Signaling with ions: the keystone for apical cell growth and morphogenesis in pollen tubes

50 Pages·2016·1.68 MB·English

Plant Physiology Preview. Published on November 28, 2016, as DOI:10.1104/pp.16.01561
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1 Running title: Ion transport in pollen tubes
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3 Corresponding Author:
4 José A. Feijó
5 Professor
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7 University of Maryland
8 Dept. of Cell Biology and Molecular Genetics
9 0118 Bioscience Research Building, 4066 Campus Dr.
10 College Park, MD 20742-5815
11 Phone [phone]
12 Fax [phone]
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14 Email: [email protected]
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20 Research Area: Special Issue, Pollen Biology, Alice Y Cheung (editor)
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24 Keywords: Arabidopsis, pollen, ion transport
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26 Title: Signalling with ions: the keystone for apical cell growth and morphogenesis in
27 pollen tubes
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32 Erwan Michard, Alexander A Simon, Bárbara Tavares, Michael M Wudick, José A
33 Feijó1*
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36 University of Maryland Dept. of Cell Biology and Molecular Genetics, 0118 Bioscience
37 Research Building, 4066 Campus Dr. College Park, MD 20742-5815, U.S. (EM, AAS,
38 MMW, JAF); Instituto Gulbenkian de Ciência, Oeiras, 2780-901, Portugal (BT)
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41 One-sentence Summary: Ion homeostasis and signalling are crucial to regulate pollen
42 tube growth and morphogenesis, and affect upstream membrane transporters and
43 downstream targets
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46 Footnotes:
47 1 This work was supported by the National Science Foundation (MCB 1616437/2016)
48 and the University of Maryland.
49 * Corresponding author: Phone [phone], Fax [phone], Email:
50 [email protected]
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54 Abstract
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56 Pollen tubes (PTs) are one of the best characterized plant cell types in many respects.
57 The identification of key players involved in tube growth offers the perspective of an
58 integrative understanding of cell morphogenesis processes. One outstanding feature of
59 PTs is their prominent dependence on ion dynamics to promote and regulate growth.
60 Many reports have identified and characterized membrane transport proteins, such as
61 channels, transporters and pumps, as well as their regulatory mechanisms, some of
62 which themselves dependent on ions such as Ca2+ and H+. The signalling network that
63 governs growth is based on a strict spatial distribution of signalling molecules, including
64 apical gradients of Ca2+, H+ and ROS. A central role for ion homeostasis, and more
65 generally membrane transport systems, is proposed to underlie the spatio-temporal
66 establishment of the signalling network that controls the PT self-organization and
67 morphogenesis. Here, we review the latest progress on understanding tube growth
68 from the perspective of membrane transporters and ion homeostasis. The ongoing
69 molecular characterization of the Ca2+-signalling pathway, as well as the recent
70 identification of female external cues and corresponding receptors on the pollen that
71 control growth orientation, offer a firm biological context to boost the field even further.
72
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73 Pollen tubes as a tailored model for studying ion dynamics at the cell biology
74 level
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76 Pollen tubes (PTs) have long been considered outstanding models for cell biology for a
77 variety of reasons. On one hand they display dramatic features at the level of cell
78 polarity, cytoskeleton dynamics, growth rates, membrane recycling, cell-cell interaction
79 mechanisms, etc. (Michard et al., 2009; Cheung and Wu, 2007; Qin and Yang, 2011;
80 Hepler, 2016). On the other hand their study is backed-up by extensive databases on
81 transcriptomics and proteomics on practically all of its biological contexts (Pina et al.,
82 2005; Honys and Twell, 2003; Borges et al., 2008; Qin et al., 2009; Boavida et al.,
83 2011; Mayank et al., 2012; Lang et al., 2015; Pertl-Obermeyer et al., 2014). All these
84 features define a unique cell type, so evolutionarily streamlined to fast growth and
85 sperm delivery (Williams, 2008), that it remained basically conserved as the only
86 gametophyte developmental end product for male function since the cretaceous
87 (Rudall and Bateman, 2007). PTs in angiosperms can grow up to 4 µm.sec-1 and are
88 characterized by the periodic formation of callose plugs that isolate older parts of the
89 tube to die so that growth can be maintained continuously restricted to the apical end
90 (review in Boavida et al., 2005a,b). In plants, PTs share the same type of apical growth
91 mechanism with root hairs, a fact that is reflected on the molecular level by the
92 existence of an “apical signature” in transcriptomics profiles (Becker et al., 2014). Root
93 hairs, however, grow slower than PTs. In addition, root hair length is controlled through
94 a signaling network involving RSL transcription factors (Honkanen and Dolan, 2016). In
95 spite of those main differences, apical growth of root hairs and PTs is characterized by
96 apical exocytosis of new cell wall material, similar ion gradients, fluxes at the tip and a
97 mechanism depending on similar actin cytoskeleton promoting cell elongation (Gu and
98 Nielsen, 2013; Ketelaar, 2013; Mendrinna and Persson, 2015; Mangano et al., 2016).
99 With so many peculiarities and extreme adaptations to function, the applicability of PT-
100 specific mechanisms to other plant somatic cells, namely diffuse growing ones, is not
101 always straightforward. This assumption is clearly reflected in the well differentiated
102 transcriptomic profiles between PTs and those of other organs and tissues (see snail-
103 view representation in Becker et al., 2003 and Pina et al., 2005). The one feature in
104 PTs that stands out the most is their strict dependence on ion dynamics to grow and
105 sustain their functions (Michard et al., 2009). Different ions, namely calcium (Ca2+),
106 protons (H+ or pH) and chloride (Cl-) form stable/standing gradients of cytosolic
107 concentration in the clear zone (Fig.1). Of relevance, these spatial patterns and their
108 temporal and spatial variations, or choreographies, correlate remarkably well with the
109 intracellular structure of PTs, be it the grading of organelle sizes defining the so called
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110 “clear-zone”, the cytoplasmic reverse fountain (Cheung and Wu, 2007; Hepler and
111 Winship, 2015) and, to some extent, the definition of the membrane recycling domains
112 in the tip (Parton et al., 2001; Kost, 2008; Bove et al., 2008, Wang et al. 2016; Bloch et
113 al. 2016)(Fig.1). The existence of these ion gradients as a putative central coordinating
114 mechanism for cellular growth and morphogenesis in PTs has been conceptualized
115 elsewhere (Feijó et al., 1995, Feijó et al., 2001; Michard et al., 2009; Daminelli et al.
116 2017). Largely believed to be generated from plasma membrane (PM) activity, the ion
117 choreography of PTs is easily traceable by non-invasive methods such as ion-specific
118 vibrating probe electrophysiology and ion-specific probe imaging to show nearly perfect
119 correlation with growth variations while also allowing to score for very subtle
120 phenotypes, hardly detectable in other non-growing cells (e.g. Michard et al., 2011).
121 Of relevance, growth rate, ion fluxes and concentrations may oscillate in PTs, and also
122 during root hair growth. Some studies present the choreography of ion fluxes and
123 intracellular ion concentrations by a relative lag time during a growth period in PT
124 (reviewed in Holdaway-Clarke and Hepler, 2003; Hepler et al., 2013) and root hair
125 (Monshausen et al., 2007, 2008). In such studies, the minimum pH or maximum
126 calcium oscillations and growth peak display a time lag of a few seconds in both PT
127 and root hairs, suggestive of similar regulation mechanisms of growth. Of notice, the
128 flux of Cl- was found to be in phase with growth (Zonia et al. 2002). Nevertheless, the
129 different estimates of advances and delays have been collected in a variety of
130 biological systems (lily, tobacco, petunia, less in Arabidopsis), imaging techniques
131 (DIC, wide-field or confocal fluorescence) and electrophysiology methods in such ways
132 that comparisons of the published delays and proposed sequences of events are
133 subject to potential distortions (Portes et al. 2015; Damineli et al. 2017). Last but not
134 least, correlation does not imply causation and not much can be deduced from those
135 studies, particularly because we do not know the kinetic properties of key reactions
136 within the networks, such as molecular diffusion, protein phosphorylation, exocyotis
137 etc. (Damineli et al. 2017)
138
139
140 (FIGURE 1 HERE)
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142 Figure 1 highlights this peculiar correlation between ion dynamics and cell structure.
143 Spatial correlation between features of the cytosolic gradients (Fig.1B, C and E), and
144 other cellular structures are conspicuous, and easily observed at the level of zonation
145 of organelles along the clear zone (Fig.1A) or the actin cytoskeleton (Fig.1D).
146 Characterizing the transport molecules that generate these gradients may be a first
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147
148 step into their manipulation and eventually test the hypothesis that spatial correlations
149 are not just a mere phenomenological coincidence, but may actually be causal and part
150 of a network of regulatory feed-back loops. One first step into that direction is the
151 establishment of a functional correlation between the transport molecules and the
152 predicted outcome of their activity in terms of ion dynamics, whether at the level of
153 cytosolic concentration, or at the level of membrane transport. One such example is
154 offered in Figure 1, where the localization of the H+- ATPase NtAHA1 (F) correlates
155 perfectly with the existence of intracellular pH domains (E) and extracellular H+ fluxes
156 (G)(Certal et al., 2008; Michard et al., 2008). The fact that this crucial pump is
157 segregated from the tip PM triggers a numbers of testable models, and by itself already
158 defines an experimental paradigm offered uniquely by PTs.
159 In Arabidopsis, more than 800 transporter transcripts have been identified in pollen
160 using the ATH1 mRNA microarray (Pina et al., 2005; Bock et al., 2006), and this over-
161 representation is confirmed by RNAseq in Arabidopsis and lily (Loraine et al., 2013;
162 Lang et al., 2015). This is perhaps one of the reasons why PTs have been widely
163 explored in recent years for phenotyping an increasing repertoire of channels,
164 transporters and pumps, rendering the vegetative cell of the PT likely one of the best
165 studied cells in plants in terms of ion dynamics. Figure 2 and Table 1 summarize this
166 accumulated knowledge. It incorporates not only genes that have been already
167 characterized, but also genes that can be predicted from transcriptomics, proteomics
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168 and comparative physiology to play roles in PT growth. In this review we focus on
169 organizing or systematizing this growing repertoire, focusing on 3 different angles: (1)
170 is this knowledge sufficient to define regulatory mechanisms around a specific ion? (2)
171 What are the downstream targets of specific ions? And (3) how do cellular process
172 feed-back to regulate ion transport?
173
174 (FIGURE 2 HERE)
175
176
177 Opposing forces: turgor and cell wall deposition
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179 When growing PTs or root hairs stop in response to an osmotic shock, the exocytosis
180 of vesicles ensuring cell wall deposition continues at the tip (Schroeter and Sievers,
181 1971; Li et al., 1996; Zerzour et al., 2009). So, apparently the main control of the
182 apical growth process does not depend on turgor as much as it does in other plant
183 cell growth mechanisms (Cosgrove, 2014; Ali and Traas, 2016). Quantification of the
184 opposing growth forces in lily PTs led to a difference of two orders of magnitude
185 between the internal turgor pressure (ca. 0.3 MPa) and the cell wall elasticity (ca. 20-
186 90 MPa; Vogler et al., 2012) clearly bringing other growth control mechanisms than
187 turgor to the board. Supporting this concept, growth can be arrested by non-related
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188 turgor means, such as caffeine treatment (Li et al., 1996). Yet, despite the fact that
189 there is no correlation between turgor and growth rate, a minimal turgor pressure of
190 ca. 0.3 Mpa is necessary to sustain PT growth in lily (Benkert et al., 1997). The
191 general consensus is that turgor drives the PT growth by providing a minimal
192 mechanical force necessary for cell wall elongation at the tip, but that it plays no or a
193 minor regulatory role. Various theoretical approaches have tried to bridge these
194 opposing forces at work by modeling anisotropic-viscoplastic properties (Dumais et
195 al., 2006), incorporation of new cell wall material, particularly pectine-esters, as a key
196 factor in softening the wall by affecting polymer cross-links (Rojas et al., 2011) and
197 finite element analysis methods (Fayant et al., 2010; Vogler et al., 2012). The
198 discussion of the opportunities and caveats of these models is beyond the scope of
199 this review, and here we focus on the facts that (1) turgor is a direct consequence of
200 water transport driven by small solutes, notably ions, and (2) ions such as Ca2+ and
201 H+ are involved in the mechanical maturation of cell walls.
202 PTs can appropriately adjust turgor pressure by adapting to changes in external
203 osmolarity (Benkert et al., 1997) but no osmosensor has yet been characterized.
204 Mechanosensitive ion channels like the cation channel AtOSCA1 (Yuan et al., 2014)
205 or the anion channel AtMSL8 (Hamilton et al., 2015) offer a conceptual basis for a
206 sensor, but so far the reported ion currents and phenotypes of these channels do not
207 warrant that they may be acting in PT growth.
208 Several arguments can be raised on the role of aquaporins in facilitating water
209 transport in PTs (Obermeyer, 2017). Pollen aquaporins of the SIP and TIP clade have
210 been located at endomembranes (Ishikawa et al., 2005; Wudick et al., 2014) while
211 NIP aquaporins were localized in the pollen PM (Lang et al., 2015). Heterologous
212 overexpression of AtPIP aquaporins yielded an increase of the water permeability of
213 lily pollen, but no evident functional phenotype (Sommer et al., 2008). Aquaporins
214 from the SIP, TIP and NIP families were shown to transport water and/or solutes and
215 appear to be involved in PT growth (Ishikawa et al., 2005; Soto et al., 2008; Di
216 Giorgio et al., 2016) and fertilization (Wudick et al., 2014). Interestingly, though not
217 expressed in pollen, it was reported recently that AtPIP2;1 also shows a non-selective
218 cation channel activity (Byrt et al., 2016), a feature that might be found for other
219 members of the aquaporin family.
220 Ion-driven osmotic changes induce electric potential shifts at the PM in addition to
221 external pH along the PT and osmoregulation depends on an active transport system
222 driven by the proton pump, through 14-3-3 protein regulation (Pertl et al., 2010).
223 Several H+-ATPase pumps are expressed in pollen (Pina et al., 2005; Bock et al.,
224 2006), with AtAHA8 being the most highly expressed and 6, 7 and 9 being pollen-
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225 specific (table 1). In tobacco, a close homolog of AtAHA6 and 9, NtNHA1 was found
226 to be localized on the PM, but segregated from the tip, and involved in tube growth
227 and callose plug formation (Certal et al., 2008). These pumps are likely to energize
228 the transport of other molecules that underlie turgor in PTs, such as sucrose (Stadler
229 et al., 1999; Goetz et al., 2001). While not necessarily affecting only PT growth, the
230 Arabidopsis AtSUC1 and rice OsSUT1 sucrose transporters have defective male
231 gametophyte phenotypes (Sivitz et al., 2008; Hirose et al., 2010). In cucumber, the
232 hexose transporter CsHT1 is necessary for PT growth (Cheng et al., 2015). Despite
233 probably related to microsporogenesis and exin pattern formation, mutants of the PM-
234 localized sucrose transporter AtRPG1/AtSWEET8 display fertility defects (Guan et al.,
235 2008; Chen et al., 2010; Sun et al., 2013).
236 However, and importantly, ion fluxes such as anions (Zonia et al., 2002) or K+ may
237 participate in turgor generation. K+ inward conductivities have been recorded by
238 patch-clamp and voltage-clamp in lily and Arabidopsis (Mouline et al., 2002;
239 Griessner and Obermeyer, 2003; Becker et al., 2004). The inward rectifier AtSPIK
240 channel is involved in PT growth (Mouline et al., 2002) and AtTPK4 mediates non-
241 rectifying currents and may also participate in osmotic regulation of the PT (Becker et
242 al., 2004). For anions, major solutes associated with water movement and turgor in
243 animals and plants, only AtSLAH3 has been presently characterized in PTs
244 (Gutermuth et al. 2013; see discussion below), but it only accounts for a small
245 percentage of the total anion flux. Yet the demonstration of a role for anion channels
246 in stomatal turgor regulation offers an analogy that could eventually serve as a
247 conceptual template to screen for their identity in PTs (see Text Box 1).
248
249 (TEXT BOX 1)
250
251 Taming turgor, the force that sustains growth: lessons from guard cells
252 PT growth is sustained by a minimum turgor (0.3 MPa in lily; Benkert et al., 1997). The
253 relative importance of turgor and wall mechanics for growth is wrapped in controversy,
254 (Winship et al. 2011 vs. Zonia and Munnik, 2011) but consensus exists around turgor
255 providing the force underlying wall elongation. Turgor implies accumulation of solutes,
256 namely ions. So how are they transported? And how do ions regulate water flux and
257 turgor?
258 Guard cells are arguably the best-studied cellular system of turgor regulation in plants,
259 relying on H+, anions and K+ to move water in and out of the cell. Evidence for the
260 same exists for PTs: H+ fluxes are well documented (Feijó et al., 1999, Certal et al.,
261 2008), large anion fluxes of up to 60 nmol/cm2/s (Zonia et al., 2002), anion conductivity
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262 (Tavares et al., 2011) and a cytosolic anion gradient (Gutermuth et al. 2013) all have
263 been characterized. Furthermore, K+ currents and the underlying channels have long
264 been observed in pollen (Obermeyer and Kolb, 1993; Becker et al., 2004; Mouline et
265 al. 2002). So is it possible to establish a parallel between what is known about turgor
266 regulation in guard cells and PT growth?
267 Similar events leading to the stomata OPENING are observable in pollen growth, with
268 the efflux of H+ in the SHANK coupling anion and K+ influxes (Zonia et al., 2002; Dias
269 et al., unpublished), which may indicate the water influx point. Conversely, as in
270 stomatal CLOSURE, large effluxes of anions, and the influx of H+ and Ca2+ occur in the
271 pollen TIP, the combined effect of which should result in the depolarization of the PM
272 (Michard et al., 2009). We hypothesize that water flows at the pollen tip following the
273 osmotic potential of anions (Cl- / NO3-) and K+. This comparison implies that the
274 mechanisms behind the TEMPORAL sequence of events in guard cells, leading to a
275 transient water flow, could have been co-opted in the PT, in the form of a SPATIAL
276 segregation of transport molecules between shank and tip, leading to regulation of
277 water transport and turgor necessary for growth (Fig.3). This concept generates
278 various testable hypotheses and gives ground to new channel discovery in PTs based
279 on similarities with guard cells.
280
281 (FIGURE 3 HERE)
282 (END OF TEXT BOX 1)
283
284 High turgor pressure typically induces the bursting in both hyphea – another tip-
285 growing cell (Money and Hill, 1997) - and PTs (Benkert et al., 1997; Amien et al.,
286 2010). The rupture point always being the tip, suggests anisotropy in the cell wall
287 mechanical properties, characterized by a stronger shank. In hyphae, the turgor
288 pressure plays a minor role in polarization, rather the apical localization of lytic
289 enzymes that loosen the cell wall determines the growth rate and polarity (Money and
290 Hill, 1997). Similarly, PT growth is sustained by the deposition of primary cell wall
291 material at the apex; once deposited at the tip, the wall is subject to a maturation
292 process that stiffens it, creating a gradient of viscosity/ elasticity between the growing
293 tip and the non-growing tube (Hepler et al., 2013; Cosgrove, 2016). Despite
294 discrepancies over the quantification of the mechanical properties of the cell wall
295 (Fayant et al. 2010; Vogler et al. 2012), many biochemical data demonstrate its
296 anisotropic composition and suggest a viscosity gradient along the tube (Steer and
297 Steer, 1989; Geitmann, 2010; Chebli et al., 2012; Hepler et al., 2013). The primary
298 cell wall of the PT deposited at the apex is essentially composed of pectin, plus 2-3%
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