bioRxiv preprint doi: https://doi.org/10.1101/2019.12.17.879577; this version posted December 18, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
1 TITLE:
2 Idioblasts as pathways for distributing water absorbed by leaf surfaces to the mesophyll in
3 Capparis odoratissima
4 Juan M. Losada1,3,4*, Miriam Díaz2, N. Michele Holbrook3,4
5 1Instituto de Hortofruticultura Subtropical y Mediterránea La Mayora. Avda. Dr. Wienberg s/n,
6 Algarrobo-Costa, 29750, Málaga, Spain.
7 2Centro de Investigaciones en Ecología y Zonas Áridas (CIEZA), Universidad Nacional
8 Experimental Francisco de Miranda, Venezuela.
9 3Department of Organismic and Evolutionary Biology. 16 Divinity Avenue Cambridge, MA
10 02138, USA.
11 4Arnold Arboretum of Harvard University. 1300 Centre St. Boston, MA 02131.
12 *Author for correspondence: [email protected]
13
14
15
16
17
18
19
1
bioRxiv preprint doi: https://doi.org/10.1101/2019.12.17.879577; this version posted December 18, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
20 ABSTRACT
21 Capparis odoratissima is a tree species native to semi-arid environments of the northern coast of
22 South America where low soil water availability coexists with frequent nighttime fog. Previous
23 work with this species demonstrated that C. odoratissima is able to use water absorbed through
24 its leaves at night to enhance leaf hydration, photosynthesis, and growth.
25 Here, we combine detailed anatomical evaluations of the leaves of C. odoratissima, with water
26 and dye uptake experiments in the laboratory. We used immunolocalization of pectin and
27 arabinogalactan protein epitopes to characterize the chemistry of foliar water uptake pathways.
28 The abaxial surfaces of C. odoratissima leaves are covered with overlapping, multicellular
29 peltate hairs, while the adaxial surfaces are glabrous but with star-shaped “structures” at regular
30 intervals. Despite these differences in anatomy, both surfaces are able to absorb condensed
31 water, but this ability is most significant on the upper surface. Rates of evaporative water loss
32 from the upper surface, however, are coincident with cuticle conductance. Numerous idioblasts
33 connect the adaxial leaf surface and the adaxial peltate hairs, which contain hygroscopic
34 substances such as arabinogalactan proteins and pectins.
35 The highly specialized anatomy of the leaves of C odoratissima fulfills the dual function of
36 avoiding excessive water loss due to evaporation, while maintaining the ability to absorb liquid
37 water. Cell-wall related hygroscopic compounds present in the peltate hairs and idioblasts create
38 a network of microchannels that maintain leaf hydration and promote the uptake of aerial water.
39 KEYWORDS: arabinogalactan proteins-AGPs, foliar water uptake-FWU, idioblast, pectins,
40 peltate hairs, sclerenchyma.
41
2
bioRxiv preprint doi: https://doi.org/10.1101/2019.12.17.879577; this version posted December 18, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
42 INTRODUCTION
43 Water-energy dynamics intrinsically drive global patterns of plant diversity (Kreft and
44 Jetz, 2007), but the global increase of aridity will likely make plants more vulnerable to the lack
45 of water in the soil (Choat et al., 2018; Olson et al., 2018). In arid and semiarid environments,
46 the traditional soil-atmosphere continuum concept of hydraulic redistribution in plants (Nobel,
47 2009), is compromised due to prolonged soil drought. Xerophyllous plants thrive in these
48 ecosystems thanks to leaf anatomical adaptations that reduce rates of water loss, such as smaller
49 areas, lower stomata index and impermeable coating structures, like cuticles and trichomes (see
50 Shields, 1950). Surprisingly, some of the structures that prevent evaporative water loss may also
51 facilitate aerial water uptake into the leaves, decoupling soil water uptake from that of the
52 canopy (Benzing et al., 1978; Gouvra and Grammatikopoulos, 2003).
53 Foliar water uptake (FWU) is a widespread phenomenon of vascular plants, known for
54 three centuries, and empirically tested in at least 53 plant families (Dawson and Goldsmith,
55 2018). FWU has direct consequences on plant function, relaxing tension in the water column of
56 the xylem, enhancing turgor-driven growth, and influencing the hydraulic dynamics of agro and
57 ecosystems (Mayr et al., 2014; Steppe et al., 2018; Aguirre-Gutiérrrez et al., 2019). The key
58 conditions for FWU are met in fog-dominated environments (Tognetti, 2015; Weathers et al.,
59 2019), where high atmospheric humidity enhances nighttime dew formation on leaf surfaces,
60 thus increasing the possibility of foliar water absorption. Indeed, studies in montane cloud forests
61 of Brazil (Eller et al., 2016), coastal California redwood forests in the USA (Burgess and
62 Dawson, 2004), or cloud forests in Mexico (Gotsch et al., 2014), have demonstrated that aerial
63 water is an important input contributing to plant function, especially during the dry periods of the
64 year. In contrast, foliar uptake in semiarid areas of the tropics is less studied, despite being a
3
bioRxiv preprint doi: https://doi.org/10.1101/2019.12.17.879577; this version posted December 18, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
65 pivotal water source that enhances biomass productivity (Díaz and Granadillo, 2005; Limm et
66 al., 2009). Better characterization of the pathways of foliar absorption will enhance
67 understanding of the mechanisms semiarid plants use to hydrate leaves with aerial water.
68 Some species absorb water through the natural leaf openings, such as stomata (Burkhardt
69 et al., 2012; Berry et al., 2014) or hydathodes (Martin and von Willert, 2000). Stomatal water
70 uptake alone conflicts with CO2 dynamics between leaves and the atmosphere (Boanares et al.,
71 2019). Other structures that are supposedly impermeable to water further participate in the aerial
72 water uptake, including cuticles (Vaadia and Waisel 1963; Yates and Hutley,. 1995; Fernández et
73 al., 2017; Schuster et al., 2017), and trichomes (Franke 1967; Benzing et al. 1978; reviewed by
74 Berry et al., 2019). While the use of these wall-thickened, sealing structures appears as a good
75 strategy to capture atmospheric water condensed on the leaf surfaces, demonstration of their
76 hygroscopic capacity requires a careful examination of their cell wall biochemistry, so far
77 lacking for most plant species. Early works in this line showed that the absorptive capacity of
78 leaves relates with the presence of polysaccharides under the cuticle (Kerstiens, 1996). However,
79 the role of ubiquitous compounds of the cell walls, such as pectins and glycoproteins, on foliar
80 water uptake, has been scarcely reported (Boanares et al., 2018). The question remains on
81 whether sealing structures such as trichomes or sclerenchymatous structures displayed such
82 hygroscopic compounds.
83 This gap of knowledge affects particularly to xerophyllous species, which display unique
84 anatomies with abundant sclerenchymatous tissues, such as idioblasts, highly specialized
85 structures that are understudied from a functional perspective. Idioblasts (Schwendener, 1874)
86 are thick walled cells typically buried in the mesophyll of leaves of vascular plants (Bailey and
87 Nast, 1945; Tomlinson and Fisher, 2005), and recurrently found in the leaves of xerophyllous
4
bioRxiv preprint doi: https://doi.org/10.1101/2019.12.17.879577; this version posted December 18, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
88 species (Foster, 1956; Heide Jorgensen, 1990). They have been traditionally explored from the
89 perspective of morphology, ontogeny, and taxonomic value (Foster,1944; 1945a,b; Bloch, 1946;
90 Foster, 1955a,b; Rao and Mody, 1961). However, important functions adscribed to idioblasts are
91 merely inferred from their putative stiffness, such as support and defense (Foster, 1947; Tucker,
92 1964; Rao and Sharma, 1968), or from their topology, such as a possible role in leaf capacitance
93 (Heide-Jorgensen, 1990). Some noteworthy exceptions suggest that idioblasts could affect the
94 physiology of the leaves more than previously thought, such as serving as light guides
95 (Karabourniotis, 1998). The diverse array of anatomies of xerophyllous species is exemplified in
96 the genus Capparis (Rao and Mody, 1961; Gan et al., 2013). Capparis odoratissima is a species
97 native to the semiarid tropical environments of the South American continent with a remarkable
98 capacity to produce new biomass using canopy irrigation as the only water source (Díaz and
99 Granadillo, 2005), likely through foliar water uptake. However, the mechanisms of this uptake
100 are unknown.
101 Here we focus on C. odoratissima with the goal of understanding the relationship between
102 anatomy and function in xerophylous leaves. We show that the leaves of C. odoratissima are
103 highly specialized structures that perform a dual function: first, they are armed organs that
104 minimize water loss, but they further contain an intricate network of hygroscopic pathways that
105 enhances s water uptake, thus maintaining leaf hydration upon water condensation on the leaf
106 surface.
107
108 MATERIALS AND METHODS
109 Plant material
5
bioRxiv preprint doi: https://doi.org/10.1101/2019.12.17.879577; this version posted December 18, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
110 Five stems with a minimum of 10 leaves of Capparis odoratissima were shipped in a plastic bag
111 with a wet paper tissue from the University of Francisco de Miranda at the state of Falcón
112 (Venezuela), and three days later received for evaluation in the laboratory of the Arnold
113 Arboretum of Harvard University in Boston (MA, USA).
114 Evaluation of the external leaf anatomy
115 Five to six fully expanded leaves were first scanned with a scale using a CANON CanoScan
116 LiDE 400 scanner, and leaf areas were calculated with the Image J1.51d software (National
117 Institutes of Health, Bethesda, MD, USA). These same leaves were then used to photograph the
118 details of both adaxial and abaxial surfaces with a Zeiss Discovery v12 Dissecting microscope
119 (objective 0.63x PanApo). Adaxially, the idioblast tips were easily visualized by their
120 translucency, and, abaxially, the peltate hairs were counted using their central parts as references.
121 Both idioblasts and peltate hairs were counted in 1mm2 areas at five different positions in each
122 leaf surface. To count the number of stomata, five leaves were fixed in FAA (formalin:acetic
123 acid, Johansen 1940), washed three times in distilled water, one hour each, stained with Feulgen
124 reaction (modified from Barell and Grossniklaus, 2005). They were finally cleared with a
125 solution containing ethanol:benzoyl benzoate 3:1 (v/v) for six hours, ethanol:benzoyl benzoate
126 1:3 (v/v), and finally benzoyl benzoate: dibutyl pftalate 1:1 (v/v) for several days (Crane and
127 Carman, 1987). Following removal of the peltate hairs, 1mm2 samples with guard cells stained in
128 red, were imaged with a Zeiss LSM700 Confocal Microscope connected to a AxioCam 512
129 camera and the Zen Blue 2.3 software, objective was 20x/0.8 M27 Plan-Apochromat. Note that
130 the area occupied by the rounded base of the peltate hairs had not stomata in the abaxial
131 epidermis (3.45%), and hence it was subtracted from the total area of leaves to get a more
132 accurate picture of stomatal frequency.
6
bioRxiv preprint doi: https://doi.org/10.1101/2019.12.17.879577; this version posted December 18, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
133 Evaluation of the internal leaf anatomy: idioblast characterization
134 To better understand the structure of the idioblasts in the mesophyll, hand transverse thin
135 sections (i.e. perpendicular to the leaf surface) of the individual leaves were obtained and stained
136 with a solution of 0.01% w/v calcofluor white in 10mM CHES buffer with 100mM KCl (pH=10)
137 (Hughes & McCully, 1975), that stains cellulose, and then with a uramine-O in 0.05 M Tris/HCl
138 buffer, (pH=7.2), was used to detect cutinized lipids (Heslop-Harrison and Shivanna, 1977).
139 These sections were visualized with a Zeiss Axioskop microscope equipped with
140 epifluorescence, and imaged with an AxioCam HRc camera, attached to the Zen Blue software
141 with multichannel. For calcofluor white, the filter used was DAPI narrow (Zeiss 48702)
142 excitation G 365, bandpass 12 nm; dichroic mirror FT 395; barrier filter LP397; for auramine O,
143 the filter used was AF488/FITC/GFP (Zeiss Filter set 9) excitation 450-490; dichroic mirror 510;
144 emission filter LP515. Then, we isolated the idioblasts from other leaf tissues by macerating
145 1mm2 leaf pieces in a solution containing acetic acid: hydrogen peroxide 1:1 (v/v) at 60º for 2
146 days. The debris of these samples was then mounted in distilled water or glycerol, and visualized
147 with a Zeiss LSM700 Confocal Microscope. Rendering images were obtained without staining
148 with the Objective: 63x/1.40 Oil DIC M27 Plan-Apochromat.
149 Samples obtained from five previously fixed leaves in FAA were washed in distilled water three
150 times, 1h each, and dehydrated with an increasing gradient of aqueous ethanol concentrations
151 (10%, 30%, 50%, 70%, 100% (x3)). They were then soaked in the infiltration solution of
152 Technovit7100 (Electron Microscopy Sciences, Hetfield, PA, USA) for several weeks, and
153 polymerized under high nitrogen concentration conditions. Blocks with samples oriented in a
154 paradermal way (i.e. parallel to the leaf surface), were serially sectioned at 4µm in with a Leica
155 RM2155 rotary microtome (Leica Microsystems, Wetlar, Germany). Sections were then stained
7
bioRxiv preprint doi: https://doi.org/10.1101/2019.12.17.879577; this version posted December 18, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
156 with an aqueous solution of 0.025% toluidine blue for general structure of the leaf tissues (Feder
157 and O’Brien, 1968), and finally imaged with a Zeiss AxioImager A2 Upright microscope
158 microscope equipped with a AxioCam 512 camera and a Zen Blue Pro with multichannel
159 software.
160 Water and dye uptake experiments
161 We first monitored cuticle transpiration in five full expanded leaves by letting them dry with
162 their petiole covered with parafilm at room temperature (average 23±3ºC), and 30±3% of
163 relative humidity (RH), weighting them every hour with a four digit precision scale. We then
164 adjusted the weight loss to a linear regression function (r2=0.98; slope = -0.0012). After that, we
165 calculated the relative leaf water content (RWC) using the formula RWC=[(FWt-DW)/(DW-
166 FW0)], were FWt is the fresh weight at time t, DW is the dry weight, and FW0 is the fresh weight
167 at time 0 (Fig. 5A). We further calculated the initial water content of the leaves (Wi) as the
168 difference between the fresh weight minus the dry weight by leaf unit area.
169 Five more leaves were weighed (Wt0) before being submerged in distilled water with the petiole
170 sealed -but not submerged- for 15min (wet cycle). To eliminate the surface water, these leaves
171 were centrifuged at 1800rpm for 2min using a Sorvall RC6 Ultracentrifuge, and immediately
172 weighed afterwards with a precision scale (Wt1). Leaves were left to dry at room temperature for
173 15min (dry cycle), and weighed again before immersion (Wt2). The cycles were repeated five
174 times in a row. Based on these data, and assuming that cuticle evapotranspiration is 0 when
175 leaves are submerged, we first calculated the net water absorbed after the wet cycle as to:
176 1) ABSt1= (Wt1-Wt0)/ LA,
8
bioRxiv preprint doi: https://doi.org/10.1101/2019.12.17.879577; this version posted December 18, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
177 where ABSt1 is the net water absorbed at time t (wet cycle, uneven numbers), and LA is leaf area
178 in cm2.
179 After that, and considering similar cuticle transpiration in both leaf sides, the expected cuticle
180 evapotranspiration (CEVT) for the dry cycle was calculated as to:
181 2) CEVT = [(-0.0012 * t )*2]+ Wt0, where t is time in hours.
182 3) ABSt* = (Wt* - Wt*-1 + CEVT)/ LA,
183 where ABSt* is the calculated water absorbed after the dry cycles (at even numbers), and Wt* the
184 weight of the leaves after the dry cycles. We then evaluated the observed water balances for each
185 alternate period as to:
186 4) ∆ABS = (ABSt+ ABSt-1) for each alternate period.
187 The calculated water lost during the dry cycles corresponded with the observed values,
188 suggesting that weight loss due to cuticle conductance was so low that it did not offset the water
189 absorbed by leaves. Thus, we then plotted the net water absorbed only during the wet cycles, and
190 adjusted it to a linear regression function (r2=0.96; slope = 0.073) (Fig. 5B), revealing a linear
191 water uptake when leaves are submerged.
192 The question remained on whether each leaf side contributed equally to water uptake. To
193 understand it, five leaves were loaded with 350uL of distilled water evenly distributed in 35
194 droplets (10uL each) on the adaxial surface, and in five more leaves loaded on the abaxial
195 surface. When the droplets disappeared from the sufaces, the leaves were weighted with a
196 precision scale, loaded again with the same amount of water, and this cycle repeated for a total
197 period of 9 hours. While the atmospheric humidity was around 30%, it is worth noting that we
9
bioRxiv preprint doi: https://doi.org/10.1101/2019.12.17.879577; this version posted December 18, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
198 replicated the experiment at high humidity (70%) within a chamber, but the water droplet
199 disappearance from the leaf surfaces was so slow that made the experiment impracticable in a
200 continuous fashion. However, it suggested that cuticle conductance is much lower at natural
201 conditions, which display atmospheric humidities ranging from 50-70% (Díaz and Granadillo,
202 2005). We then calculated the water gains as described below for the dry cycles, but assuming
203 cuticle transpiration in only one side of the leaves [CEVT = (-0.0012* t) + Wt0], and finally
204 adjusted the results to a linear regression curve. For the adaxial surface, r2=0.81; slope = 0.0012
205 (Fig. 5C).
206 To understand the pathways of water uptake, we used a 1% aqueous solution of the apoplastic
207 fluorescent dye tracer Lucifer Yellow (LY; CH dilithium salt; Sigma). 10uL droplets of the dye
208 were applied in either the adaxial or the abaxial surfaces within a humidified chamber, and
209 waited until they disappeared from the surface. A paper tissue was then used to wipe the traces of
210 the dye from the leaf surface prior to obtaining transverse sections with a double edge razor
211 blade. Sections were mounted in an aqueous solution of 70% glycerol, and immediately observed
212 with a Zeiss LSM700 Confocal Microscope (wavelength 488nm). Similar sections of the same
213 leaves, but in areas without the dye were used as negative controls.
214 Immunolocalization of pectin and arabinogalactan glycoprotein epitopes
215 A preliminary test with the general dye Ruthenium red was used in freshly dissected leaves to
216 evaluate the presence of neutral pectins (Colombo and Rascio, 1977). Similarly, a preliminary
217 test for arabinogalactan protein (AGP) presence was applied to cleared leaves with a 2% solution
218 of the chemical reagent beta glucosyl Yariv reagent in 0.15M NaC1 (Yariv, 1967). The β-GlcYR
219 reacts with arabinogalactan proteins giving a red color upon precipitation.
10
bioRxiv preprint doi: https://doi.org/10.1101/2019.12.17.879577; this version posted December 18, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
220 To detect the presence of pectic and AGP epitopes in the leaves of Capparis odoratissima, two
221 monoclonal antibodies that detect highly sterified pectins (JIM5), and AGPs (JIM8)
222 (Carbosource services, Georgia, USA), were used. 4µm cross and paradermal sections of
223 embedded leaves were incubated with 1XPBS thrice for five minutes each, then in 5% bovine
224 albumin serum (BSA), then in 1XPBS five minutes, and with the undiluted primary monoclonal
225 antibody for 45 min. They were washed three times in 1XPBS, and finally incubated again with
226 an anti-Rat secondary antibody Alexa 488 conjugated with a fluorescent compound Fluorescein
227 Isiothianate. Three final washes with 1XPBS preceded observations with a confocal microscope
228 (see details below). Negative controls were treated in the same way, but substituting the primary
229 antibody by a solution of 1%BSA in PBS.
230 RESULTS
231 Extrinsic leaf anatomy of Capparis odoratissima
232 Detailed evaluations of the oblong, astomatous leaves of C. odoratissima showed a dark green
233 color in the adaxial surface (Fig. 1A). Numerous translucent spots were located in concave areas
234 of the surface, which corresponded with the tips of the idioblasts (Fig. 1B). Idioblast tips
235 interspersed between the epidermal cells and, after staining and clearing the tissue, displayed a
236 star-like shape when observed from above (Fig. C). At higher magnification, each idioblast
237 projected a narrow pore toward the adaxial surface of the leaf (Fig. 1D). Abaxially, the leaves
238 showed a pale green color (Fig. 1E), which resulted from the total coverage of the lamina by an
239 imbricated carpet of peltate hairs (Fig. 1F). These non-glandular hairs had variable size, but a
240 uniform multicellular, umbrella-like base, with thick-walled filiform cells (Fig. 1G), and a
241 central shield on top that protrudes to the exterior. Despite having very thick walls, this central
242 part revealed symplasmic connections between its cells (Fig. 1H).
11
bioRxiv preprint doi: https://doi.org/10.1101/2019.12.17.879577; this version posted December 18, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
243 Intrinsic leaf anatomy of Capparis odoratissima: cuticles, peltate hairs and idioblasts.
244 Cross-sections of the leaves revealed that the idioblasts are thick-walled columnar structures that
245 connect the adaxial and the abaxial surfaces of the leaves (Fig. 2A, drawn by Metcalfe and
246 Chalk, 1950). Idioblasts did not stain for lipids, but the auramine O staining revealed a thick
247 cuticle layer in the adaxial surfaces of the leaves (Fig. 2B), which was thinner in the concave
248 cavities on top of the idioblasts. In contrast, the thick columnar walls of the idioblasts stained
249 intensely for cellulose with calcofluor white (Fig. 2C), which further delivered partial
250 lignification upon phluoroglucinol staining. The abaxial surface of the leaf showed a more
251 compact rugose cuticle under the peltate hairs (Fig. 2D). At the anchoring areas of the peltate
252 hairs, the abaxial epidermis invaginated and contained a hyper accumulation of cutin (Fig. 2E).
253 Idioblasts exposed a sophisticated structure with a clear bipolar pattern from the adaxial to the
254 abaxial side of the leaf (Fig. 3A). A thin projection that protruded apoplastically between cells of
255 the upper epidermis connected with the surface, and was sustained underneath with four to five
256 filiform anchors attached to the base of the epidermal cells (Fig. 3B). In the area of the palisade
257 parenchyma, the idioblasts had a columnar shape, with a very thick wall, a narrow lumen, and
258 crenations that connected the lumen with the palisade parenchyma (Fig. 3C). In the spongy
259 mesophyll, the irregular shape of idioblasts was characterized by numerous protuberances and
260 crenations that connected the lumen of the idioblasts with the mesophyll cells (Fig. 3D). While
261 idioblasts thinned down and were irregularly shaped in the abaxial side of the leaf, they
262 anastomosed and converged toward the base of the peltate hairs, yet keeping narrow lumens that
263 connected the lumens of the idioblasts with the lumens of the peltate hairs (Fig. 3E). The average
264 rate of idioblast anastomosing by peltate hair was 4:1 (Fig. 4), whereas the frequency of abaxial
265 stomata under the peltate hairs was significantly lower.
12
bioRxiv preprint doi: https://doi.org/10.1101/2019.12.17.879577; this version posted December 18, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
266 Leaf water uptake in Capparis odoratissima
267 The initial water content of detached leaves was approximately 10 mg cm-2. Weight loss of
268 leaves exposed to room temperature conditions was used to understand cuticle
269 evapotranspiration (Fig. 5A). The percentage of water retained by leaves was higher than 80%
270 during the first 21 hours of exposure, but then decreased sharply, being completely dry 143 hours
271 after the initial measurements. In contrast, the cumulative water uptake by submerged leaves was
272 sixty orders of magnitude higher than water losses during the first 10 hours after immersion,
273 surpassing cuticle evapotranspiration even though the cycles for dry and wetting periods were
274 similar (Fig. 5B). Given the asymmetric anatomies between both leaf surfaces, the contribution
275 of each side to water uptake was evaluated by loading them individually with water droplets. As
276 expected, this assay evidenced that water uptake from droplets deposited on only one leaf surface
277 was slower than from submerged leaves, given the effect of cuticle evapotranspiration (Fig.5C).
278 Additionally, while there was a net uptake of water from both sides, the water balance was
279 positive for the leaves loaded on the abaxial surface only the first two hours after exposure,
280 decreasing thereafter. In contrast, weight gain was maintained for at least ten hours when
281 droplets were applied on the adaxial surface. Most strikingly, the slope of the absorption curve
282 on the adaxial side was the same as the water lost by cuticle transpiration, suggesting that the
283 adaxial surface is responsible for most of the cuticle transpiration, or, in other words, cuticle
284 transpiration is almost suppressed at the abaxial surface.
285 The pathways of leaf water uptake in Capparis odoratissima
286 Dye loading in the abaxial surface resulted in uptake mediated by the central anchoring area of
287 the peltate hairs (Fig. 6A), evidenced by the intense staining that remained in the thick walls of
288 this central area of the peltate hairs after dye absorption (Fig. 6B). Fluorescence was further
13
bioRxiv preprint doi: https://doi.org/10.1101/2019.12.17.879577; this version posted December 18, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
289 observed in the mesophyll of the leaves when loading the dye in either the adaxial or the abaxial
290 surface, pointing to the dual role of the leaf surfaces in water uptake. Strikingly, the structures
291 which fluoresced the most were the walls of the idioblasts, compared with the areas of the leaf
292 without dye (Fig. 6C, D), suggesting that these walls had some degree of hygroscopicity. Not
293 only the external walls, but the lumen of the idioblasts was loaded with the fluorescent dye,
294 including the crenations (Fig. 6E, F), revealing them as the bridges of water uptake from the
295 surface toward the mesophyll.
296 Pectins and arabinogalactan proteins in the leaves of Capparis odoratissima
297 Immunolocalization of pectin and arabinogalactan protein epitopes with monoclonal antibodies
298 showed a dual topological pattern. On one hand, pectin epitopes were present in the cell walls of
299 both adaxial and abaxial epidermis (Fig. 7A, B), in the external walls of the cells composing the
300 spongy mesophyll (Fig. 7C), and the external cell walls of the peltate hairs (Fig. 7D), but not in
301 the idioblasts.
302 On the other hand, the presence of arabinogalactan proteins was roughly detected with the beta
303 glucosyl Yariv Reagent, which showed an intense red color at the areas of the leaves with
304 idioblasts. More specifically, the epitopes recognized by the JIM8 monoclonal antibody, labelled
305 a narrow area that corresponded with the lumen of the idioblasts, from their adaxial projection
306 (Fig. 7E), through the columnar lumen in the palisade parenchyma (Fig. 7F), to the lumens of the
307 anchoring areas of the peltate hairs (Fig. 7G). As a result, AGPs coated the continuum of lumens
308 between the peltate hairs and idioblasts.
309 DISCUSSION
310 Water balance and leaf permeability in Capparis odoratissima
14
bioRxiv preprint doi: https://doi.org/10.1101/2019.12.17.879577; this version posted December 18, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
311 Previous studies of C. odoratissima showed that, unlike other species coexisting in the same
312 environment, C. odoratissima increased its biomass with the only source of water being from the
313 atmosphere (Díaz and Granadillo, 2005). However, the magnitude of water uptake by individual
314 leaves is missing so far. Our detailed evaluations of water evaporation through time (i.e. cuticle
315 evapotranspiration), and entry of water (measured as the increase of leaf weight) from detached
316 leaves served to understand the extreme values of water inputs and outputs. First, we
317 demonstrate a high capacitance of the C. odoratissima leaves, which kept a high water content
318 (80%) when left to dry with the petiole sealed, in line with the idea of low cuticular permeability
319 for species from xerophylous environments compared with temperate species (reviewed by
320 Kerstiens, 1996). For example, Fagus sylvatica leaves showed a more continuous dehydration
321 that responded to water pressure deficit (Gardinen and Grace, 1992). The dehydration rate of C.
322 odoratissima leaves is likely lower in natural conditions, not only because the leaves are not
323 detached, but also because the values of relative humidity of the atmosphere oscillates between
324 50% and 90% (Diaz and Granadillo, 2005). To understand whether water uptake is possible with
325 zero cuticular conductance, we exposed the leaves to a water-saturated environment, revealing a
326 linear uptake of water without cuticle evapotranspiration, gaining up to 40% of the initial water
327 content of the leaves. Leaf water absorption has been widely studied in the major groups of
328 angiosperms, including magnoliids (8 genera), monocots (7 genera), and eudicots (67 genera)
329 (reviewed by Berry et al., 2019 and Dawson and Goldsmith, 2018), pointing to FWU as a key
330 factor affecting plant function in most ecosystems (Weathers et al., 2019). However, this effect is
331 stronger in dry or semi-dry environments (Schreel et al., 2019), such as the dryland tropical areas
332 where C. odoratissima grows, where the water in the soil is a limiting resource, and aerial water
333 becomes pivotal for plant growth and survival.
15
bioRxiv preprint doi: https://doi.org/10.1101/2019.12.17.879577; this version posted December 18, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
334 The major barriers for water absorption and/or evaporation are the leaf coatings, such as cuticles.
335 Our anatomical evaluations reveal two different cuticle layers in the two surfaces of C.
336 odoratissima leaves. On the upper surface, a thick cuticle layer becomes thinner at some concave
337 areas. Thick lipid coatings such as cuticles associate with avoidance of leaf dehydration (Shields,
338 1950). Yet, the leaf cuticle is gradually revealing as a permeable layer to absorb water and
339 electrolytes (Fernández et al., 2014; 2017). However, cuticle thickness has not a direct
340 relationship with impermeability (Schuster et al., 2017), but its biochemistry appears to be more
341 important for water exchange between the mesophyll and the atmosphere. The compact and
342 rugose appearance of the cuticle in the lower side of the leaves of C. odoratissima suggests a
343 higher surface area and probably a better isolation of leaf tissues. Stomata embedded in this
344 cuticle are gates of water escape through transpiration, but they are covered by a carpet of peltate
345 hairs, which dramatically reduce evapotranspiration. Pubescence is a typical character of
346 xerophylous species, and functionally related with protection against desiccation and thus
347 temperature regulation (Shields, 1950; Fahn, 1986).
348 Asymmetric foliar water uptake and anatomical specializations in Capparis odoratissima
349 Our experiments with water droplets in each leaf surface revealed an asymmetry regarding the
350 functional role of leaf surfaces in water uptake. While both surfaces initially showed a positive
351 gain of water, only the adaxial surface maintained this competence longer in time. Water
352 condenses most likely on the adaxial surface of leaves, and, indeed, most works spraying water
353 on the leaf surfaces evidenced that the upper side is more permeable to water (Gardingen and
354 Grace, 1992; Fernández et al., 2014). In C. odoratissima, the reasons behind this asymmetric
355 behavior correlate with the markedly distinct anatomy between leaf surfaces. Abaxially,
356 trichomes are hygroscopic, participating in foliar water uptake. Hygroscopic peltate hairs have
16
bioRxiv preprint doi: https://doi.org/10.1101/2019.12.17.879577; this version posted December 18, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
357 been reported in some angiosperm species (Gramatikopoulos and Manetas, 1994; Bickford,
358 2016; Eller et al., 2016; Pina et al., 2016; Vitarelli et al., 2016), being most outstanding in
359 epiphytic bromeliads (Benzing and Burt, 1970; Benzing, 1976; Benzing et al., 1978; Benz and
360 Martin, 2006; Ohrui et al., 2007). However, the presence of peltate hairs is not indicative of
361 foliar water uptake (Bickford, 2016), since remarkable examples with peltate hairs such as in
362 Olea europaea, showed no evidence of FWU (Arzeee, 1953). The ability to uptake water by
363 peltate hairs is offset by cuticle evapotranspiration of the adaxial surface (not loaded with water
364 droplets). Thus, a tradeoff between cuticle transpiration and water entrance from the atmosphere
365 appears to be rather positive when water is condensed on the adaxial surface. Oddly, our
366 observations revealed unique and numerous microscopic openings projecting to the atmosphere
367 in the upper leaf surfaces of C. odoratissima. Natural openings (i.e. not created by
368 microorganisms) are unusual in this surface, except for amphistomous leaves, or leaves with
369 hydathodes (Martin and von Willert, 2000). We hereby expose unique micropores projecting
370 toward concave areas on the adaxial leaf surfaces, thus allowing symplasmic water transport of
371 water from the atmosphere to the leaf. These openings connect with the lumen of columnar
372 idioblasts, pointing to these structures as significant players in the water budgets of the leaves. In
373 addition, the lumen of the idioblasts formed a continuum with the lumen of the peltate hairs,
374 traversing the cross sectional area of the leaves. As a result, an intricate network of micro
375 channels linked the surface with the mesophyll. Trichome-idioblast associations are commonly
376 found in species from arid environments such as those from the family Euphorbiaceae
377 (Solereder, 1908; Metcalfe and Chalk, 1950), Olea europaea (Arzeee, 1953), or Androstachys
378 johnsoni (Alvin, 1987). Idioblasts have rarely been demonstrated as vectors of foliar water
379 uptake in angiosperms, with some exceptions such as Hakea suaveolens (Heide-Jorgensen,
17
bioRxiv preprint doi: https://doi.org/10.1101/2019.12.17.879577; this version posted December 18, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
380 1990). However, their topology in the numerous forest species where they have been described -
381 including gymnosperms (Hooker, 1864; Sterling, 1947), and more than eighty eudicot families
382 (Solereder, 1908; Foster, 1955a,b; Rao and Mody, 1961; Zhang et al., 2009; Vitarelli et al.,
383 2016), suggested a role in leaf capacitance. As thick-walled sclerenchymatous tissues (Evert,
384 2006), idioblasts evolved multiple shapes and dispositions within leaves, but the columnar type
385 of idioblasts displayed in the leaves of C. odortatissima have only been described in a couple of
386 species so far: Hakea suaveolens (Heide-Jorgensen, 1990), and Mouriria huberi (Foster, 1947).
387 These adaptations cannot be related to xeric environments, since other species from the same
388 genus and adapted to dry climates have completely different anatomies, such as the
389 Mediterranean Capparis spinosa (Rhizopoulou, 1990; Rhizopoulou and Psaras, 2003; Gan et al.,
390 2013).
391 The biochemistry of foliar water uptake in Capparis odoratissima
392 The pathway of atmospheric water entry in the leaves of C. odoratissima involves hygroscopic
393 materials deposited in the leaf coatings (Gouvra and Gamatipoulus, 2003). In the current work,
394 we first describe the thick walled structures composing the multicellular peltate hairs, which
395 project pectins to the external part, suggesting their involvement in the initial water capture when
396 condensation happens in the abaxial side. A few reports have revealed the presence of pectins in
397 trichomes, such as species from semi-arid forests like Crombretum leprosum (Pina et al., 2016),
398 or the tropical species Drymis brasiliensis (Eller et al., 2013). In addition, we revealed that the
399 cell walls of both epidermal cells and spongy mesophyll cells show a high concentration of low
400 sterified pectins in their cell walls. This is in line with the ubiquity of pectins within the leaf
401 mesophyll, typically acting as a pulling force for water uptake from the soil. In the leaves of C.
18
bioRxiv preprint doi: https://doi.org/10.1101/2019.12.17.879577; this version posted December 18, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
402 odoratissima, their tight association with the numerous idioblasts is likely acting as a pulling
403 force for water deposited on the leaf surfaces, using idioblasts as carriers.
404 The idioblasts of C. odoratissima are highly hygroscopic, with cellulosic walls that contain polar
405 molecules for water attachment, but also partially lignified, suggesting a secondary role on
406 defense and structural support. A finely tuned water uptake in C. odoratissima is revealed by our
407 experiments with the apoplastic dye tracer Lucifer Yellow. Water entering from the surrounding
408 atmosphere to the idioblasts flows through an intricate network of crenations and branches
409 within the idioblasts that connect all leaf tissues and the exterior. Most strikingly is the fact that
410 epitopes belonging to arabinogalacatan proteins are specifically located within these channels.
411 AGPs are highly branched proteins with a short aminoacid backbone attached to the plasma
412 membrane of cells, and a large saccharidic part that has been related with nutritive and/or
413 signaling functions between cells (Ellis et al., 2010). AGPs play different roles in plant
414 development, including mate recognition and support during reproduction, proper early seedling
415 development, and so many others (Majewska-Sawka and Nothnagel, 2000; Vaughn et al., 2007;
416 Pereira et al., 2016). However, the role of AGPs on plant hydration has been scarcely studied.
417 Remarkably, works evaluating the compositon of the cell walls in the resurrection plant
418 Craterostigma wilmsii, which completely losses water, regaining it thereafter, pointed to the
419 hygroscopic properties of AGPs as critical players in this rapid and effective rehydration (Vicré
420 et al, 2004). AGP-related proteins have been previously related with the tensile strength of stems
421 due to their participation in secondary cell wall composition, such as in the vessels or fibers (Ito
422 et al., 2005; Liu et al., 2013). But the presence of AGPs have never been reported before in
423 idioblasts with such specific pattern as in the current work. What it reveals is the biochemical
424 complexity of sclerenchymatous tissues, which are possible effectors enabling hydration of
19
bioRxiv preprint doi: https://doi.org/10.1101/2019.12.17.879577; this version posted December 18, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
425 tissues during periods where water deficits in the soil combines with water saturation in the
426 atmosphere (Fig. 8). Thus, AGPs may be secluded in the lumen of the idioblasts during
427 development, serving as bridges of water uptake between the atmosphere and the leaf tissues.
428 Future works will elucidate the possible analogy between AGPs in other sclerenchymatous
429 tissues of other species, as well as their putative role on leaf capacitance.
430 CONCLUSIONS
431 Our data strongly suggest that the pathway for water uptake in Capparis odoratissima is
432 mediated by the idioblasts and peltate hairs, which are interconnected. We propose a unique
433 model in angiosperms (Fig. 8) that involves apertures of the leaves toward the adaxial surface,
434 which are involved in water uptake, but may facilitate evapotranspiration. Hygroscopic materials
435 belonging to arabinogalactan proteins serve to capture water to the lumen of the idioblasts, and
436 pectins in the mesophyll and the epidermis may further facilitate the incorportation of water to
437 the leaf tissues. This cascade of biochemical and physical events allows Capparis odoratissima
438 trees to make use of atmospheric water resources.
439 ACKNOWLEDGEMENTS
440 We are grateful to Ned Friedman, Kea Wodruff, and Faye Rosin from the Arnold Arboretum of
441 Harvard University for sharing facilities. We thank Mr. Omar Fernando Sierra for his help with
442 plant material. Juan M. Losada is a ComFuturo Researcher at the IHSM-CSIC-UMA, funded by
443 the Fundación General CSIC (FGCSIC), and within the project RTI2018-102222A100. This
444 work was supported by the National Science Foundation (EAGER 1659918, IOS 1456845 and
445 DMR 14-20570).
446 LITERATURE CITED
20
bioRxiv preprint doi: https://doi.org/10.1101/2019.12.17.879577; this version posted December 18, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
447 Aguirre-Gutiérrez, C. A., Holwerda, F., Goldsmith, G. R., Delgado, J., Yepez, E., Carbajal, N., 448 Escoto-Rodríguez M., Arredondo, J. T. (2019). The importance of dew in the water balance of a 449 continental semiarid grassland. Journal of Arid Environments, 168, 26–35. 450 Alvin, K. L. (1987). Leaf anatomy of Androstachys johnsonii Prain and its functional 451 significance. Annals of Botany, 59(5), 579–591. 452 Arzeee, T. (1953). Morphology and ontogeny of foliar sclereids in Olea europaea. I. Distribution 453 and structure. American Journal of Botany, 680–687. 454 Bailey, I. W., & Nast, C. G. (1945). Morphology and relationships of Trochodendron and 455 Tetracentron, I. Stem, root, and leaf. Journal of the Arnold Arboretum, 26(2), 143–154. 456 Barrell, P., Grossniklaus U. (2005). Confocal microscopy of whole ovules for analysis of 457 reproductive development: the elongate1 mutant affects meiosis II. The Plant Journal 43, 309– 458 320. 459 Benz, B. W., Martin, C. E. (2006). Foliar trichomes, boundary layers, and gas exchange in 12 460 species of epiphytic Tillandsia (Bromeliaceae). Journal of Plant Physiology, 163(6), 648–656. 461 Benzing, D. H. (1976). Bromeliad trichomes: structure, function, and ecological significance. 462 Selbyana 1: 330–348. 463 Benzing, D. H., Burt, K. M. (1970). Foliar permeability among twenty species of the 464 Bromeliaceae. Bulletin of the Torrey Botanical Club, 269–279. 465 Benzing, D. H., Seemann, J., Renfrow, A. (1978). The foliar epidermis in Tillandsioideae 466 (Bromeliaceae) and its role in habitat selection. American Journal of Botany, 65(3), 359–365. 467 Berry, Z. C., Emery, N. C., Gotsch, S. G., Goldsmith, G. R. (2019). Foliar water uptake: 468 Processes, pathways, and integration into plant water budgets. Plant, cell and 469 Environment, 42(2), 410–423. 470 Berry, Z. C., White, J. C., Smith, W. K. (2014). Foliar uptake, carbon fluxes and water status are 471 affected by the timing of daily fog in saplings from a threatened cloud forest. Tree Physiology, 472 34(5), 459–470. 473 Bickford, C. P. (2016). Ecophysiology of leaf trichomes. Functional Plant Biology, 43(9), 807– 474 814. 475 Bloch, R. (1946). Differentiation and pattern in Monstera deliciosa. The idioblastic development 476 of the trichosclereids in the air root. American Journal of Botany, 33(6), 544–551. 477 Boanares, D., Ferreira, B. G., Kozovits, A. R., Sousa, H. C., Isaias, R. M. S., França, M. G. C. 478 (2018). Pectin and cellulose cell wall composition enables different strategies to leaf water 479 uptake in plants from tropical fog mountain. Plant Physiology and Biochemistry, 122, 57–64. 480 Boanares, D., Kozovits, A. R., Lemos Filho, J. P., Isaias, R. M., Solar, R. R., Duarte, A. A., 481 Vilas-Boas T., França, M. G. (2019). Foliar water uptake strategies are related to leaf water
21
bioRxiv preprint doi: https://doi.org/10.1101/2019.12.17.879577; this version posted December 18, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
482 status and gas exchange in plants from a ferruginous rupestrian field. American journal of 483 botany. 484 Breshears, D. D. McDowell N.G., Goddard K.L., Dayem K.E., Martens S.N., Meyer C., Brown 485 K.M. (2008). Foliar absorption of intercepted rainfall improves woody plant water status most 486 during drought. Ecology 89, 41–47. 487 Bickford, C. P. (2016). Ecophysiology of leaf trichomes. Functional Plant Biology, 43(9), 807– 488 814. 489 Burkhardt, J., Basi, S., Pariyar, S., & Hunsche, M. (2012). Stomatal penetration by aqueous 490 solutions–an update involving leaf surface particles. New Phytologist, 196(3), 774–787. 491 Burgess, S. S. O., Dawson, T. E. (2004). The contribution of fog to the water relations of 492 Sequoia sempervirens (D. Don): foliar uptake and prevention of dehydration. Plant, Cell and 493 Environment, 27(8), 1023–1034. 494 Choat, B., Jansen, S., Brodribb, T. J., Cochard, H., Delzon, S., Bhaskar, R., Bucci S., Feild T.S., 495 Gleason S.M., Hacke U.G., Jacobsen A.L., Lens F., Maherali H., Martinez-Vilalta J., Mayr S., 496 Mecuccini M., Mitchell P.J., Nardini A., Pittermann J., Pratt R.B., Sperry J.S., Westoby M., 497 Wright I.J. Zanne A.E.. (2012). Global convergence in the vulnerability of forests to 498 drought. Nature, 491(7426), 752. 499 Colombo, P. M., & Rascio, N. (1977). Ruthenium red staining for electron microscopy of plant 500 material. Journal of Ultrastructure Research, 60(2), 135–139. 501 Crane, C. F., Carman, J. G. (1987). Mechanisms of apomixis in Elymus rectisetus from eastern 502 Australia and New Zealand. American Journal of Botany, 74(4), 477–496. 503 Dawson, T. E., Goldsmith, G. R. (2018). The value of wet leaves. New Phytologist, 219(4), 504 1156–1169. 505 Díaz, M., Granadillo, E. (2005). The significance of episodic rains for reproductive phenology 506 and productivity of trees in semiarid regions of northwestern Venezuela. Trees, 19(3), 336–348. 507 Liu D., Tu L., Li Y., Wang L., Zhu L., Zhang X. (2008). Genes encoding fasciclin-like 508 arabinogalactan proteins are specifically expressed during cotton fiber development. Plant 509 Molecular Biology Reporter 26, 98–113. 510 Eller, C. B., Lima, A. L., Oliveira, R. S. (2013). Foliar uptake of fog water and transport 511 belowground alleviates drought effects in the cloud forest tree species, D rimys brasiliensis (W 512 interaceae). New Phytologist, 199(1), 151-162. 513 Eller, C. B., Lima, A. L., Oliveira, R. S. (2016). Cloud forest trees with higher foliar water 514 uptake capacity and anisohydric behavior are more vulnerable to drought and climate change. 515 New Phytologist, 211(2), 489–501. 516 Ellis, M., Egelund, J., Schultz, C. J., Bacic, A. (2010). Arabinogalactan-proteins: key regulators 517 at the cell surface? Plant Physiology, 153(2), 403–419.
22
bioRxiv preprint doi: https://doi.org/10.1101/2019.12.17.879577; this version posted December 18, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
518 Evert, R. F. (2006). Esau's plant anatomy: meristems, cells, and tissues of the plant body: their 519 structure, function, and development. John Wiley & Sons. 520 Fahn, A. (1986). Structural and functional properties of trichomes of xeromorphic leaves. Annals 521 of Botany, 57(5), 631–637. 522 Feder, N. E. D., O'brien, T. P. (1968). Plant microtechnique: some principles and new methods. 523 American Journal of Botany, 55(1), 123–142. 524 Fernández, V., Bahamonde, H. A., Javier Peguero–Pina, J., Gil-Pelegrín, E., Sancho-Knapik, D., 525 Goldbach, H. E., Gil, L., Eichert, T. (2017). Physico-chemical properties of plant cuticles and 526 their functional and ecological significance. Journal of Experimental Botany, 68(19), 5293– 527 5306. 528 Fernández, V., Sancho-Knapik, D., Guzmán, P., Peguero-Pina, J. J., Gil, L., Karabourniotis, G., 529 Khayet, M., Fasseas, C., Heredia-Guerrero, J. A., Heredia, A., Gil-Pelegrín, E. (2014). 530 Wettability, polarity, and water absorption of holm oak leaves: effect of leaf side and age. Plant 531 Physiology, 166(1), 168–180. 532 Foster, A. S. (1944). Structure and Development of sclereids in the petiole of Camellia japonica 533 L. Bulletin of the Torrey Botanical Club, 71(3), 302–326. 534 Foster, A. S. (1945a). Origin and development of sclereids in the foliage leaf of Trochodendron 535 aralioides Sieb. & Zucc. American Journal of Botany, 32(8), 456–468. 536 Foster, A. S. (1945b). THE FOLIAR SCLEREIDS OF TROCHODENDRON ARALIOIDES 537 SIEB. & ZUCC. Journal of the Arnold Arboretum, 26(2), 155–162. 538 Foster, A. S. (1947). Structure and ontogeny of the terminal sclereids in the leaf of Mouriria 539 huberi Cogn. American Journal of Botany, 34(9), 501–514. 540 Foster, A. S. (1955a). Comparative morphology of the foliar sclereids in Boronella Baill. Journal 541 of the Arnold Arboretum, 36(2/3), 189–198. 542 Foster, A. S. (1955b). Structure and ontogeny of terminal sclereids in Boronia 543 serrulata. American Journal of Botany, 42(6), 551–560. 544 Foster, A. S. (1956). Plant idioblasts: remarkable examples of cell specialization. Protoplasma, 545 46(1), 184–193. 546 Franke, W. (1967). Mechanisms of foliar penetration of solutions. Annual Review of Plant 547 Physiology, 18(1), 281–300. 548 Gan, L., Zhang, C., Yin, Y., Lin, Z., Huang, Y., Xiang, J., Fu, C. (2013). Anatomical adaptations 549 of the xerophilous medicinal plant, Capparis spinosa, to drought conditions. Horticulture, 550 Environment, and Biotechnology, 54(2), 156–161. 551 Gardingen, P. R. V., Grace, J. (1992). Vapour pressure deficit response of cuticular conductance 552 in intact leaves of Fagus sylvatica L. Journal of Experimental Botany, 43(10), 1293–1299.
23
bioRxiv preprint doi: https://doi.org/10.1101/2019.12.17.879577; this version posted December 18, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
553 Grammatikopoulos, G., Manetas, Y. (1994). Direct absorption of water by hairy leaves of 554 Phlomis fruticosa and its contribution to drought avoidance. Canadian Journal of Botany, 555 72(12), 1805–1811. 556 Gotsch, S. G., Asbjornsen, H., Holwerda, F., Goldsmith, G. R., Weintraub, A. E., Dawson, T. E. 557 (2014). Foggy days and dry nights determine crown level water balance in a seasonal tropical 558 montane cloud forest. Plant, Cell and Environment, 37(1), 261–272. 559 Gouvra, E., & Grammatikopoulos, G. (2003). Beneficial effects of direct foliar water uptake on 560 shoot water potential of five chasmophytes. Canadian Journal of Botany, 81(12), 1278–1284. 561 Heide-Jorgensen, H. S. (1990). Xeromorphic leaves of Hakea suaveolens R. Br. IV. Ontogeny, 562 structure and function of the sclereids. Australian Journal of Botany, 38(1), 25–43. 563 Heslop-Harrison, Y., & Shivanna, K. R. (1977). The receptive surface of the angiosperm stigma. 564 Annals of Botany, 41(6), 1233–1258. 565 Hughes, J., McCully, M. E. (1975).The use of an optical brightener in the study of plant 566 structure. Stain Technology (50), 319–329. 567 Ito, S., Suzuki, Y., Miyamoto, K., Ueda, J., and Yamaguchi, I. (2005). AtFLA11, a fasciclin-like 568 arabinogalactan-protein, specifically localized in sclerenchyma cells. Bioscience and 569 Biotechnological Biochemistry, 69 (10), 1963–1969. 570 Johansen, D. A. 1940. Plant microtechnique. McGraw Hill Book, New York, New York, USA. 571 Karabourniotis, G. (1998). Light-guiding function of foliar sclereids in the evergreen sclerophyll 572 Phillyrea latifolia: a quantitative approach. Journal of Experimental Botany, 49(321), 739–746. 573 Kerstiens, G. (1996). Cuticular water permeability and its physiological significance. Journal of 574 Experimental Botany, 47(12), 1813–1832. 575 Kreft, H., & Jetz, W. (2007). Global patterns and determinants of vascular plant 576 diversity. Proceedings of the National Academy of Sciences, 104(14), 5925–5930. 577 Limm, E. B., Simonin, K. A., Bothman, A. G., Dawson, T. E. (2009). Foliar water uptake: a 578 common water acquisition strategy for plants of the redwood forest. Oecologia, 161(3), 449–459. 579 Liu, H., Shi, R., Wang, X., Pan, Y., Li, Z., Yang, X., Zhang G., Ma, Z. (2013). Characterization 580 and expression analysis of a fiber differentially expressed Fasciclin-like arabinogalactan protein 581 gene in Sea Island cotton fibers. PloS one, 8(7), e70185. 582 Majewska-Sawka, A., Nothnagel, E. A. (2000). The multiple roles of arabinogalactan proteins in 583 plant development. Plant Physiology, 122(1), 3–10. 584 Colombo M.P., Rascio N. (1977). Ruthenium red staining for electron microscopy of plant 585 material. Journal of Ultrastructure Research, 60(2), 135–139.
24
bioRxiv preprint doi: https://doi.org/10.1101/2019.12.17.879577; this version posted December 18, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
586 Martin, C. E., von Willert D.J. (2000). Leaf epidermal hydathodes and the ecophysiological 587 consequences of foliar water uptake in species of Crassula from the Namib Desert in southern 588 Africa. Plant Biology, (2), 229–242. 589 Mayr S., Schmid P., Laur J., Rosner S., Charra-Vaskou K., Dämon B., Hacke U.G. (2014) 590 Uptake of water via branches helps timberline conifers refill embolized xylem in late winter. 591 Plant Physiology, (164), 1731–1740. 592 Metcalfe, C. R., chalk, L., 1950. Anatomy of the dicotyledons 1 & 2. Clarendon Press. Oxford. 593 Nobel PS, 2009. Physicochemical and Environmental Plant Physiology (4th edition). Elsevier 594 Inc. New York. 595 Olson, M. E., Soriano, D., Rosell, J. A., Anfodillo, T., Donoghue, M. J., Edwards, E. J., León- 596 Gómez C., Dawson T., Camarero Martínez J.J., Castorena M., Echeverría A., Espinosa C.I., 597 Fajardo A., Gazol A., Isnard S., Lima R.S., Marcati C.R., Méndez-Alonzo R. (2018). Plant 598 height and hydraulic vulnerability to drought and cold. Proceedings of the National Academy of 599 Sciences, 115(29), 7551–7556. 600 Ohrui, T., Nobira, H., Sakata, Y., Taji, T., Yamamoto, C., Nishida, K., Yamakawa T., Yaguchi 601 Y., Tanekaga H., Tanaka, S. (2007). Foliar trichome-and aquaporin-aided water uptake in a 602 drought-resistant epiphyte Tillandsia ionantha Planchon. Planta, 227(1), 47–56. 603 Pereira, A. M., Lopes, A. L., Coimbra, S. (2016). Arabinogalactan proteins as interactors along 604 the crosstalk between the pollen tube and the female tissues. Frontiers in plant science, 7, 1895. 605 Pina, A. L., Zandavalli, R. B., Oliveira, R. S., Martins, F. R., Soares, A. A. (2016). Dew 606 absorption by the leaf trichomes of Combretum leprosum in the Brazilian semiarid region. 607 Functional Plant Biology, 43(9), 851-861. 608 Rao, T. A., Mody, K. J. (1961). On terminal sclereids and tracheoid idioblasts. Proceedings of 609 the Indian Academy of Sciences-Section B, 53(5), 257–262. 610 Rao, A. R., Sharma, M. (1968). The terminal sclereids and tracheids of Bruguiera gymnorhiza 611 Blume and the cauline sclereids of Ceriops roxburghiana Arn. Proceedings of the Indian 612 Academy of Sciences-Section B, (34), 267–275. 613 Rhizopoulou, S. (1990). Physiological responses of Capparis spinosa L. to drought. Journal of 614 Plant Physiology, 136(3), 341–348. 615 Rhizopoulou, S., Psaras, G. K. (2003). Development and structure of drought tolerant leaves of 616 the mediterranean shrub Capparis spinosa L. Annals of Botany, 92(3), 377–383. 617 Schreel, J. D., von der Crone, J. S., Kangur, O., Steppe, K. (2019). Influence of drought on foliar 618 water uptake capacity of temperate tree species. Forests, 10(7), 562. 619 Schuster, A. C., Burghardt, M., Riederer, M. (2017). The ecophysiology of leaf cuticular 620 transpiration: are cuticular water permeabilities adapted to ecological conditions? Journal of 621 Experimental Botany, 68(19), 5271–5279.
25
bioRxiv preprint doi: https://doi.org/10.1101/2019.12.17.879577; this version posted December 18, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
622 Shields, L. M. (1950). Leaf xeromorphy as related to physiological and structural influences. The 623 Botanical Review, 16(8), 399–447. 624 Solereder, H. (1908). Systematic Anatomy of the Dicotyledons. Oxford. 625 Steppe K., Vandegehuchte M.W., van de Wal B,A,E,, Hoste P., Guyot A., Lovelock C.E., 626 Lockington D.A. (2018). Direct uptake of canopy rainwater causes turgor-driven growth spurts 627 in the mangrove Avicennia marina. Tree Physiology, 38, 979–991. 628 Schwendener, S. (1874). Das mechanische Prinicip im aiiatomischen Bau der Moniocotylen. 629 Tognetti, R. (2015). Trees harvesting the clouds: fog nets threatened by climate change. Tree 630 Physiology, 35(9), 921–924. 631 Tomlinson, P. B., Fisher, J. B. (2005). Development of non-lignified fibers in leaves of Gnetum 632 gnemon (Gnetales). American Journal of Botany, 92(3), 383–389. 633 Tucker, S. C. (1964). The terminal idioblasts in magnoliaceous leaves. American Journal of 634 Botany, 51(10), 1051–1062. 635 Vaadia, Y., Waisel, Y. (1963). Water absorption by the aerial organs of plants. Physiologia 636 Plantarum, 16(1), 44–51. 637 Vaughn, K. C., Talbot, M. J., Offler, C. E., McCurdy, D. W. (2007). Wall ingrowths in 638 epidermal transfer cells of Vicia faba cotyledons are modified primary walls marked by localized 639 accumulations of arabinogalactan proteins. Plant and Cell Physiology, 48(1), 159–168. 640 Vicré, M., Lerouxel, O., Farrant, J., Lerouge, P., Driouich, A. (2004). Composition and 641 desiccation induced alterations of the cell wall in the resurrection plant Craterostigma wilmsii. 642 Physiologia Plantarum, 120(2), 229–239. 643 Vitarelli, N. C., Riina, R., Cassino, M. F., Meira, R.M. S. A. (2016). Trichome-like emergences 644 in croton of Brazilian highland rock outcrops: evidences for atmospheric water uptake. 645 Perspectives in plant ecology, evolution and systematics, 22, 23–35. 646 Weathers, K. C., Ponette-González, A. G., Dawson, T. E. (2019). Medium, vector, and 647 connector: fog and the maintenance of ecosystems. Ecosystems, 1–13. 648 https://doi.org/10.1007/s10021-019-00388-4 649 Yates, D. J., Hutley, L. B. (1995). Foliar uptake of water by wet leaves of Sloanea woollsii, an 650 Australian subtropical rainforest tree. Australian Journal of Botany, 43(2), 157–167. 651 Yariv J., Lis H., Katchalski E. (1967). Precipitation of arabic acid and some seed 652 polysaccharides by glycosylphenylazo dyes. The Biochemical Journal 105, 1C – 2C . 653 Zhang, W., Hu, Y., Li, Z., Wang, P., Xu, M. (2009). Foliar sclereids in tea and its wild allies, 654 with reference to their taxonomy. Australian Systematic Botany, 22(4), 286–295. 655
26
bioRxiv preprint doi: https://doi.org/10.1101/2019.12.17.879577; this version posted December 18, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
656
657
658
659
660
661
662
663 FIGURES AND LEGENDS
27
bioRxiv preprint doi: https://doi.org/10.1101/2019.12.17.879577; this version posted December 18, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
664
665 Figure 1. Leaf surface anatomy of Capparis odoratissima. A Elongated leaf shape with a
666 bright green adaxial color. B Conspicuous pattern of translucent idioblast tips through the entire
667 adaxial leaf area. C Cleared tissue showing a star-like appearance at the tip of these structures. D
668 Closer view, showing a narrow pore in the center of the idioblasts. E Abaxial surface of the
669 leaves with a pale green color. F. A continuous layer of imbricated peltate hairs covers the
670 abaxial surface. G Peltate hairs are composed of two different units: the bigger one is
671 underneath, with filiform projections that elongate laterally, and have thicker walls towards the
672 center. H. In the center of each peltate trichome, a compact cup of thick walled filiform
673 structures showed ample lumens. Dissecting scope images (A-D, adaxial; E-H, abaxial), and
28
bioRxiv preprint doi: https://doi.org/10.1101/2019.12.17.879577; this version posted December 18, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
674 confocal images of cleared leaves stained with the Feulgen reaction (C,D, adaxial; G,H,
675 abaxial).Scale bars: A,E = 500µm; B,F = 200µm; C,G = 100µm; D,H = 20µm.
676
677 Figure 2. The internal anatomy of the leaves in Capparis odoratissima. A Cross-section of the
678 leaf displaying one of the multiple ididoblasts that traverse the mesophyll and connects the
679 adaxial and abaxial leaf surfaces. B Thick cuticle layer (white arrowheads) on the adaxial
680 epidermis of the leaf, but thinner in the idioblas tips. C The thick walls of the idioblasts contain
681 massive cellulose (blue color). D The cuticle in the abaxial surface (arrows) is thinner, rugose
682 and accumulates massively at the base of the peltate hairs. E The idioblasts directly connect with
683 the center of the peltate hairs in the abaxial surface of C. odoratissima leaves. Ep, epidermis; pp,
29
bioRxiv preprint doi: https://doi.org/10.1101/2019.12.17.879577; this version posted December 18, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
684 palisade parenchyma; id, idioblast; sp, spongy parenchyma; ph, peltate hair. Scale bars: A,D,E =
685 50µm; B,C = 20µm.
30
bioRxiv preprint doi: https://doi.org/10.1101/2019.12.17.879577; this version posted December 18, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
686
31
bioRxiv preprint doi: https://doi.org/10.1101/2019.12.17.879577; this version posted December 18, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
687 Figure 3. Characterization of the idioblasts of Capparis odoratissima. A Maximum 3D
688 projection of an isolated idioblast showing their typical structure from a small tubular projection
689 towards the adaxial epidermis (top) and the irregular walls at the bottom. B Cells of the adaxial
690 epidermis are thick and have substantial extracellular materials that intersperse with the thick
691 adaxial cuticle (white arrowheads). C The idioblasts are columnar at the area of the palisade
692 parenchyma, with thick refringent walls and narrow lumens (arrow). D At the level of the spongy
693 mesophyll, numerous canals connect the lumen of the idioblast with the cells of the leaf. E At the
694 abaxial epidermis, adjacent idioblasts anastomose and form an irregular structure that coincides
695 with the center of the peltate hairs. C, cuticle; ep, epidermis; pp, palisade parenchyma; id,
696 idioblast; sp, spongy parenchyma; ph, peltate hair. Dotted arrows in A indicate the plane of
697 section of images on the right. Scale bars: A = 100µm; B-E = 20µm.
698
32
bioRxiv preprint doi: https://doi.org/10.1101/2019.12.17.879577; this version posted December 18, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
699 Figure 4. Frequency of idioblasts, stomata and peltate hairs in the leaves of Capparis
700 odoratissima. Idioblasts constitute a significant part of the leaf structures, being more numerous
701 than stomata. Interestingly, there are on average ten times more peltate hairs than idioblasts.
33
bioRxiv preprint doi: https://doi.org/10.1101/2019.12.17.879577; this version posted December 18, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
702 34
bioRxiv preprint doi: https://doi.org/10.1101/2019.12.17.879577; this version posted December 18, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
703 Figure 5. Water uptake by leaves of Capparis odoratissima. A Relative water content (%) of
704 C. odoratissima leaves drying under lab conditions (temperature 22°C; RH 30%), calculated
705 according to van Gardingen and Grace, 1992: RWCt=[(Fresht-Dry)/(Fresh0-Dry)]*100. B
706 Cumulative water uptake by C. odoratissima leaves immersed for periodis of 15 minutes
707 followed by quick centrifugation, and then bench dry for 15 min. C Foliar water uptake by
708 surfaces of C. odoratissima leaves; water droplets on the adaxial surface (black) increased leaf
709 weight in a logarithmic fashion, whereas water droplets in the abaxial surface gained water very
710 modestly (grey dots).
35
bioRxiv preprint doi: https://doi.org/10.1101/2019.12.17.879577; this version posted December 18, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
711
36
bioRxiv preprint doi: https://doi.org/10.1101/2019.12.17.879577; this version posted December 18, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
712 Figure 6. Fluorescent dye loading in the leaves of Capparis odoratissima. A Negative control
713 showing absence of autofluorescence in the cell walls of the idioblasts with a 488nm wavelength.
714 D Following abaxial loading of the dye, idioblasts showed a strong fluorescence on their walls. E
715 Maximum projection of the lumen of an idioblast filled with LY dye after adaxial deposition. F
716 Same as E, but when the dye was loaded abaxially. A. Abaxial deposition of 1µL droplets LY
717 revealed strong fluorescence towards the center of the peltate hairs. B Detail of the small external
718 part of the peltate hairs showing fluorescence in the cell walls (arrows). Id, idioblast. LY addax,
719 adaxial load of Lucifer yellow; LY abax, abaxial load of Lucifer Yellow. Scale bars: A = 100µm;
720 B-F = 20µm.
37
bioRxiv preprint doi: https://doi.org/10.1101/2019.12.17.879577; this version posted December 18, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
721
38
bioRxiv preprint doi: https://doi.org/10.1101/2019.12.17.879577; this version posted December 18, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
722 Figure 7. Immunolocalization of pectins (JIM7 mAb) and arabinogalactan proteins (JIM8
723 mAb) in Capparis odoratissima leaves. A, C, F, G, cross-setions of the leaves. B, D, E.
724 Paradermal sections of the leaves. A, B Pectins were strongly present in the apoplast of the
725 epidermal cells, particularly between the cuticle and the cell walls of the epidermis (arrows). C
726 In contrast, AGPs epitopes were present in the lumen of all the idioblasts (arrows). D Pectins
727 also abounded in the external cell walls of the spongy mesophyll in contact with the idioblasts
728 (arrows). E Arabinogalactan protein epitopes showed a consistent presence along the lumen of
729 idioblasts (arrow). F The external walls of the peltate hairs showed a consistent presence of
730 pectins (arrows). G The lumens of the peltate hairs further contained AGPs , consistent with their
731 presence along the idioblast-hair continuum. C, cuticle; ep, epidermis; pp, palisade parenchyma;
732 id, idioblast; sp, spongy parenchyma; ph, peltate hair.
733
734 Figure 8. Simplified model of water balances in the leaves of Capparis odoratissima. A. In
735 the absence of high humidity of the surrounding atmosphere, the water potential of the leaves is
39
bioRxiv preprint doi: https://doi.org/10.1101/2019.12.17.879577; this version posted December 18, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
736 higher than the surrounding environment, thus there is a net loss of water (blue arrows). B. The
737 opposite situation happens with submerged leaves, which have lower water potentials than the
738 environment, thus gaining water (blue arrows) through the lumen of the continuum peltate hairs-
739 idoblasts. C. When water condenses in the upper surface, the inner leaf potential is lower, thus
740 allowing water entrance through he lumens of the idioblasts (blue arrow), and allowing a
741 minimal evapotranspiration in the lower surface due to the presence of an extra layer of peltate
742 hairs covering the stomata (dotted arrow). D. When water condenses in the lower surface, water
743 enters through the lumen of the peltate hairs and the idioblasts (thin blue arrow), but
744 evapotranspiration of the upper surface appears to be higher than water entrance (thick blue
745 arrow).
40