker line (Man49-mCherry) and pollen that was expressing GFP (Lat52pro::GFP). In all cases, the Golgi marker remained randomly distributed throughout the synergid cells, even after pollen tube rupture (Fig 2C-D, Movies S3 and S4). These results indicate that the movement of NTA-GFP during pollen tube reception is not linked to mass redistribution of the Golgi apparatus.
Distribution of cellular compartments in synergid cells during pollen tube reception
A signal from the arriving pollen tube seems to trigger the movement of NTA-GFP out of the Golgi-associated compartments. It is possible that pollen tube arrival triggers other changes to synergid subcellular organization. We previously reported the localization of synergid-expressed markers for the ER, peroxisome, endosome and the trans-Golgi Network (TGN) in unfertilized ovules in vivo using confocal laser scanning microscopy (Jones et al., 2018). Before pollen tube arrival, SP-mCherry-HDEL (an ER-associated marker), mCherry-SKL (a peroxisome-associated marker), and mCherry-RabA1g (a recycling endosome-associated marker) were all distributed evenly throughout synergid cells (Fig S2 and 3A; (Jones et al., 2018). The TGN-associated marker SYP61 exhibited two types of distribution patterns before pollen tube arrival: in type 1 synergids, the marker accumulated near the filiform apparatus, whereas type 2 synergids displayed a punctate distribution pattern throughout the cells (Fig S2; (Jones et al., 2018). During pollen tube reception, no change was seen in the TGN marker distribution in either type of synergids (Fig S2, C-D, Movies S5 and S6). Likewise, the ER and peroxisome markers maintained a diffuse distribution throughout the synergids and did not accumulate at the filiform apparatus region (Fig S2, A-B and E-F, Movies S7-10). In contrast, we detected a more dynamic behavior of the endosome marker during pollen tube reception. Endosomes are membrane-bound compartments that are involved in the endocytic membrane transport pathway from the plasma membrane to the vacuole. Endosomes also transport molecules from the Golgi and either continue to vacuole or recycle back to the Golgi (Stoorvogel et al., 1991). We previously reported that mCherry-RabA1g is distributed throughout synergid cells and had some overlap with NTA-GFP in synergids of unpollinated ovules (Jones et al., 2018). Using the semi-in vivo system, we confirmed that before pollen tube arrival, mCherry-RabA1g distributed throughout synergid cells (Fig 3A). Interestingly, as pollen tubes approached, the endosome marker started to accumulate in the filiform apparatus region of the synergid cells (Fig 3A and B). By the time that pollen tube reception was completed, most of the endosome signal was concentrated at or near the filiform apparatus (Fig 3B-D and S3, Movies S11 and S12). These results indicate that the RabA1g endosome compartments have a distinct response to pollen tube arrival and may play a role in facilitating the intercellular signaling pathway that occurs between the synergids and the pollen tube.
The CaMBD is important for NTA’s function in pollen tube reception
The timing of the NTA redistribution during pollen tube arrival is similar to the start of synergid [Ca2+]cyto oscillations that are triggered by the pollen tube in a FER-dependent manner (Iwano et al., 2012; Hamamura et al., 2014; Ngo et al., 2014). MLO proteins have the potential to bind Ca2+ through calmodulin binding domains (CaMBD) in their C-terminal cytoplasmic tails following the seventh transmembrane domain (Devoto et al., 1999; Kim et al., 2002a; Panstruga, 2005; Consonni et al., 2006). Like all MLO proteins, NTA has a predicted CaMBD in its C-terminal tail (Fig 4A). In nta-1 mutants, pollen tube-triggered [Ca2+]cyto oscillations occur but at a lower amplitude (Ngo et al., 2014). Domain swaps with MLO8 revealed that the C-terminal tail domain of NTA is necessary and sufficient for MLO-mediated pollen tube reception and important for NTA’s subcellular distribution in synergids (Jones et al., 2017). Together, these data suggest that the subcellular distribution of NTA before and during pollen tube reception could be influenced by [Ca2+]cyto levels sensed by the CaMBD. Previous studies have found that CaM-binding activity of the CaMBD influences MLO function in powdery mildew susceptibility (Kim et al., 2002a).
We hypothesized that CaM-binding activity is also important for NTA’s function during pollen tube reception. To test this, a truncation removing the C-terminal tail including the CaMBD (NTAΔ450), a point mutation in a conserved tryptophan (NTAW458A) necessary for CaM-binding function in other CaMBDs (Arazi et al., 1995; Yamada et al., 1995; Kim et al., 2002b), and a truncation removing the tail immediately following the CaMBD (NTAΔ481), all fused to GFP, were generated and expressed under the synergid-expressed MYB98 promoter in the nta-1 background (Fig 4). Ovule counts in homozygous lines revealed that all three constructs had significant reductions in unfertilized ovules compared to nta-1. However, only NTAΔ481 rescued at similar levels as full-length NTA (Fig 4B), indicating that the C-terminal tail after the CaMBD is dispensable for NTA function. Both NTAΔ450 and NTAW458A partially rescued nta-1, suggesting that either removal or disruption of the CaMBD have a similar impact on NTA’s function.
A functional CaMBD facilitates NTA accumulation at the filiform apparatus during pollen tube arrival
Prior to pollen tube arrival, NTA is distributed throughout the entire synergid cell where it is localized primarily within Golgi (Fig 4C) (Kessler et al., 2010; Jones et al., 2017). When expressed in synergid cells, related MLO proteins that localize within Golgi can rescue nta-1 whereas those that localize elsewhere do not function in pollen tube reception (Jones et al., 2017). The partial complementation of nta-1 by the CaMBD-disrupted variants could be due to disrupted localization patterns before and/or after pollen tube arrival. In virgin ovules, both NTAΔ481 and NTAW458A were distributed throughout the synergid cell in punctate compartments and were predominantly excluded from the filiform apparatus (Fig 4E and F). NTAΔ450 accumulated in punctate compartments throughout the synergid, but was also detected near the filiform apparatus and in the vacuole (Fig 4D). Although the two variants with disrupted CaMBDs (NTAΔ450 and NTAW458A) both partially rescued the nta-1 unfertilized ovule phenotype at similar levels, they had different distributions in the synergid cell. This suggests that differences in localization between these two variants may not be functionally relevant to pollen tube reception. Due to this, we focused primarily on NTAW458A for our downstream analyses and comparisons with wildtype NTA so as to not further complicate the interpretation of our results.
With “NTA-like” distribution in the synergid cell, we suspected that the NTAW458A variant would accumulate within Golgi-associated compartments similar to wildtype NTA. NTAW458A was co-expressed with fluorescent markers for Golgi (LREpro::Man49-mCherry, (Liu et al., 2016)) and the TGN (MYB98pro:Syp61-mCherry,(Jones et al., 2017)) in synergid cells and virgin ovules were analyzed via CLSM (Fig S4). The NTAW458A variant partially co-localized with the Golgi maker (Fig S4A) and had no overlap with the TGN marker (Fig S4B), similar to wildtype NTA localization(Jones et al., 2017). These data demonstrate that neither NTA’s distribution within the synergid cell, where it is maintained out of the filiform apparatus prior to pollen tube arrival, nor its localization within Golgi-associated compartments are dependent on a functional CaMBD.
During pollen tube reception, NTA is actively redistributed toward the filiform apparatus region. The NTAW458A variant maintains a wildtype distribution and localization pattern in synergids without pollen tubes but does not fully rescue nta-1’s unfertilized ovule phenotype. To test whether the CaMBD is important for NTA’s redistribution in response to pollen tube arrival, the semi-in vivo system described above was used to monitor NTAW458A movement in the nta-1 background. In this background, the partial rescue by NTAW458A leads to some ovules having normal pollen tube reception, while others exhibit pollen tube overgrowth and a failure of pollen tube rupture. In the semi-in vivo system, NTAW458A ovules that did not attract a pollen tube, maintained distributions outside of the filiform apparatus, consistent with unpollinated flowers above (Fig S5A and C). In ovules with successful pollen tube reception, NTAW458A redistributed to the filiform apparatus region, but in many cases this redistribution was not as complete as with the wild-type NTA-GFP protein, with some GFP signal remaining outside the filiform apparatus region (Fig 5 and Fig S5, B and D). In ovules where pollen tube reception was not successful due to pollen tube overgrowth, NTAW458A did not accumulate at the filiform apparatus region of the synergids (Fig 5C). These data suggest that an active CaMBD enhances NTA’s redistribution to the filiform apparatus region during pollen tube reception and that NTA redistribution is correlated with pollen tube rupture.
Premature distribution of NTA to the filiform apparatus region is not toxic to synergid cells
The selective targeting of NTA-GFP from the Golgi apparatus to the filiform apparatus region of the synergid cells during pollen tube arrival (Fig 1-2) and the link between NTAW458A redistribution and pollen tube reception (Fig 5) suggests that NTA accumulation at the pollen tube/synergid interface is important for the intercellular communication process that occurs between the pollen tube and synergids. In nta-1 mutants, around 30% of ovules display pollen tube overgrowth and fail to complete double fertilization, but the other 70% are fertilized normally (Kessler et al., 2010). This indicates that NTA is not absolutely required for pollen tube reception, but may function as a modifier of the FER signaling pathway. Since FER signaling in the synergids leads to cell death as pollen tube reception is completed (Huck et al., 2003; Rotman et al., 2003; Escobar-Restrepo et al., 2007), NTA trafficking to the filiform apparatus from could be a mechanism to regulate this death and would thus require sequestration of “toxic” NTA in the Golgi before pollen tube arrival. In order to test this hypothesis, we took advantage of sequence variation leading to differential subcellular localization of MLO proteins to manipulate the subcellular localization of NTA. When expressed in synergids, other proteins from the Arabidopsis MLO family have different subcellular localization patterns, indicating that specific sequences within the MLOs direct them to different parts of the secretory system (Jones and Kessler, 2017). MLO1-GFP localizes in the filiform apparatus region of the synergids when it is ectopically expressed under control of the synergid-specific MYB98 promoter and cannot complement the nta-1 pollen tube reception phenotype (Jones et al., 2017), also see Fig 6C). Domain swaps between different regions of NTA and MLO1 revealed that the C-terminal cytoplasmic tail including the CaMBD of MLO1 (NTA-MLO1CTerm, see diagram in Fig 6B) was sufficient to direct the fusion protein to the filiform apparatus region of the synergids, in a pattern very similar to MLO1-GFP (Fig 6A-C). Quantification of the GFP signal along the length of the synergids from the chalazal end to the filiform apparatus in the NTA-GFP, NTA-MLO1Cterm-GFP, and MLO1-GFP confirmed that the MLO1 tail was sufficient to move the NTA protein to the filiform apparatus end of the cell (Fig 6D). In all MLO1-GFP and NTA-MLO1Cterm-GFP ovules, the majority of GFP signal was detected in the lower 20-40% of the synergids and most of the signal overlapped with the diffuse FM4-64 staining in the filiform apparatus (Fig 6E). In contrast, NTA-GFP is excluded from the filiform apparatus (Fig 6A and (Jones et al., 2017).
The successful manipulation of NTA subcellular localization provided a tool for determining the functional relevance of NTA redistribution. We transformed the NTA-MLO1CTerm-GFP construct into nta-1 plants and used the percentage of unfertilized ovules as a measurement for the ability of the fusion construct to complement the nta-1 phenotype of unfertilized ovules caused by pollen tube overgrowth (Kessler et al., 2010). Two independent NTA-MLO1CTerm-GFP insertions were able to rescue the nta-1 phenotype when expressed in synergids (Fig 6F). These data indicate that premature targeting of NTA to the filiform apparatus is not toxic to synergid cells.
Discussion
Synergids respond to a signal from the approaching pollen tube
Successful pollination and production of seeds requires a series of signaling events between the male gametophyte (pollen tube) and both sporophytic and gametophytic cells of the female. In this study, we used live imaging to characterize dynamic subcellular changes that occur in the synergid cells of the female gametophyte in response to the arrival of the pollen tube. We showed that both the NTA protein and endosomes are actively mobilized to the filiform apparatus region where male-female communication occurs during pollen tube reception (Fig 7). Disruption of NTA’s CaMBD partially compromised NTA redistribution and function in pollen tube reception, revealing that Ca2+ may play a role the synergid response to the signal from the pollen tube.
The polar accumulation of the RabA1g endosome marker near the filiform apparatus during pollen tube reception suggests a change in synergid secretory system behavior that is triggered by the approaching pollen tube. Our results with the ER, Golgi, TGN, and peroxisome markers indicate that the mobilization of the RabA1g endosomes toward the approaching pollen tube is not just a symptom of FER signaling triggering synergid cell death that leads to mass disruption of subcellular compartments. Trans-Golgi Network/Early endosomes (TGN/EEs) have been shown to be involved in the trafficking of both secretory and endocytic cargo (Viotti et al., 2010). RabA1g is present in endosomes that are highly sensitive to Brefeldin A in roots, suggesting that they could function as recycling endosomes (Geldner et al., 2009). While the resolution of our live imaging system did not allow us to determine whether NTA completely co-localizes with this compartment, it is tempting to speculate that the RabA1g endosomes are mediating the polar movement of NTA to the filiform apparatus region. Alternatively, these endosomes could be transporting other signaling molecules either to or from the filiform apparatus.
Signal-mediated protein trafficking
Signal-mediated regulation of protein trafficking is an elegant mechanism to control the delivery of molecules to the precise location where they are needed for critical signaling events that occur over relatively short time frames. Selective protein targeting similar to NTA movement in response to pollen tube arrival has also been observed during cell-to-cell communication between the egg and sperm cells in Arabidopsis. After pollen tube reception and release of the sperm cells, a signal from the sperm and/or the degenerated synergid cell causes the egg cell to secrete EGG CELL 1 (EC1) peptides that have been stored in punctate compartments in the egg cytoplasm toward the sperm cells. The sperm cells perceive the EC1 signal and, in turn, mobilize the gamete fusogen HAPLESS2/GENERATIVE CELL SPECIFIC1 (HAP2/GCS1) from a cytoplasmic compartment to the cell surface (Sprunck et al., 2012). This controlled movement of proteins that have already been translated and stored allows for a quick response that allows the egg and sperm to become activated for fertilization. Likewise, NTA mobilization to the filiform apparatus region of the synergids as the pollen tube arrives could play a role in sending a signal to the pollen tube that leads to the mobilization of pollen tube proteins that allow the pollen tube to rupture and release the sperm cells. For example, proteins that regulate the integrity of the tip of the pollen tube could be quickly delivered after the “arrival” signal from the synergid is perceived. Recent work on the role of the pollen-expressed ANXUR1 and 2 and BUDDHA PAPER SEAL1 and 2 receptor-like kinases in pollen tube tip integrity support this hypothesis. During pollen tube growth through the female tissues, RALF4 and RALF19 peptides that are secreted from pollen tubes act as ligands for ANX1/2 and BPS1/2 to promote tip growth, while RALF34 secreted from the synergids displaces RALF4 and 19 from the receptors leading to subcellular changes that result in pollen tube rupture (Ge et al., 2017).
Calcium and NTA movement
Our study revealed that an intact CaMBD facilitates the movement of NTA from the Golgi to the filiform apparatus in response to the stimulus from the approaching pollen tube. This result provides an intriguing link to Ca2+ since the polar movement of NTA-GFP to the filiform apparatus region occurs in a similar time frame to [Ca2+]cyto spiking in the synergids during pollen tube reception (Denninger et al., 2014; Ngo et al., 2014). Subcellular Ca2+ spiking occurs in plant responses to both biotic and abiotic external stimuli. Notably, Ca2+ oscillations occur during pollen tube-synergid interactions, egg-sperm interactions, and in biotrophic interactions between plant cells and both beneficial and harmful microbes (reviewed in (Chen et al., 2015). In most cases, the mechanism for decoding Ca2+ spikes into a cellular response is not known, but Ca2+-binding proteins such as calmodulin (CaM) and calmodulin-like proteins could play a role in relaying Ca2+ signals (Chin and Means, 2000). In nta-1 mutants, the [Ca2+]cyto oscillations still occur, but at a lower magnitude than in wild-type synergids, suggesting that NTA could be involved in modulating Ca2+ flux (Ngo et al., 2014). The source of Ca2+ during these oscillations is not known, but it is possible that NTA regulates Ca2+ channels to regulate the flow of Ca2+ions into or out of the apoplast near the filiform apparatus.
Ca2+ has also been linked to regulation of endomembrane trafficking (reviewed in (Himschoot et al., 2017). In animals, calmodulin plays a role in regulating vesicle tethering and fusion (Burgoyne and Clague, 2003), and in plants calmodulin-like proteins are associated with endosomal populations (Ruge et al., 2016). Thus, it is possible that the CaMBD in NTA is critical for the precise targeting of NTA in response to pollen tube arrival. Another mechanism that has been proposed for Ca2+regulation of protein targeting is that electrostatic interactions between Ca2+ and anionic phospholipids in specific domains of the plasma membrane regulate vesicle fusion and differential recruitment of proteins to these domains (Simon et al., 2016; Platre et al., 2018). The filiform apparatus of the synergids is distinctive from the plasma membrane in other parts of the synergid and likely has a unique phospholipid composition that could play a role in recruiting NTA to this domain. Whether NTA movement is a cause or consequence of [Ca2+]cyto spiking requires more live imaging experiments at a higher time resolution to determine if NTA redistribution happens before or after the initiation of [Ca2+]cyto spiking.
Cell death and pollen tube reception
MLOs were first discovered in barley as powdery mildew resistance genes (Buschges et al., 1997). mlo mutants in both monocots and dicots are resistant to powdery mildew infection, indicating that the MLO proteins are required for infection. These mutants also have ectopic cell death, indicating that one function of MLO proteins is to negatively regulate cell death (Panstruga, 2005). Pollen tube reception is catastrophic for both the pollen tube and the receptive synergid cell: both cells die as a result of successful male-female signaling and delivery of the male gametes. The timing of synergid degeneration remains under debate, with some studies suggesting that it occurs upon pollen tube arrival at the synergid and others concluding that it occurs concurrently with pollen tube rupture (Sandaklie-Nikolova et al., 2007; Hamamura et al., 2011; Leydon et al., 2015). Our live imaging experiments with both NTA-GFP and the subcellular compartment reporters suggest that the synergid cells are still alive and regulating their secretory systems up to the point of pollen tube rupture. Given the function of the “powdery mildew” members of the MLO gene family in preventing cell death, it is possible that one role of NTA is to prevent early degeneration of the synergids. An interesting parallel between powdery mildew infection and pollen tube reception is that, in both cases, an MLO protein gets redistributed to the site of interaction with a tip-growing cell. In the powdery mildew system, active transport of proteins and lipids to penetration site leads to membrane remodeling and establishment of the extrahaustorial membrane which separates the plant cytoplasm from the invading fungal hyphae (Huckelhoven and Panstruga, 2011). Although the relationship between the arriving pollen tube and the filiform apparatus has not been established, it is possible that similar reorganization occurs in the filiform apparatus during signaling with the pollen tube. In both cases, perhaps an MLO protein is needed in these special membrane regions to stabilize the cell and prevent precocious cell death. Our result that premature delivery of NTA (in the NTA-MLO1CTerm domain swap construct) to the filiform apparatus region of the cell does not disrupt pollen tube reception is consistent with this hypothesis, since other signaling processes occurring in the synergids during communication with the arriving pollen tube would likely not be triggered by simply moving NTA to the filiform apparatus in the absence of a pollen tube.
In this study, we showed that signals from an approaching pollen tube trigger the movement of NTA out of the Golgi and to the region of the filiform apparatus and that this redistribution is correlated with pollen tube reception. However, localization of the NTA-MLO1CTerm fusion protein was able to complement the nta-1 mutant phenotype, indicating that the final localization of the NTA protein may be more important than the active trafficking from the Golgi compartment. Future work will focus on determining the mechanism through which NTA becomes polarly redistributed and on identifying the signals from the pollen tube that lead to important subcellular changes in the synergids during pollen tube reception.
Materials and methods
Plant materials and growth conditions
Arabidopsis thaliana lines expressing NTApro::NTA-GFP, MYB98pro::NTA-GFP, MYB98pro::MLO1-GFP Golgi-associated marker (Man49-mCherry), ER-associated marker (SP-mCherry-HDEL), TGN-associated marker (SYP61-mCherry), peroxisome-associated marker (Peroxisome-mCherry) and recycling endosome-associated marker (mCherry-RabA1g), were generated and described in previous publications (Kessler et al., 2010; Liu et al., 2016; Jones et al., 2017; Jones et al., 2018). Pollen tube marker lines, ACA9::DsRed (Boisson-Dernier et al., 2008) and Lat52::GFP (Palanivelu and Preuss, 2006) were generously provided by Dr. Aurelien Boisson-Dernier and Dr. Ravi Palanivelu. Seeds were sterilized and plated on ½-strength Murashige and Skoog (MS) plates. All plates were sealed and stratified at 4°C for two days, and then transferred to the growth chamber (long day conditions, 16 h of light and 8 h of dark at 22°C) for germination and growth. After 5-7 days, seedlings were transplanted to soil. Seeds from transformed lines were sterilized and plated on ½ MS plate with 20 mg/L hygromycin for selection of transgenic seedlings, which were then transplanted to soil and grown in long days.
Live Imaging of Pollination Using a Semi-in vivo Pollen Tube Guidance Assay
The semi-in vivo system of pollen tube reception was modified from (Palanivelu and Preuss, 2006). Approximately 150 μL of pollen germination media (5 mM KCl, 1 mM MgSO4, 0.01% (w/v) H3BO3, 5 mM CaCl2, 20% sucrose, 1.5% agarose, and adjusted pH to 7.5 with KOH) was poured into a Glass Bottom Culture Petri Dish (MatTek Corporation, P35G-1.0-20-C) and spread out using a pipette. Pistils were emasculated and 2 d later were hand pollinated with ACA9::DsRed or Lat52::GFP pollen and returned to the growth chamber. Approximately 1 h after pollination, pistils were removed from plants and placed on double sided tape on a glass slide. Stigmas were cut using single-sided razor blade and placed on pollen germination media using forceps. 8-10 ovules were arranged around the cut style and the petri dish was returned to the growth chamber. After 4-6 h, pollen tubes grew through the stigma and style and emerged near the ovules. Imaging commenced when the pollen tubes approached ovules. Time-lapse images were acquired at 5 min intervals by spinning disk confocal microscopy using an Andor Revolution XD platform with a Yokogawa CSU-X1-A1 scanner unit mounted on an Olympus IX-83 microscope and a 20X/0.5 NA objective (Olympus). An Andor iXon Ultra 897BV EMCCD camera was used to capture GFP fluorescence (488-nm excitation) and red fluorescent protein (dsRed or mCherry) fluorescence (561-nm excitation).
For each experimental condition, at least 60 ovules were imaged over the time course from pollen tube approaching the ovule to completion of pollen tube reception (pollen tube rupture to release the sperm cells). Neighboring ovules that did not attract pollen tubes were imaged at the same time and served as controls for photoxicity.
Confocal laser scanning microscopy (CLSM)
CLSM to examine MLO variant subcellular localization was performed on ovules dissected 2 d after emasculation. FM4-64 staining was performed according to the protocol described in (Jones et al., 2017). CLSM was done using either a Nikon A1Rsi inverted confocal microscope according to (Yuan and Kessler, 2019) or a Leica SP8 upright confocal microscope according to (Jones et al., 2017).
Quantification of fluorescence signal intensity
Two-channel images were adjusted for brightness and contrast using Fiji (Schindelin et al., 2012). Then, they were input to NIS-Elements software (Ver. 5.02) to measure the fluorescence signal intensity. A line that spanned from the chalazal end to the filiform apparatus end of the synergid was used for the signal intensity measurements. For a more accurate representation of the total area of the synergid, signal intensities were recorded along the same length line at five parallel position within the synergid cell. Finally, all the measurement data were output as Excel files. Graphs and statistical analysis were performed with Prism software (www.graphpad.com).
Video processing
Images were filtered to remove the noise and cropped using Fiji (Version 2.0.0). QuickTime Player (Version 10.5) was used for movie editing and time-lapse analyses.
Cloning and Generation of Transgenic Lines
PCR amplification with PHUSION High-Fidelity Polymerase (NEB, M0535S) or Q5 High-Fidelity DNA Polymerase (NEB, M0419S) were used to generate the following constructs in this study. Genes were amplified with primers that had attB1 and attB2 sites for recombination via BP reaction into the Gateway-compatible entry vector pDONR207. Full-length NTA entry vectors used in this study was generated as described previously (Jones et al., 2017). NTA truncations were amplified using NTA full-length entry vector as a template with forward primer NTA-FattB1 and the following reverse primers: NTA450-RattB2 and NTA481-RattB2 (See all primer sequences in Table S1). The NTAW458A point mutation was generated using the same NTA template and amplifying two fragments of NTA with desired point mutations introduced into the primers: NTA-FattB1 + NTAW458A-R and NTAW458A-F + NTA-RattB2. The two fragments were purified and pasted together with overlaps using a PCR-pasting protocol. The NTA-MLO1CTerm construct was generated using the full-length entry vectors of NTA and MLO1 used in previous study (Jones et al., 2017) as templates and amplifying two fragments of NTA and MLO1 using the two pairs of primers: NTA-FattB1 +NTA-R19 and MLO1-F + MLO1-RattB2. The two fragments were purified and pasted together with overlaps using a PCR-pasting protocol. The coding sequence from both truncations and the point mutation and the fusion sequence were fully sequenced in entry vectors. All entries were then recombined via LR reactions into the pMDC83 backbone with the MYB98pro (Muller et al., 2016) to drive expression of each NTA variant in synergid cells with a C-terminal GFP fusion.
For nta-1 complementation assay and co-localization analyses, expression vectors were transformed into the Agrobacterium tumefaciens strain GV3101 and transformed into the nta-1 mutant background or the Col-0 background (co-localization – Col-0 was stably expressing the Golgi or TGN synergid secretory markers) via the floral-dip method (Bent, 2006). Stable transgenic lines were selected using their respective selections described above. Homozygous T2 lines were used in nta-1 complementation assay and co-localization imaging in the synergid was done in a T1 analysis.
nta-1 Complementation Assays
3-4 independent insertion lines for each construct were taken to the T2 generation and screened for homozygosity using fluorescence microscopy to ensure transgene expression in synergids of every ovule. Unfertilized vs. fertilized ovule counts from self-pollinated flowers were assessed in at least three plants of each insertion line and compared to nta-1, Wassilewskija (Ws-2; wildtype), and the previously published full-length NTA (MYB98pro::NTA-GFP in nta-1 background, (Jones et al., 2017). Ovule counts were statistically analyzed using Prism (www.graphpad.com) with significance determined using a Student’s t-test. Comparisons of the NTA variants were made to full-length NTA and the nta-1 mutant.
Author Contributions
JY, YJ, DSJ, and SAK conceived and designed the experiments. JY, YJ, DSJ, NL, and WZ performed the experiments. JY, YJ, DSJ, and SAK analyzed the data. JY, YJ, DSJ, and SAK wrote the manuscript, and all authors revised and approved the final manuscript.
Competing Interests
The authors declare no competing interests.
Supplemental Materials
Supplementary figure 1. NTA consistently redistributes to the filiform apparatus region during pollen tube arrival.
Supplementary figure 2. Subcellular marker behavior during reception of Lat52::GFP labeled pollen tubes.
Supplementary figure 3. Additional examples of RabA1g endosome markers during pollen tube reception.
Supplementary figure 4. NTAW458A co-localizes with a Golgi marker in synergid cells.
Supplementary figure 5. NTA and NTAW458A distribution in the synergid at pollen tube arrival.
Movies
Supplementary movie 1. NTA-GFP (green signal) redistributes to filiform apparatus region as ACA9::DsRed labeled pollen tube (magenta signal) approaches.
Supplementary movie 2. NTA-GFP (green signal) redistributes to filiform apparatus region during pollen tube reception (GFP channel only, same movie as S1).
Supplementary movie 3. Golgi-mCherry signals (magenta signal) are evenly distributed along the length of the synergid as Lat52::GFP labeled pollen tube (green signal) approaches.
Supplementary movie 4. Golgi-mCherry signals (magenta signal) are evenly distributed along the length of the synergid during pollen tube reception (mCherry channel only, same movie as S3).
Supplementary movie 5. The trans-Golgi marker SYP61-mCherry (magenta signal) is localized toward the micropyle region of synergid cells both before and after pollen tube (green signal) arrival.
Supplementary movie 6. The trans-Golgi marker SYP61-mCherry (magenta signal) is localized toward the micropyle region of synergid cells during pollen tube reception (mCherry channel only, same movie as S5).
Supplementary movie 7. Before and after pollen tube (green signal) arrival, the ER marker SP-mCherry-HDEL (magenta signal) is distributed throughout synergid cells.
Supplementary movie 8. The ER marker SP-mCherry-HDEL (magenta signal) is distributed throughout synergid cells (mCherry channel only, same movie as S7).
Supplementary movie 9. The peroxisome marker mCherry-SLK (magenta signal) does not redistribute to the filiform apparatus region after pollen tube (green signal) arrival.
Supplementary movie 10. The peroxisome marker mCherry-SLK (magenta signal) does not redistribute to the filiform apparatus region during pollen tube reception (mCherry channel only, same movie as S9).
Supplementary movie 11. RabA1g-mCherry endosome marker (magenta signal) accumulates at the filiform apparatus region in response to pollen tube (green signal) arrival.
Supplementary movie 12. RabA1g-mCherry endosome marker (magenta signal) accumulates at the filiform apparatus region during pollen tube reception (mCherry channel only, same movie as S11).
Supplementary table 1. List of primers used for cloning.
Figure 4 Source Data: Seed count data from NTA truncation and point mutation complementation experiments.
Figure 6 Source Data: Seed count data from NTA-MLO1CTerm complementation experiments.
Acknowledgements
We thank Patrick Day for technical assistance and Rachel Flynn and Thomas Davis for helpful discussions and comments on the manuscript. This work was supported by funds from NSF IOS-1733865 to SAK, Purdue University Start-up funds to SAK, and a grant from the Oklahoma Center for the Advancement of Science and Technology #PS14-008 to SAK. Spinning disk confocal microscopy in the Staiger laboratory was supported, in part, by an award from the Office of Science at the US Department of Energy, Physical Biosciences Program, under contract number DE-FG02-09ER15526 to CJS.
Footnotes
Supplemental figures and movies added.