CORNICHON sorting and regulation of GLR channels underlie pollen tube Ca2+ homeostasis

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Science  04 May 2018:
Vol. 360, Issue 6388, pp. 533-536
DOI: 10.1126/science.aar6464

Multiple, diverse, and complex

Calcium currents characterize the developing pollen tube in the small mustard plant Arabidopsis and correlate with growth at the tip of the pollen tube. This system constitutes a practical model for screening for Ca2+-signaling mechanisms in plants. Wudick et al. analyzed multiple variants of glutamate receptor–like (GLR) channels and discovered that some work alone and others work in pairs or trios. Subcellular localization of GLRs is a complex response to CORNICHON sorting proteins, which leave some GLRs at the plasma membrane and ferry others to internal calcium reservoirs. The calcium current at the tip of the growing pollen tube apparently integrates multiple intracellular currents.

Science, this issue p. 533


Compared to animals, evolution of plant calcium (Ca2+) physiology has led to a loss of proteins for influx and small ligand–operated control of cytosolic Ca2+, leaving many Ca2+ mechanisms unaccounted for. Here, we show a mechanism for sorting and activation of glutamate receptor–like channels (GLRs) by CORNICHON HOMOLOG (CNIH) proteins. Single mutants of pollen-expressed Arabidopsis thaliana GLRs (AtGLRs) showed growth and Ca2+ flux phenotypes expected for plasma membrane Ca2+ channels. However, higher-order mutants of AtGLR3.3 revealed phenotypes contradicting this assumption. These discrepancies could be explained by subcellular AtGLR localization, and we explored the implication of AtCNIHs in this sorting. We found that AtGLRs interact with AtCNIH pairs, yielding specific intracellular localizations. AtCNIHs further trigger AtGLR activity in mammalian cells without any ligand. These results reveal a regulatory mechanism underlying Ca2+ homeostasis by sorting and activation of AtGLRs by AtCNIHs.

Plants use Ca2+ signaling despite lacking many components known to control the cytosolic Ca2+ concentration in mammalian cells (1). The gene family encoding glutamate receptor–like channels (GLRs) has been implicated in Ca2+ transport in various settings, namely, male gamete function (2, 3), stomatal closure (4), immunity (5), wound signaling (6), and root initiation (7). Evolutionary diversification of this family in Arabidopsis (AtGLR) resulted in 20 genes, distributed into three clades (8). To study AtGLR function in pollen tubes, whose growth and function depend on Ca2+ influx (9), we used reverse genetics, generating multiple AtGLR mutants (fig. S1, A and B).

In general, pollen tubes carrying AtGLR mutations showed conspicuous branching (Fig. 1, A to D), preceded by emergence of a new Ca2+ gradient, which reestablished the gradient onto the growing tip (Fig. 1, F and G, and movie S1). We also observed that sperm cells were typically shunted into the growing branch (Fig. 1E), suggesting that tip Ca2+ signaling established growth polarity and guided sperm nuclei toward the growing tip, thereby providing a basis for the unaffected fertility of most mutants.

Fig. 1 Characterization of AtGLR loss-of-function lines.

Differential interference contrast images of wild-type (A), glr1.4-1 (B), glr3.3-1 (C), and glr1.4-1/3.3-1 (D) pollen. (E) Time-lapse series of migrating vegetative (magenta) and sperm (green) nuclei in a branching glr3.3-1 tube. The tube outline is dotted; the asterisk indicates the growing branch. (F) Steep Ca2+ gradient in an YC3.6-expressing wild-type pollen tube with highest Ca2+ concentration ([Ca2+]) at the tip. (G) Time series (t0 to t4, movie S1) of a branching YC3.6-expressing glr1.4-1 tube with oscillatory [Ca2+] at the newly emerging tip (asterisk). Bars, 10 μm. (H) Pollen tube growth rate (upper panel) and Ca2+ influx (lower panel) across the tip plasma membrane of wild-type (Col-0) and glr mutants. Dot-dashed lines represent wild-type means of growth rate or Ca2+ influx, respectively. *P < 0.05, ·P < 0.1. (I) YC3.6-measured cytosolic Ca2+ signatures (YFP/CFP fluorescence values) were significantly lower in glr1.1/1.2 than in wild type, both at the tip (*P < 0.05) and shank (*P < 0.05). (J) Average length of wild-type and glr1.2/glr1.4-1 pollen tubes 6 hours after pollination (HAP, n = 15) on wild-type pistils. Asterisk indicates significant difference from wild type (P < 0.05); dotted line represents mean. (K) Comparison of normalized growth rates/fluxes, in mutants with (AtGLR3.3 not mutated) or without AtGLR3.3 (AtGLR3.3 mutated). Numbers refer to the lines in Fig. 1H. *P < 0.01.

We measured extracellular net Ca2+ fluxes of growing pollen tube tips using a Ca2+-selective vibrating probe. Tubes from glr1.2, glr2.1, glr1.1/glr1.2, glr1.2/glr1.4-1, and glr1.2/1.4-1/2.2/3.3-1 mutants showed only half the flux of wild type (Fig. 1H, lower panel). Lower cytosolic Ca2+ concentrations in tip and shank were found in glr1.1/glr1.2 tubes (Fig. 1I) as quantified with the ratiometric Ca2+ sensor Yellow Cameleon 3.6 (YC3.6) (2, 10), supporting the view of AtGLRs as plasma membrane channels (1). Additionally, glr1.2/glr1.4 pollen tubes were shorter than those of wild type (~100 μm; Fig. 1J and fig. S2A), suggesting that mutations in AtGLRs affect various pollen-mediated phenotypes. Mutations that did not alter Ca2+ fluxes often experienced genetic compensation. For instance, in glr1.1/1.4-1 pollen, AtGLR1.2 mRNA was overexpressed (fig. S1C).

A diminished growth rate did not correlate with Ca2+ flux reductions. Double and triple mutants involving AtGLR3.3 showed greater Ca2+ fluxes than mutants not involving AtGLR3.3. Whereas glr1.2 alone decreased Ca2+ flux, the glr1.2/3.3 double mutant restored it. Nonetheless, in all mutant combinations, pollen tubes grew more slowly than wild type (Fig. 1H, upper panel). Thus, a correlation between Ca2+ influx and growth (11) did not hold. Cluster analysis revealed a group of mutants with wild-type or higher Ca2+ fluxes but slower-than-normal growth rates, composed of double or triple mutants that include AtGLR3.3 (fig. S2B). We defined a “Ca2+ use growth efficiency” metric as the ratio of growth rate to Ca2+ flux (Fig. 1K). In mutants involving glr3.3, this efficiency decreased as a result of high fluxes, and we hypothesized that mechanisms other than channel conductance could regulate Ca2+ homeostasis.

So far, AtGLRs, like their mammalian counterparts, have been assumed to localize to the plasma membrane (1, 7). However, the antagonistic effects of glr1.2 and glr3.3 could be explained by their localization in different membranes. Therefore, we analyzed the subcellular localization of AtGLR3.3 and AtGLR2.1, the AtGLR most highly expressed in pollen. AtGLR2.1-GFP (green fluorescent protein) localized to the complex vacuolar system (Fig. 2A and fig. S2, C to F), whereas AtGLR3.3-GFP localized to the sperm plasma membrane and endomembranes but was undetectable in the pollen tube plasma membrane (Fig. 2B and fig. S2, G to I), indicating that the secretory pathway must sort and target both AtGLRs.

Fig. 2 Subcellular localization of AtGLRs and AtCNIHs, their interaction, and functional yeast complementation.

Wild-type (wt) pollen tubes expressing AtGLR2.1-GFP (A) or AtGLR3.3-GFP (B) localizing to the tonoplast or sperm cell plasma membrane and endomembranes, respectively. Tube contour indicated by dotted line. (C) Yeast mating-based split ubiquitin system (mbSUS) assay revealing interaction on control (row 1) or selective media (rows 2 and 3) of AtGLR3.3/AtCNIH1 (column 1) or AtCNIH4 (column 2) in the absence (0 μM) or presence (500 μM) of methionine. Negative (NubG) and positive controls (NubWT) are in columns 3 and 4, respectively. AtGLR3.3/AtCNIH1 and AtGLR3.3/AtCNIH4 interactions were corroborated by LacZ activation (bottom row). (D) RFP-AtCNIH1 and (E) RFP-AtCNIH4 labeling ER exit site–like structures (ERES, arrowheads), and the tube plasma membrane [(E), arrows]. Bars, 10 μm. (F) Fluorescence (upper row) and merged bright-field images (lower row) of an BYT45ervpΔ yeast expressing ScNha1p-GFP and cotransformed with the AtCNIHs. Arrows indicate the peri-nuclear ER. Bars, 5μm.

We queried a membrane-based interactome database (12) for CORNICHON homologs, because they mediate the trafficking of ionotropic glutamate receptors (iGluRs) in animal cells (13). Arabidopsis contains five CORNICHONS (AtCNIH1-5; fig. S3, A and D). AtCNIH1 interacted with various AtGLRs (12). AtCNIH1-5 all share characteristic features, including a cornichon motif (fig. S3, A and B) and an “IFRTL”-like Sec24-interacting motif (fig. S3, A, B, E, and F). RNA of all AtCNIHs was detected in pollen, being highest for AtCNIH4 (fig. S3C). Both AtCNIH1 and AtCNIH4 interact with AtGLR3.3 (Fig. 2C). In other species, CORNICHONS are essential for sorting and trafficking proteins from the endoplasmic reticulum (ER). We confirmed this for AtCNIHs by complementing the erv14pΔ yeast Saccharomyces cerevisiae (Sc) CORNICHON mutant (14) and observing tolerance toward Na+ and ScNha1p localization (Fig. 2F and fig. S3G). Plasma membrane localization of ScNha1p in the erv14pΔ mutant was rescued by AtCNIH1, 3, and 4, but not by AtCNIH2 or 5, and restored Na+ tolerance to wild-type levels, permitting growth on 800 mM NaCl.

In pollen, red fluorescent protein–tagged AtCNIHs (RFP-AtCNIHs) localized to endomembranes and punctate structures (Fig. 2, D and E, and fig. S4, A to C) that colocalized with markers for the ER (fig. S4, L to S) but not the cis or medial Golgi (fig. S4, D to K). We also observed pollen tube plasma membrane localization of RFP-AtCNIH4 (Fig. 2E) and RFP-AtCNIH3 (fig. S4C). The presence of AtCNIHs in ER foci (Fig. 2, D and E) was consistent with their localization at ER exit sites (ERES) (15). Indeed, AtCNIHs colocalized with the ERES marker AtSec24 (fig. S4, T to W). cnih1, cnih4, and the double cnih1/cnih4 mutants (fig. S5, A and B) showed reduced pollen tube tip Ca2+ fluxes but wild-type–like growth rates (fig. S5, C and D).

We next analyzed the impact of AtCNIHs on cargo trafficking. Although AtGLR3.3-GFP localized to sperm in cnih1 or cnih4 pollen (Fig. 3, A and B), it accumulated in reticulate and punctate structures in cnih1/cnih4 (Fig. 3C) and other cnih double mutants (fig. S6, A to E). Neither overexpression of AtGLR3.3-GFP in cnih1 and cnih4 pollen, nor its endomembrane retention in cnih1/cnih4 mutants, changed the Ca2+ flux phenotypes observed in corresponding lines lacking AtGLR3.3-GFP (fig. S7A). Confirming a role in ER sorting, AtGLR2.1-GFP was similarly retained in endomembranes in cnih1/cnih4, but not in any single mutant (fig. S6, F to H).

Fig. 3 Expression of AtGLR3.3-GFP and reference proteins in control and cnih pollen.

(A) cnih1 and (B) cnih4 pollen expressing AtGLR3.3-GFP in sperm cells (arrows). (C) AtGLR3.3-GFP localization in cnih1/cnih4 pollen, labeling endomembranes. (D) Expression of AtAHA6-GFP or (E) AtACA9-YFP in wild-type (wt, upper panels) or cnih1/cnih4 pollen (lower panels). (F) Localization of the cis or medial Golgi marker AtCGL1-CFP in wild-type (wt, left panel) and cnih1/cnih4 pollen (right panel). Expression of mCherry-AtCNGC9 in wild-type (wt) (G) or cnih1/cnih4 pollen (H) coexpressing AtGLR3.3-GFP (I). (J) Corresponding merged image. (K) Expression of AtTIP1;3-GFP, (L) AtTIP5;1-mCherry, (M) AtTET12-GFP, (N) Mus musculus Lyn24 (MmLyn24-GFP), (O) AtLIP2-RFP, and (P) R-GECO1 in wild-type (wt, left panels) and cnih1/cnih4 pollen (right panels). Arrows indicate the tube plasma membrane, arrowheads the sperm cells. Bars, 10 μm.

We next addressed the AtCNIH cargo specificity in wild type and cnih1/cnih4 by comparing two P-type ATPases (adenosine triphosphatases) with comparable intramembrane domain topology. Similarly to AtGLR2.1 and 3.3, the autoinhibited H+-ATPase 6 (AtAHA6-GFP) was retained in endomembranes in cnih1/cnih4 (Fig. 3D), whereas the autoinhibited Ca2+-ATPase 9 (AtACA9-YFP) was unaffected in this (Fig. 3E) or other cnih double mutants (fig. S6, I and J), suggesting cargo specificity for pairs of AtCNIHs. Indeed, marker proteins for the cis or medial Golgi (Fig. 3, F to J, and fig. S6, K to M), vegetative and sperm vacuoles (Fig. 3, K and L), and coat protein complex II (COPII) vesicles (fig. S6, N and O) remained correctly targeted in cnih1/cnih4 pollen, as were proteins with AtGLR3.3-like localization (Fig. 3, M to O), or a cytoplasmic protein (Fig. 3P). Thus, ER sorting of certain integral membrane proteins, but not of other soluble or membrane-attached proteins, was dependent on AtCNIH pairs. Although we found that AtCNIH homomers may be formed (Fig. 4, A and B), AtGLR trafficking was dependent on the formation of AtCNIH heteromers (Fig. 4C and fig. S6, P to R).

Fig. 4 Interaction of AtCNIH1/AtCNIH4 and their effect on AtGLR3.3 current properties.

Transient tobacco epidermis leaf expression of cYFP-AtCNIH1 + nYFP-AtCNIH1 (A) and cYFP-AtCNIH4 + nYFP-AtCNIH4 (B) caused YFP fluorescence reconstitution, indicating the formation of homomers (A and B) or heteromers (C). Insets represent merged bright-field/ fluorescence images. Bars, 20 μm. (D) Typical currents in COS-7 cells expressing AtCNIHs, AtGLR3.3, or a combination thereof. (E) Stationary current/voltage relationships from recordings shown in (D). (F) Typical currents recorded in cells coexpressing AtGLR3.3 and AtCNIH1/AtCNIH4 in bath solution (control) or after Gd3+ addition (Gd3+ 500 μM). (G) Stationary normalized current/voltage relationships from recordings shown in (F). Error bars represent SE.

We next challenged AtCNIH regulation of AtGLR channel activity, as documented for animal glutamate receptors (13). Patch-clamp experiments in COS-7 cells expressing AtCNIH1, AtCNIH4, AtGLR3.3, or AtCNIH1/AtGLR3.3 revealed control-like currents (Fig. 4, D and E, and fig. S7B). However, cells coexpressing AtGLR3.3/AtCNIH4 showed larger currents, and coexpression of AtGLR3.3/AtCNIH1/AtCNIH4 led to a twofold increase, even without ligand (Fig. 4, D and E). Currents were ohmic-like, Gd3+-sensitive, and without selectivity for Na+ over Ca2+, as revealed by the reversal potential close to 0 mV, despite asymmetric solutions (pipette: 150 mM Na+; bath: 20 mM Ca2+, 10 mM Na+; Fig. 4, E to G). We next addressed AtCNIH specificity for eliciting currents by using AtGLR3.2, a pollen tubeexpressed AtGLR3.3 homolog. Coexpressing AtGLR3.2 with either AtCNIH1 or AtCNIH4 yielded activation of currents similar to those for AtGLR3.3/AtCNIH1/AtCNIH4 (fig. S7, C to F), albeit up to fivefold higher than the currents measured for any other AtGLR in heterologous systems (7, 16). In COS-7, AtGLR3.2 plasma membrane localization was independent of AtCNIH1 or 4 (fig. S7, G to H), suggesting they were more relevant for channel activity than targeting in this system.

We conclude that AtCNIHs are essential for sorting, trafficking, and localizing AtGLRs in planta, and the interaction between proteins of these two families enhances AtGLR channel activity. These additive effects support increased cationic currents driven by AtGLRs at a magnitude not previously observed (2, 7, 16). Ionic selectivity of AtGLRs is not understood, but Physcomitrella patens GLR1 (PpGLR1) conducts Ca2+ (3). Multiple mechanisms linked to cytosolic Ca2+ homeostasis may contribute to the phenotypes that we observed. Some AtGLR members, like AtGLR1.2, may work as plasma membrane Ca2+ channels, but we posit that sorting of other AtGLRs to internal Ca2+ reservoirs (ER, vacuole, mitochondria) contributes to cytosolic Ca2+ homeostasis. When perturbed by multiple mutations, Ca2+ homeostasis is disrupted and growth is affected. Therefore, our results suggest that specific AtCNIHs regulate quantity, location, and activity of AtGLRs, affecting the concentration of cytosolic Ca2+ and, by interacting with other protein families, possibly the concentration of different ions (15).

Supplementary Materials

Materials and Methods

Supplementary Text

Figs. S1 to S7

Table S1

Movie S1

References (1747)

References and Notes

Acknowledgments: We thank L. Boavida, F. Brandizzi, A. Costa, U. Grossniklaus, J. Harper, M. Iwano, I. Heilmann, M. Palmgren, L.-J. Qu, and Y. Zhang for sharing materials; S. Wolniak and N. Andrews for sharing facilities; A. Beaven for assisting image acquisition; A. David and T. Maié for lab help; L. Boavida for discussions; and A. A. Simon for reviewing the manuscript. Funding: NSF (MCB 1616437/2016 and MCB1714993/2017), UMD, and FCT (PTDC/BEX-BCM/0376/2012, PTDC/BIA-PLA/4018/2012) to J.A.F.; CONACYT (220085) and DGAPA-UNAM (IN-203817) to O.P.; and postdoctoral fellowships SFRH/PD/70739/2010 and SFRH/PD/70820/2010 to M.M.W. and M.T.P., respectively. Author contributions: M.M.W. contributed to the conceptualization, methodology, validation, formal analysis, investigation, resources, data curation, writing, editing, supervision, and vizualization. M.T.P. and E.M. contributed to the methodology, validation, formal analysis, investigation, data curation, writing, reviewing, and visualization. P.R.-S. contributed to the methodology, validation, investigation, resources, reviewing, and visualization. M.A.L. contributed to the methodology, investigation, resources, reviewing, and visualization. C.C. contributed to the methodology, investigation, resources, and review. C.O.N., J.C.C., and P.T.L. contributed to the methodology, validation, investigation, and review. D.S.C.D. contributed to the conceptualization, methodology, software, validation, formal analysis, data curation review, and visualization. O.P. contributed to methodology, review, supervision, and funding aquisition. J.A.F. contributed to the conceptualization, methodology, writing, editing, supervision, project administration, and funding aquisition. Competing interests: None declared. Data and materials availability: Accession numbers for all genes referred to in this manuscript and all data needed to evaluate the conclusions in the paper are present in the paper or the supplementary materials.
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