ANXUR Receptor-Like Kinases Coordinate Cell Wall Integrity with Growth at the Pollen Tube Tip Via NADPH Oxidases

1It is well established that growing animal cells control the biogenesis, deposition, and remodeling of their extracellular matrix (ECM). In vivo the ECM contributes to the bulk, shape, and strength of many tissues and, therefore, plays a central role in development. However, it is often underappreciated that the ECM also controls intracellular activities far beyond providing mechanical stability. For example, the ECM is under continuous cellular surveillance in order to monitor the loss of adhesion to the surrounding matrix, which leads to apoptosis. Consequently, disruption of signaling between the ECM and the cell is associated with tumorigenicity. Similarly, growing plant cells direct the deposition of the primary cell wall (CW): the plants rigid, carbohydrate-rich ECM that resists turgor pressure, yet is flexible enough to allow cell expansion. Growing plant cells tightly coordinate the loosening and pressure-driven deformation of the CW with the addition of new membrane and CW materials through exocytosis. Thus, the cell must be kept informed about any environmental changes modifying the CW properties in order to avoid growth arrest or rupture. To circumvent these catastrophic scenarios, it has become increasingly evident that plant cells have developed mechanisms to sense CW integrity, which relay information about CW performance to the internal growth machinery. The molecular nature of this relay mechanism, however, remains largely unknown.

2Since the first reports on THESEUS1 (THE1 ) and FERONIA (FER ), these Arabidopsis receptor-like kinases (RLKs) of the Catharanthus roseus RLK1-like subfamily (CrRLK1L) have received increasing attention as putative sensors that coordinate cellular growth and CW integrity (reviewed in –). How this coordination precisely works and which molecular players of the growth machinery are involved remained elusive, although Rho GTPases of plants (ROPs) and the production of NADPH oxidase-dependent reactive oxygen species (ROS) have emerged as putative downstream components. The role of NADPH oxidases, the ROS-producing enzymes that, based on their homology to the catalytic glycoprotein subunit of the mammalian phagocyte oxidase (gp91phox), are also called “respiratory burst oxidase homologues (Rboh)”, has been firmly established in various fundamental processes. These include localized lignin deposition, stomatal closure, pathogen responses, and root hair growth. NADPH oxidases are plasma membrane (PM)-bound enzymes with six trans-membrane domains, an N-terminal region that contains EF-hands, and a C-terminal oxidase domain responsible for oxidizing O2 to produce superoxide radicals in the apoplast (reviewed in,). The latter can quickly be dismutated, enzymatically or otherwise, into H2O2 that can freely diffuse back from the apoplast into the cytosol.

3Connections between members of the CrRLK1L and NADPH oxidase families have been proposed or established for THE1 and FER, respectively. For example, THE1 has been reported to be a positive regulator of CW damage-induced ROS production in seedlings, possibly through RbohD, while FER is both a negative regulator of H2O2 production in unchallenged leaves and of ROS in guard cells. Furthermore, in root hairs that elongate by tip-growth, FER is a positive regulator of ROS production through the ROP2-RbohC pathway. Similar to the rbohC loss-of-function mutant (also called root hair defective2 [rhd2]), disruption of FER leads to an impairment of ROS production and defective root hairs that burst,. Disruption of the redundant CrRLK1Ls ANXUR1 (ANX1) and ANX2, the two closest homologues of FER, triggers the rupture of pollen tubes (PTs), the tip-growing male gametophytes of flowering plants, resulting in male sterility,. Similar to fer root hairs, anx1 anx2 double mutant pollen form bulges and burst, failing to maintain their integrity during growth. This indicates that the FER and ANX RLKs could be cell-surface receptors that control CW integrity in tip-growing cells. In PTs, genetic evidence for the involvement of NADPH oxidases is lacking, but several studies have revealed a role for ROS during PT growth that remains to be precisely characterized. For example, it has been shown that either the use of ROS scavengers or the NADPH oxidase inhibitor diphenylene iodonium (DPI), or the down-regulation of a NADPH oxidase, reduces PT growth in tobacco. In addition, the application of DPI at higher concentrations has also been reported to induce PT rupture in lily.

4Because of the difficulty to image the dynamics of ROS production with good spatial and temporal resolution, and because of its multi-faceted impacts on CW properties and the activation of intracellular signaling, it is unknown how NADPH oxidase-dependent ROS control polar growth,. It was first reported that RbohC/RHD2 is required for calcium influx via the stimulation of Ca2+ channels and for the generation of a tip-focused gradient of cytosolic free calcium [Ca2+]cyt, which is essential for polar growth,. Later, Monshausen and colleagues reported that, under certain conditions, rbohC root hairs still display a tip-focused Ca2+ gradient, showing that RbohC was not essential for its establishment. Moreover, they showed that artificially increasing or decreasing apoplastic ROS leads to growth cessation and root hair bursting, respectively, consistent with a role for ROS in regulating CW properties. Finally, oscillations in apoplastic ROS levels just behind the tip were reported during root hair growth and correlated with growth rate, leading the authors to propose a model in which ROS rigidify the CW behind the tip, such that growth would be restricted to the tip. However, due to the irreversible nature of the ROS-sensitive oxidation of the dye they used, the observed oscillations are unlikely to reflect the true nature of ROS dynamics. Nonetheless, both models—namely the growth-promoting effect at the tip related to intracellular Ca2+ signaling and the growth-inhibiting effect behind the tip by rigidifying the CW—are not mutually exclusive as they could recruit different forms of ROS at different times and in different locations.

5In this study we show that over-expression of the ANX RLKs inhibits PT growth by the over-activation of exocytosis and the over-accumulation of secreted membrane and CW materials. Genetic interaction studies coupled with a phenotypic characterization of loss-of-function mutants of two partially redundant, pollen-expressed NADPH oxidases, RbohH and RbohJ, demonstrate that the ANX RLKs function upstream of these NADPH oxidases. Furthermore, analyses of the genetically encoded H2O2-sensitive HyPer and Ca2+-sensitive YC3.60 sensors in NADPH oxidase-deficient pollen revealed that NADPH oxidases generate tip-localized, pulsating ROS that are responsible—possibly through activation of Ca2+ channels—for maintaining a steady, tip-focused Ca2+ gradient.

6We have previously shown that ANX1-yellow fluorescent protein (YFP) and ANX2-YFP protein fusions are polarly localized in the PM at the tip of growing PTs in independent T1 transgenic Arabidopsis lines. Although in T1 lines, which contain a mixture of untransformed and transformed pollen grains, no obvious fertilization-related phenotypes could be detected, in vitro pollen germination and growth assays of homozygous lines that carry a single insertion of the constructs in the T3 generation revealed that ANX1-YFP and, to a lesser extent, ANX2-YFP inhibit pollen germination and PT growth compared to the wild type (WT) (Figures 1A, 1B, and S1). To investigate whether these phenotypes are due to over-expression or non-functionality of the fusion proteins, we transformed anx1-2/anx1-2 anx2-2/ANX2 and anx1-1/anx1-1 anx2-1/ANX2 plants with ANX1-YFP and ANX2-YFP fusions, respectively. In all T1 anx1-2/anx1-2 anx2-2/ANX2 lines expressing ANX1-YFP and anx1-1/anx1-1 anx2-1/ANX2 expressing ANX2-YFP PT rupture was reduced compared to the corresponding untransformed genotype. Moreover, in all T3 homozygous lines with good ANX1/2-YFP expression in the anx1 anx2 double mutant background, pollen germination, PT rupture, and PT length was indistinguishable from the WT (Figures 1A, 1B, S1, and S2). Thus, both ANX1-YFP and ANX2-YFP fusion proteins are functional, and the phenotypes observed in WT pollen expressing these fusion proteins are due to over-expression. Hereafter, independent homozygous lines expressing the ANX-YFP fusion proteins in the anx1 anx2 background will be called either complemented lines or ANX-YFP in anx1 anx2, while homozygous lines expressing the same fusion proteins in a WT background will be referred to as ANX-OX or ANX-YFP in WT.

7Pollen of ANX-OX lines germinated poorly and produced shorter and wider PTs than pollen of either WT or complemented lines (Figure 1A, 1B, and S1). To check whether these in vitro phenotypes impact the fitness of PTs in vivo, the male transmission efficiency (TEM) was assayed for each of the ANX-OX lines. Male transmission of ANX-YFP fusions was significantly decreased for all but one ANX2-OX line, showing that PTs over-expressing ANX-YFP fusion proteins are not as competitive as untransformed WT PTs (Figure 1C). Interestingly, the difference in the severity of these phenotypes between ANX1-OX lines or between ANX2-OX lines nicely correlated with the difference in the level of YFP fluorescence imaged at the PM of growing ANX-OX PTs (Figure 1D). The two strongest ANX1-OX lines (#1 and #4) and one ANX1-YFP complemented line were selected for further investigations.

8Time-lapse imaging of YFP fluorescence in growing PTs 6 hours after incubation showed that all PTs of the complemented line display the previously reported asymmetric distribution of YFP in the PM at the PT tip and were growing normally (n>100, Figure 2A, left panels). In contrast, only 43% to 47% of ANX1 over-expressing PTs grew and exhibited the same YFP distribution (n>100 PTs, ANX1-OX #4 and #1, respectively). The remaining ANX1-OX PTs (53% to 57% of all PTs) had ceased to elongate and displayed PM invaginations at the PT tip as observed with both YFP and the lipophilic dye FM4-64 (Figure 2A, right panels). Intriguingly, in the ANX1 over-expressing PTs that had ceased to elongate, the PM at the tip kept growing inwards, creating tunnel-like structures, instead of outwards as normally observed for tip-growing cells (Figures 2B and S3A; Video S1). Invaginations can start early as they were observed even in pollen grains that did not yet produce a tube (Figure S3B). The PM invagination phenotype was also observed in ANX2 over-expressing lines, while we never saw it in any of the ANX1-YFP or ANX2-YFP complemented lines (n>100 PTs, two independent lines for each fusion protein). PM invaginations were accompanied by thick extracellular deposits of CW material (Figure 2A, asterisk), which were pectinaceous as revealed by Ruthenium red staining (Figure 2C). This finding indicates that secretion of CW material still occurred at the site of PM invagination. In addition, detailed observations of ANX-OX PTs showed that apical CW thickening occurred before the invagination of the apical membrane.

9Since the surface of both the PM and the secreted CW material increase at the tip, we hypothesize that the balance between endocytosis and exocytosis rates might be tilted towards exocytosis at the tip of ANX-OX PTs. This could be achieved by a decrease or increase in the rate of endocytosis or exocytosis, respectively, or by a combination of both. For example, CW accumulation and PM invaginations have also been reported for tobacco PTs that over-express the phosphatidylinositol-4-phosphate 5-kinases PIP5K4, PIP5K5, and PIP5K6 –. The PIP5K-OX phenotypes originate from an over-initiation of aborted endocytosis in PIP5K-OX PTs, which show a dramatic inhibition of FM4-64 uptake,. To investigate this phenotype further, we conducted two types of experiments on growing PTs of complemented and ANX1-OX lines before they start to show PM invaginations and apical CW accumulation. First, we labeled PTs for 5 min with FM4-64, a styryl dye that quickly labels the PM and is internalized via endocytosis. In growing PTs of complemented lines (as in WT), FM4-64 is observed at the PM and in the apical cytoplasm as an inverted cone that presumably contains both endocytotic and secretory vesicles (Figure 3A and Video S2, upper panels). In growing ANX1-OX PTs, the same distribution was observed (Figure 3A and Video S2, lower panels), indicating that, in contrast to PIP5K-OX PTs, FM4-64 uptake and thus endocytosis was not impaired,. However, FM4-64 fluorescence intensity at the PM versus the apical cytoplasm was significantly lower than in complemented PTs, suggesting that there were globally more endocytotic and secretory vesicles in ANX1 over-expressing PTs (Figure 3A and 3B; n>25 each, p<0.01).

10As evidenced by Brefeldin A (BFA) treatment, a well-known inhibitor of exocytosis, ANX1-YFP is inserted at the apical PM via exocytosis (Figure S4A). Thus, we performed fluorescence recovery after photobleaching (FRAP) experiments for ANX1-OX and complemented PTs to analyze exocytosis dynamics in growing PTs as described previously,. Photobleaching was applied to the tip of growing PTs and measurements of the recovery of YFP fluorescence in the apical PM of the PT tips were carried out every 4 seconds. For ANX1 complemented PTs, the relative fluorescence recovery in the PM 10 seconds after photobleaching (I10sec) reached on average 47%±9% of the maximum relative fluorescence with a PT growth rate of 4.02±1.41 µm min−1 (n = 18; Figure 3C and 3D, left panels; Table S1; Video S3). No correlation was observed between I10sec and the fluorescence intensity pre-bleaching (R2 = 0.0092; Figure S4B), suggesting that the secretion rate of new ANX1-YFP fusion protein in the PM is independent of the amount of fusion protein originally present in the PM. Furthermore, no correlation was observed between I10sec and PT growth rate (R2 = 0.0001; Figure S4C), indicating that exocytosis and PT growth rate do not share a direct linear relationship.

11Interestingly, for PTs of both ANX1 over-expressing lines, I10sec was significantly higher than in the complemented line (57%±10%, p<0.01 for line #1; 57%±13%, p<0.05 for line #4), while their PT growth rate was significantly decreased to 1.34±0.68 µm min−1 and 1.78±0.53 µm min−1, respectively (n = 17 and n = 20 for ANX1-OX lines #1 and #4, respectively, p<0.01; Figure 3C and 3D right panels; Table S1; Video S3). The faster fluorescence recovery is unlikely to be due to a secondary effect of slow PT growth, because all the mutant PTs tested so far in FRAP experiments, namely DN-ROP1-OX (dominant negative ROP1), CA-ROP1-OX (constitutively active ROP1), RIC3-OX, RIC4-OX, and PIP5K6-OX grow slower than controls and show an inhibition of fluorescence recovery,. Thus, increased rates of fluorescence recovery at the apical PM indicate that the rate of exocytosis is increased at the apical PM of growing ANX1-OX PTs as compared to controls.

12Altogether, our results support the hypothesis that ANX over-expression tilts the balance of exo- to endocytosis towards more exocytosis, which progressively leads to CW accumulation. PT growth slows down as the apical CW thickness increases. When the latter reaches a certain threshold where the CW is not deformable anymore, expansion ceases and apical PM grows inwards due to continuing exocytosis.

13A better understanding of how the ANX RLKs regulate exocytosis requires the identification of downstream components of the ANX-dependent pathway. Recently, FER, which is the closest homologue of the ANX RLKs in Arabidopsis, has been shown to function as an upstream regulator of the ROP2/NADPH oxidase RbohC signaling pathway that controls ROS-dependent root hair growth. Moreover, down-regulation of a pollen-expressed NADPH oxidase and application of ROS scavengers inhibit PT growth in tobacco. Thus, we hypothesized that pollen-expressed NADPH oxidases could be downstream components of the ANX RLK pathway that coordinates CW integrity and PT growth. In Arabidopsis, NADPH oxidases belong to a family with ten members, two of which, RbohH (At5g60010) and RbohJ (At3g45810), sharing 81% amino acid identity, define a subgroup that is preferentially expressed in pollen (Figure S5C),. We isolated two independent single T-DNA insertional mutants for each of these NADPH oxidases, namely rbohH-1 (GABI_028G04), rbohH-3 (SALK_136917), rbohJ-2 (SAIL_31_D07), and rbohJ-3 (SALK_050665), which show little or no expression of the corresponding gene (Figure S5B). Pollen germination assays showed that PTs of single rbohJ-2 and rbohJ-3 mutant plants behaved like WT (∼8.5% bursting), while around 57% of PTs of single rbohH-1 and rbohH-3 mutant plants ruptured in vitro (Figure 4A). However, this mild PT rupture phenotype did not significantly reduce seed set or TEM in vivo (Figure 4A; Table S2). To investigate whether RbohH and RbohJ are redundant, single mutants were crossed to generate independent double mutant rbohH-1 rbohJ-2 and rbohH-3 rbohJ-3 plants. First, double homozygous mutant plants were only rarely found in the progeny of rbohH-1/RbohH rbohJ-2/rbohJ-2 and rbohH-3/RbohH rbohJ-3/rbohJ-3 (Table S3). Secondly, PTs of both independent rbohH-1 rbohJ-2 and rbohH-3 rbohJ-3 double mutants ruptured up to 80% in vitro (Figure 4A and 4B). The remaining germinating grains produced longer PTs, but they eventually also burst (Figure 4B). As a consequence, double homozygotes for rbohH-1 rbohJ-2 and rbohH-3 rbohJ-3 were partially sterile, producing only five to seven seeds per silique as compared to ∼60 seeds in WT or single mutant plants (Figure 4A and 4C). As evidenced by aniline blue staining after reciprocal crosses of rbohH rbohJ with WT, this sterility was due to the double mutant pollen being unable to grow sufficiently in vivo to reach and fertilize the female gametophytes (Figure 4D). This was further supported by analyses of male and female transmission efficiencies (TEs) of the rbohH-1 rbohJ-2 and rbohH-3 rbohJ-3 mutations, which showed a greatly reduced TEM while TEF was not significantly affected (Table S2).

14Taken together, these results provide compelling evidence that disruption of both RbohH and RbohJ leads to spontaneous PT rupture, preventing PTs to reach and fertilize the female gametophytes in vivo. Interestingly, all the above mentioned phenotypes are reminiscent of the anx1 anx2 double mutant phenotype,. Moreover, our results show partial functional redundancy between pollen-expressed NADPH oxidases, with RbohH being able to perfectly substitute for the loss of RbohJ, while the latter can only partially substitute for the loss of RbohH.

15To test whether RbohH and RbohJ are indeed downstream effectors of the ANX RLK-dependent pathway, the strong ANX1-OX line (#4) was crossed to rbohH-1 rbohJ-2 double mutant plants. Partially male sterile plants homozygous for rbohH-1 rbohJ-2 and homozygous for ANX1-YFP were retrieved in the F2 generation. Intriguingly, rbohH-1 rbohJ-2 pollen strongly expressing ANX1-YFP behaved exactly like rbohH-1 rbohJ-2 pollen with germination and PT rupture rates of ∼70% and 82%, respectively (Figure 5A). Furthermore, none of the growing PTs (n>100) of rbohH-1 rbohJ-2 plants homozygous for ANX1-YFP displayed CW accumulation or PM invagination, phenotypes observed in ANX1-OX PTs. To independently confirm these results, we directly transformed rbohH-1 rbohJ-2 mutant with ANX1-YFP fusion. Four independent, partially male sterile rbohH-1 rbohJ-2 transgenic lines homozygous for ANX1-YFP were recovered in the T2 generation. Again, neither CW accumulation nor PM invagination was observed in growing PTs (n>100 PTs for each), which eventually ruptured similar to the rbohH-1 rbohJ-2 PTs without the ANX1-OX construct. Furthermore, FRAP analyses also showed that the fast recovery rate at the apical PM observed in ANX1-OX PTs was suppressed in the rbohH rbohJ background as I10sec for rbohH rbohJ PTs over-expressing ANX1-YFP was similar to the complemented line (45%±9%, n = 23; Figure 5B and 5C; Table S1). Interestingly, a few rbohH rbohJ PTs over-expressing ANX1-YFP did not recover 80% of the initial fluorescence, a phenomenon that was never observed in controls or ANX1-YFP over-expressing PTs (Figure S6), indicating that exocytosis may become defective in these rbohH rbohJ ANX1-OX PTs.

16In summary, these results demonstrate that ANX1-OX phenotypes are dependent on functional RbohH and RbohJ and, consequently, that these pollen-expressed NADPH oxidases are positive downstream effectors of the ANX RLK-dependent pathway.

17To check whether disruption of RbohH and RbohJ impairs the production of ROS, we made use of the fluorescent ROS-sensitive dye 5-(and 6-)chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (CM-H2DCFDA) to stain PTs of WT and rbohH rbohJ double mutants. Fluorescence quantification of the apical cytoplasm in growing PTs treated for 5 minutes with 2 µM CM-H2DCFDA showed that PTs of the rbohH-1 rbohJ-2 and rbohH-3 rbohJ-3 double mutants displayed only 25% of the CM-H2DCFDA-derived fluorescence signal observed in WT PTs (p<0.01; Figure S7A and S7C). These low levels of CM-H2DCFDA-derived fluorescence were not due to a defect in dye uptake, as mutant and WT PTs exhibited the same level of fluorescence derived from the ROS-insensitive dye fluorescein diacetate (FDA) (Figure S7B and S7D). These results show that ROS production is indeed impaired in rbohH rbohJ PTs as expected for NADPH oxidase mutants. However, because CM-H2DCFDA oxidation is sensitive to different reactive oxygen and nitrogen species, sensitive to light, and irreversible, this dye cannot be used to monitor ROS production over time in growing PTs. Thus, we generated stably transformed Arabidopsis lines with PT expression of the genetically encoded YFP-based ratiometric sensor HyPer, which has been shown to faithfully report H2O2 production in bacteria, animal, and plant cells,. Curiously, in growing WT PTs expressing cytosolic HyPer (n = 27), the HyPer activity measured as the ratio of F488/F405 was stronger in the shank of PTs than at the tip (Figure 6A and 6B). We hypothesized that this strong shank activity could either be due to the presence of H2O2-producing organelles, such as mitochondria and/or peroxisomes in this region, an artifact of HyPer due to its pH sensitivity, or a combination of both. Indeed, it was shown that HyPer's activity artificially increases when the pH increases and that PTs display a pH gradient with an acidic tip and a alkaline shank. Interestingly, at the tip of growing PTs, HyPer activity displayed irregular oscillations originating from the tip periphery (Figures 6B and S8A; Video S4). However, oscillations of HyPer activity did not seem to correlate with growth rates (Figure S8B). In growing rbohH-1 rbohJ-2 PTs, HyPer activity was 16 and 18 times lower at the tip and in the shank, respectively, as compared to the WT (n = 22, p<0.001; Figure 6A and 6C). This indicates that membrane-bound RbohH and RbohJ are responsible for most of the H2O2 production revealed by the HyPer sensor. Moreover, since HyPer activity in the shank was also strongly reduced in rbohH-1 rbohJ-2 double mutant PTs (Figure 6A and 6C), the strong activity in the shank of WT PTs is likely due to propagation of the tip-derived H2O2 in the alkaline shanks, which artificially increases HyPer activity.

18To investigate if Rboh localization is consistent with the Rboh-dependent H2O2 production observed at the tip-periphery, we transformed partially sterile rbohH-3 rbohJ-3 plants with a green fluorescent protein (GFP)-RbohH fusion. Forty-four independent T1 transgenic lines out of 50 displayed rescue of sterility with WT-like elongated siliques (e.g., for three independent T1 lines with good GFP expression, the average of seeds per silique was 40.8±3.8, 40.9±11.1, and 43.4±6.9 as opposed to 6.2±3.5 in untransformed rbohH-3 rbohJ-3, n = 12 siliques per plant). In vitro pollen growth assays confirmed that the rbohH rbohJ bursting phenotypes were rescued by GFP-RbohH (Figure S8C) and that GFP-RbohH localized polarly to the plasma membrane at the tip of growing PTs (Figure S8D, left panels). These results show that GFP-RbohH protein fusion is functional and that its localization is consistent with both ANX1-YFP localization (Figure 2A, left panels) and Rboh-dependent H2O2 production at the tip periphery (Figure S8A). Furthermore, unlike the rbohH rbohJ complemented plants, in WT plants expressing the GFP-RbohH fusion, PM invagination and over-accumulation of CW material were also observed (Figure S8D, right panels), although these phenotypes appeared milder and less frequent than in ANX1-OX PTs.

19ROS and H2O2 have been shown to regulate calcium-permeable channels, e.g., in protoplast of root hairs and pear pollen, and a tip-focused Ca2+ gradient is essential for polar growth. Therefore, we crossed WT plants expressing the genetically encoded FRET-based Ca2+-cameleon YC3.60 in PTs with the anx1-2 anx2-2 and rbohH-1 rbohJ-2 double mutants, and partially male sterile anx1-2 anx2-2 and rbohH-1 rbohJ-2 plants homozygous for YC3.60 were recovered in subsequent generations. Cytosolic Ca2+ concentrations ([Ca2+]cyt, measured as FCFP/FVenus) were monitored over time at the PT tip and behind the tip when possible, and compared to YC3.60-expressing WT PTs grown and imaged under the same conditions. First, we attempted to study Ca2+ dynamics in anx1 anx2 bulges before bursting, young growing WT PTs, and arrested WT bulges. Bulges of anx1 anx2 never produced a growing tube, and only two out 17 burst during imaging. Interestingly, for both anx1 anx2 bulges that eventually burst, a sudden increase of [Ca2+]cyt was observed (Figure S9A, white arrow) before the first visible sign of rupture (Figure S9A, black arrow; Video S5). However, before bursting, [Ca2+]cyt in non-growing anx1 anx2 bulges was on average lower than at the tip of growing WT PTs but similar to the arrested WT bulges (Figure S9B). Because one cannot conclude if the decreased levels of [Ca2+]cyt in anx1 anx2 are due to the lack of ANX RLKs or rather to an indirect effect of arrested growth, we focused on studying Ca2+ dynamics in growing WT PTs and rbohH rbohJ pollen grains, which produce a few growing PTs that eventually burst.

20In steadily growing WT PTs, the tip-focused Ca2+ gradient (i.e., higher [Ca2+]cyt at the PT tip compared to behind the tip) was always observed and quite stable (n = 46; Figures 7A, 7C, S10A, and S10D; Video S6). Furthermore, as reported previously for in vitro grown Arabidopsis PTs,, but unlike lily PTs, we did not observe regular oscillations for either the PT growth rate or [Ca2+]cyt (Figure 7A). In growing rbohH rbohJ PTs (n = 30), [Ca2+]cyt was significantly lower than in the WT (p<0.001 for both tip and behind the tip regions; Figure 7B and 7C; Video S6). However, the tip-focused Ca2+ gradient and the PT growth rate were on average similar to that of WT PTs (Figure 7B and 7C; p = 0.054 for gradient, p = 0.84 for growth rate). But both the tip-focused Ca2+ gradient and the PT growth rate were significantly less steady over time in the rbohH rbohJ double mutant than in the WT, as evidenced by a significantly higher variance (p = 4.1280•10−13 and p = 0.008737 for [Ca2+]tip/[Ca2+]behind and growth rate, respectively; Figure S10A–S10F). The steady and jerky growth rate of WT and rbohH rbohJ PTs, respectively, were quite obvious during live-imaging of growing FM4-64 stained PTs (Video S7).

21These results indicate that disruption of the pollen-expressed NADPH oxidases RbohH and RbohJ does not abolish the tip-focused Ca2+ gradient but results in PTs that display (i) overall lower [Ca2+]cyt levels, and (ii) unstable tip-focused Ca2+ gradients and growth rates. Finally, increasing the external [Ca2+] in the germination medium from 5 mM to 15 mM or 30 mM, significantly decreased the rupture of rbohH rbohJ PTs, while lowering the external [Ca2+] had the opposite effect (p<0.05; Figure S11). These findings indicate that supplementing Ca2+ externally can partially stabilize the growth of rbohH rbohJ PTs. Conversely, decreasing external [Ca2+] strongly, increases the frequency of PTs that rupture in the WT (Figure S11), consistent with a pioneering report from the 1980s.

22In tip-growing root hairs, FER and RbohC/RHD2 have been proposed to function in the same pathway based on the facts that: (i) fer and rbohC/rhd2 display similar phenotypes with stunted, collapsed, and bursting root hairs, and (ii) that roots and root hairs of fer and one FER-OX line accumulate less and more ROS than WT, respectively,. Similarly, we show here that two independent rbohH rbohJ double mutants display anx-like phenotypes, i.e., PTs that burst, preventing them from growing to fertilize the female gametophytes (Figure 4). Consequently, both anx1 anx2 and rbohH rbohJ mutant plants are nearly male sterile. In addition, over-expression of both ANX1-YFP and GFP-RbohH triggers over-accumulation of membrane and CW materials (Figures 2 and S8D, right panels). Furthermore, we provide strong genetic evidence for the NADPH oxidases to function downstream of the ANX RLKs, by showing that the phenotypes observed in ANX-OX lines are abolished in the rbohH rbohJ mutant background (Figure 5). Therefore, the CrRLK1L-NADPH oxidase signaling module appears to be conserved in tip-growing cells. However, it is unlikely that the CW integrity pathway in pollen is a carbon copy of the root hair pathway, as the biological functions, growth habits and patterns, CW compositions, and growth environments are quite different between these tip-growing cells. For example, in root hairs FER has been shown to positively regulate RbohC-dependent ROS production through ROP2-signaling. In PTs, however, it remains unclear whether ANX RLKs also activate the NADPH oxidases RbohH and RbohJ through ROP-signaling, because over-activation of ROP-signaling leads to growth depolarization but does not trigger CW accumulation, PM invagination, or increased apical exocytosis,,, as we observed it in ANX1-OX lines (Figures 2 and 3).

23Our understanding of the role of NADPH oxidase-derived ROS signaling in plant development and in responses to abiotic and biotic stresses has improved tremendously over the past few years,,. Production of different ROS species has been imaged in different plant tissue and cell types, but because of the irreversible oxidation of the different dyes used (e.g., diaminobenzidine tetrahydrochloride, nitro blue tetrazolium [NBT], H2DCFDA and derivatives) meaningful information about the dynamics of ROS production is still scarce. GFP-based, genetically encoded sensors such as roGFPs and HyPer, which display reversible changes in fluorescence to alterations in redox/ROS levels, have been successfully developed and tested in plant cells. However, none of them have been assayed in a mutant background affecting ROS-producing enzymes. Here, we used the cytoplasmic H2O2-selective HyPer sensor expressed in PTs in a rbohH rbohJ NADPH oxidase-deficient mutant background to gain more insights into H2O2 production in tip-growing cells. HyPer activity displayed irregular oscillations at the tip of growing WT PTs (Figures 6B and S8). HyPer oscillations are unlikely due to pH oscillations reported for the tip of growing PTs, because (i) pH at the tip varies in a range where HyPer is not really pH-sensitive, and (ii) HyPer activity is completely abolished in growing rbohH rbohJ mutant PTs (Figure 6A and 6C). Moreover, HyPer activity originated from the periphery of the growing tip (Figure S8A), which is consistent with (i) the tip-preferential PM RbohH localization (Figure S8D, left panels) and the reported PM localization of other NADPH oxidases,, (ii) the NADPH oxidase activity reported at the PM,, and (iii) the extracellular, tip-localized O2. − distribution revealed by NBT staining of PTs,.

24The exact mechanism by which NADPH oxidase-dependent ROS regulate polar growth is still not fully understood. One reason for this is that quantitative information with good temporal and spatial resolution is difficult to obtain from growing CrRLK1L and/or NADPH-oxidase mutant cells (root hairs or PTs), owing to their rapid loss of cellular integrity. On one hand, the NADPH oxidase RbohC has been proposed to generate ROS that activate Ca2+-permeable channels at the PM to establish the tip-focused Ca2+ gradient and to promote expansion at the tip of root hairs,. On the other hand, a tip-focused Ca2+ gradient was observed in rbohC root hairs under certain conditions, indicating that RbohC was not essential to generate the Ca2+ gradient, but rather plays a role in restricting growth to the tip by rigidifying the CW behind the tip. On the basis of our results we propose a third alternative. Unlike anx1 anx2, a small but appreciable number (∼20%) of germinating rbohH rbohJ pollen grains are able to produce longer tubes in vitro that, however, will eventually burst, too. We took advantage of this opportunity to study [Ca2+]cyt dynamics with a good spatial and temporal resolution on growing NADPH oxidase-deficient PTs. First, the tip-focused Ca2+ gradient, visualized by the ratio between [Ca2+]tip/[Ca2+]behind, was clearly visible in growing rbohH rbohJ PTs, confirming that NADPH oxidases are not required to generate the Ca2+ gradient. However, unlike steadily growing WT PTs, which maintain a constant Ca2+ gradient over time (Figure S10A, S10D, and S10E), rbohH rbohJ PTs displayed a very unstable gradient, which could sometimes be steep but was abolished a few seconds later (Figure S10B, S10D, and S10E). This was correlated with more variable growth rates in rbohH rbohJ mutant compared to steadily growing WT PTs. Moreover, the global cytosolic Ca2+ levels were significantly lower in the growing rbohH rbohJ mutant PTs compared to WT PTs (Figure 7C). An increase in external [Ca2+] partially rescued the rupture of rbohH rbohJ PTs, while lowering the external [Ca2+] increased PT rupture in both the mutant and WT (Figure S11). This is in agreement with previous studies, which showed that lowering external [Ca2+] or limiting/blocking Ca2+ influx causes PTs and root hairs to burst,. The data reported here are consistent with NADPH oxidase-dependent ROS activating Ca2+-permeable channels for Ca2+ influx,. However, we propose that these yet unidentified channels do not generate the tip-focused Ca2+ gradient on their own; rather, they fine tune the Ca2+ gradient by stabilizing it to sustain steady growth of Arabidopsis PTs. It is noteworthy that different types of PM-localized Ca2+ channels have been characterized recently in tip-growing cells,. Among these, the Cyclic-Nucleotide-Gated Channel (CNGC) family is of particular interest, because single cngc18 or double cngc7 cngc8 mutant PTs spontaneously rupture after germination or produce kinky PTs that often burst as well,. Thus, CNGCs constitute good candidates for Ca2+ channels that are regulated by the CrRLK1L-NADPH oxidase signaling module at the PM. Annexins are also possible candidates as ANN1 has recently been shown to function as a ROS-activating Ca2+ transporter in root cells.

25One of the many roles proposed for the tip-focused Ca2+ gradient is to facilitate and stimulate exocytosis at the site of growth,, where the exocyst complex has been shown to function. Increasing external [Ca2+] leads to root hair and PT growth inhibition and CW thickening,. However, in this case it is not clear whether the accumulation of secreted CW material is due to an increase of the exocytosis rate or to uncoupling of exocytosis (that otherwise remains the same) from growth. Interestingly, we found that ANX over-expressing PTs grow slower than controls and also display CW accumulation (Figure 2). By performing FRAP analyses in the apical membrane of growing PTs of WT, anx1 anx2, and rbohH rbohJ plants expressing the ANX1-YFP fusion protein, we show that the rate of exocytosis is significantly increased in ANX1-OX PTs compared to controls. In contrast, for some of the rbohH rbohJ mutant PTs that have low calcium levels and an unsteady Ca2+ gradient, the recovery was impaired during our analysis. In agreement, the Ca2+channel blocker LaCl3, which has been shown to trigger the rupture of root hairs, appears to inhibit FRAP at the PT tip.

26Altogether our data are consistent with the following sequence of events: ANX RLKs positively regulate the NADPH oxidases RbohH and RbohJ, possibly through ROP signaling, to periodically produce ROS. Subsequently, ROS activate Ca2+-permeable channels for calcium influx to fine tune the tip-focused Ca2+ gradient, which in turn sustains secretion at the apical tip enabling PTs to elongate steadily without a loss of CW integrity. Perturbations of the pathway by over-expressing ANX RLKs would lead to a NADPH oxidase-dependent over-production of ROS and Ca2+ influx at the PT tip, which in turn would increase the secretion rate of membrane and CW materials, progressively leading to growth cessation and membrane invagination. Conversely, disrupting the ANX RLKs or NADPH oxidase would abolish NADPH oxidases-dependent ROS production and impair the opening of ROS-activated Ca2+-permeable channels, thus limiting the cell's ability to buffer [Ca2+]cyt variation that is required to maintain a steady tip-focused Ca2+ gradient. Consequently, the Ca2+ gradient and exocytosis at the PT tip would become erratic and, if not stabilized by compensatory mechanisms, the CW thickness would decrease until turgor pressure would lead to PT rupture. Finally, our model does not exclude that, in parallel to the signaling events described above, NADPH oxidase-dependent ROS and/or Ca2+ could directly alter CW properties, thereby affecting PT tip-growth. To investigate this possibility, direct measurements of the impact of ROS on CW properties during tip-growth would need to be established. We are confident that combining the continuously improving polar growth models and techniques to measure mechanical properties of growing cells, with genetic approaches, will soon uncover some of the remaining mysteries of the fascinating coordination between CW integrity and polar growth.

27After 3 h to 5 h of in vitro incubation on solid germination medium, 100 µl of liquid germination medium containing 0.01% Ruthenium red (Sigma, R-2751) or 2 µM of either FM4-64 (Molecular Probes, Invitrogen, T3166), CM-H2DCFDA (Molecular Probes, Invitrogen, C6827), or FDA (Sigma, F7378) were applied for 5 min to PTs, then washed away with fresh dye-free medium before imaging. PTs stained with Ruthenium red were imaged with a Leica DM6000 (Leica Microsystems). PTs stained with either FM4-64, CM-H2DCFDA, or FDA were imaged with a Leica SP2 or SP5 confocal microscope. For FM4-64 stained PTs, the apical PM region was defined as the first 2.5 µm along PM at the apex. A circle (2.5 µm in diameter) 3 µm away from tip was chosen for measurement of apical cytosol intensity. Relative localization of the FM4-64 dye on the PM versus the apical cytosol was calculated to illustrate the degree of FM4-64 internalization. For CM-H2DCFDA and FDA stained PTs, a circle (4 µm in diameter) 3 µm away from tip was chosen to measure apical cytosol intensity. All dye-derived fluorescence intensities were measured using the ImageJ 1.47d software after background subtraction. PTs of different genotypes were all imaged and quantified under the same conditions.

28Growing PTs expressing ANX1-YFP in an anx1-2 anx2-2 (complemented line), WT (ANX-OX, lines #1 and #4), and rbohH-1 rbohJ-2 backgrounds were used for FRAP analyses with the same imaging and quantification parameters. The apical region of PTs was photobleached using 100% power of a 514-nm laser (Leica SP5), and the recovery of fluorescence was monitored every 4 s in the following 2 min. The apical PM region was defined on the bright-field pictures at every time frame as the first 2.5 µm along PM at the apex, and fluorescence intensities were measured with ImageJ 1.47d software after background subtraction. Relative intensity of PM-localized ANX1-YFP compared with fluorescence before photobleaching was used to quantify the speed of fluorescence recovery. See Table S1 for curve fitting.

29Fluorescence in growing PTs of WT and rbohH-1 rbohJ-2 expressing either HyPer or YC3.60 were acquired (Leica SP2 confocal microscope) and quantified (ImageJ 1.47d) in the exact same conditions. For HyPer, fluorescence was acquired with the sequential mode and excitation at 488 nm and emission between 500–540 nm for F488 and excitation at 405 nm and emission between 500–540 nm for F405. Two circular regions of interest (ROIs; 4 µm in diameter), one 0.5 µm, the other 20 µm away from the apex were drawn for measurement of apical cytosol and far behind the tip intensities, respectively, for each single time point of each PT. For YC3.60, excitation was 458 nm then emission 469–501 nm for FCFP and 522–554 nm for FVenus. Two circular ROIs (4 µm in diameter), one 0.5 µm, the other 10 µm away from the apex, were drawn for measurement of apical cytosol and behind the tip intensities, respectively, for each single time point of each PT. All ratiometric measurements, i.e., F488/F405 and FCFP/FVenus, were determined with ImageJ 1.47d and its Ratio ROI Manager plugin after background subtraction. Ratiometric pictures were generated with the plugin Ratio Stack after median filtering. The YC3.60 titration curve (Figures 7D) was obtained as described before.

30All primers used in this study are listed in Table S4. Additional protocols are described in Text S1.