Manuscript Details

Manuscript number AQBOT_2018_255

Title AERENCHYMA, GAS DIFFUSION, AND CATALASE ACTIVITY IN Typha

domingensis: A COMPLEMENTARY MODEL FOR RADIAL OXYGEN LOSS

Abstract
Radial oxygen loss (ROL) is a physical phenomenon that occurs naturally in aquatic plants. Typha domingensis was
chosen as a model plant because it presents basic morphological characteristics found in many aquatic plants, such
as leaves emerging from a stem (rhizome) and from its adventitious roots. This study aimed to evaluate: the relevance
of T. domingensis anatomy on gas diffusion among organs; the influence of plant parts on ROL; the role of catalase
(CAT) in ROL; and the proposition of a novel possibility to explain the downward diffusion of oxygen (O2) through
aquatic macrophytes organs and into the environment. T. domingensis plants were cultivated using Hoagland solution
in a greenhouse under different conditions: plants with intact leaves, plants with leaves cut in half, and plants without
leaves. Furthermore, we evaluated the percentage of aerenchyma in different vegetative organs, the minimum
pressure required for ROL, the daily variations of dissolved O2, and the roots’ CAT activity. The results demonstrated
that certain cellular features contribute to decreased O2 diffusion between the organs, specifically, those found in the
leaf-rhizome and root-rhizome interfaces as well as suberin and lignin layers in these regions. Additionally, our
experiments with a CAT activator and inhibitor validated that a significant amount of the O2 released from ROL could
not, in fact, be exclusively supplied by the atmosphere. Thus, a complementary model is proposed were the CAT
activity is an important component on ROL.

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‘HIGHLIGHTS’

 Typha domingensis roots release O2 into its environment.

 Cattail not depends on the shoot to supply the O2 to radial oxygen loss.
 The catalases of T. domingensis can provide O2 to radial oxygen loss.
 Meristems and suberized tissues increase the resistance to O2 diffusion among plant
organs.
1 AERENCHYMA, GAS DIFFUSION, AND CATALASE ACTIVITY IN Typha
2 domingensis: A COMPLEMENTARY MODEL FOR RADIAL OXYGEN LOSS
3

4 Vinícius P. Duarte1, Marcio P. Pereira1, Felipe F. Corrêa1, Evaristo M. Castro1 and

5 Fabricio J. Pereira2 fabricio.pereira@unifal.mg.edu.br
6 1 Departamento de Biologia, Universidade Federal de Lavras, zip code: 37200-000,
7 Lavras, Brazil.
8 2 Instituto de Ciências da Natureza, Universidade Federal de Alfenas, zip code: 37130-
9 001, Alfenas, Brazil.
10 * Corresponding author: fabricio.pereira@unifal.mg.edu.br, phone: +55 35 3701 9685
11 Abstract
12 Radial oxygen loss (ROL) is a physical phenomenon that occurs naturally in aquatic
13 plants. Typha domingensis was chosen as a model plant because it presents basic
14 morphological characteristics found in many aquatic plants, such as leaves emerging
15 from a stem (rhizome) and from its adventitious roots. This study aimed to evaluate: the
16 relevance of T. domingensis anatomy on gas diffusion among organs; the influence of
17 plant parts on ROL; the role of catalase (CAT) in ROL; and the proposition of a novel
18 possibility to explain the downward diffusion of oxygen (O2) through aquatic
19 macrophytes organs and into the environment. T. domingensis plants were cultivated
20 using Hoagland solution in a greenhouse under different conditions: plants with intact
21 leaves, plants with leaves cut in half, and plants without leaves. Furthermore, we
22 evaluated the percentage of aerenchyma in different vegetative organs, the minimum
23 pressure required for ROL, the daily variations of dissolved O2, and the roots’ CAT
24 activity. The results demonstrated that certain cellular features contribute to decreased
25 O2 diffusion between the organs, specifically, those found in the leaf-rhizome and root-
26 rhizome interfaces as well as suberin and lignin layers in these regions. Additionally,
27 our experiments with a CAT activator and inhibitor validated that a significant amount
28 of the O2 released from ROL could not, in fact, be exclusively supplied by the
29 atmosphere. Thus, a complementary model is proposed were the CAT activity is an
30 important component on ROL.
31

32 Keywords: Intercellular spaces, antioxidant system, aquatic macrophytes, hypoxia,

33 anatomic barriers, hydrogen peroxide.

1
34 1 Introduction
35

36 Aquatic plants are an important part of wetlands, and they are distributed
37 worldwide. These plants maintain the balance of aquatic ecosystems by constituting part
38 of the local diversity (Bolduc et al., 2016). In addition, aquatic macrophytes have shown
39 potential for phytoremediation (Dai et al., 2017; Oliveira et al., 2017).
40 Among the environmental functions of aquatic macrophytes, an important but
41 less investigated trait is their capacity to oxygenate its surroundings (Pang et al., 2016).
42 To overcome the limitations imposed by low oxygen (O2) concentrations in wetlands,
43 macrophytes have developed effective adaptations, such as an efficient antioxidant
44 system (Alam and Ghosh 2018) and a tissue specialized to store and distribute gases
45 within the plant structure, the aerenchyma (Voesenek and Bailey-Serres 2015). It is well
46 known that O2 diffusion in an aqueous medium is 10,000 times slower than in a gaseous
47 medium. Thus, the plants’ internal gas diffusion reservoir plays a key role in the
48 wetland ecosystem.
49 Flooding is a potential source of reactive oxygen species (ROS) in aquatic
50 organisms. ROS participate in deleterious processes, such as damage to cell structures,
51 including lipids, membranes, proteins, and DNA (Spengler et al., 2017); therefore,
52 plants’ resistance to environmental pressures is linked to their antioxidant system.
53 Catalase (CAT) is one of the main enzymes of this system, and it actively participates in
54 the detoxification process consuming hydrogen peroxide (H2O2) (a ROS) and producing
55 O2 and H2O (Alam and Ghosh 2018). Additionally, this enzyme has been shown to be
56 particularly efficient in aquatic macrophytes, such as T. domingensis (Corrêa et al.,
57 2015).
58 Gas diffusion from intercellular spaces contributes to supply adequate amounts
59 of O2 for processes such as photosynthesis and respiration (Mizutani and Kanaoka
60 2018). In submerged tissues such as those in macrophyte roots, the three-dimensional
61 structural arrangement of cells forming the tissue is critical for gas diffusion (Ho et al.,
62 2016). Some specialized tissues such as aerenchyma exhibit high porosity; thus, it
63 favors internal gas diffusion in plants (Kordyum et al., 2017). On the other hand,
64 absence of intercellular spaces characterizes the cellular arrangement of meristematic
65 tissues, and this favors the reduction or even blockage of gas diffusion between this
66 tissue and other parts of the plant (Brulé et al., 2016).

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67 The radial oxygen loss (ROL) is defined by Armstrong (1980) as the O2 transfer
68 from the root aerenchyma of aquatic plants to their rhizosphere. According to Dai et al.,
69 (2017), ROL is an important trait of aquatic plants and promotes tolerance to
70 environments with low O2 content. Previous studies showed the importance of
71 rhizosphere aeration for the uptake and transport of heavy metals by hyperaccumulator
72 plants (White and Ganf 2000), for the maintenance of microorganisms that interact with
73 plant roots (Ma et al., 2018), and for normal plant growth (Mano et al., 2006).
74 Consequently, researchers have investigated ROL for a wide range of applications, such
75 as environmental conservation, crop plant improvement, wastewater treatment, and
76 heavy metal tolerance (Cheng et al., 2010; Rehman et al., 2017; Zhang et al., 2017).
77 There are studies that aimed to explain the origin and production of the O2 released in
78 ROL (Zhang et al., 2014) and the mechanisms involved in gas diffusion and ROL
79 (Colmer 2003).
80 Armstrong and Armstrong (1988); Armstrong (1980); Brix (1993) investigated
81 the physical principles involved in gas exchange dynamics and the mathematical models
82 that could explain them. Furthermore, Colmer (2003) and Tanaka et al. (2007) proposed
83 models for gas transport over long distances inside plants. These findings have
84 contributed to improve our understanding of gas diffusion dynamics and gas release by
85 the plant; however, these models still present issues. For instance, these models evaluate
86 only the aerenchyma tissue, instead of the entire plant anatomical structure. Studies
87 have shown the presence of some tissues such as meristems (Kaul 1974), exodermis
88 (Armstrong et al., 1992; Corrêa et al., 2015) in cattail. However, these anatomical
89 constraints have not been explored in previous studies; particularly, investigations that
90 consider the connections among different plant organs are nonexistent.
91 Therefore, we hypothesize that the presence of tissues with little intercellular
92 spaces will significantly increase the resistances for gas diffusing between the different
93 plant organs as well as into the soil; and that at least a significant part of the O2 released
94 from ROL originates from CAT activity in the roots of macrophytes, such as T.
95 domingensis.
96 Therefore, the purpose of this study was to clarify the origin of O2 involved in
97 ROL by using T. domingensis, an aquatic macrophyte distributed worldwide, to
98 investigate the anatomical barriers for O2 diffusion and the origin of the O2 released
99 from ROL. Importantly, the objectives of this study were: (1) to investigate the role of
100 anatomical traits in gas flow in T. domingensis; (2) to test the role of CAT regarding the

3
101 O2 released from ROL in T. domingensis; and (3) to propose a complementary model to
102 study the origin of O2 released from the roots in ROL.
103

104 2 Material and Methods

105 2.1 Plant material
106 Cattail plants (Typha domingensis Pers. − Typhaceae) were collected from
107 natural populations in wetlands located at Alfenas, Minas Gerais (21° 25’ 44” S, 45° 56’
108 49” W) in the southeast region of Brazil. The collected plants contained rhizomes and
109 approximately five leaves each (1.5 m in length). These plants were subjected to
110 hypochlorite 50% [commercial sodium hypochlorite (NaClO) solution and distilled
111 water (v v-1), the final NaClO concentration was 3% (w v-1)] for 10 minutes and then
112 washed with tap water before further cultivation in the greenhouse. The plants were
113 grown in 60-L plastic pots containing 10 L of a nutrient solution (Hoagland and Arnon
114 1940) at 40% ionic strength to obtain acclimatized clone plants. Hoagland and Arnon
115 nutritive solution contains the following salts: NH4H2PO4, Ca(NO3)2, Mg(NO3)2, KNO3,
116 K2SO4, FeSO4·7H2O, H2BO3, MnSO4·H2O, ZnSO4·7H2O, CuSO4·5H2O, and
117 H2MoO4.H2O. Additionally, all clone plants used in the experiments described below
118 were of matching size and age (15 cm tall and 60 days old).
119 2.2 Experiments
120 Different experiments were conducted to investigate: (1) the role of T.
121 domingensis anatomy in the gas diffusion between different plant organs, (2) the
122 influence of plant parts in ROL, and (3) the role of CAT in ROL.
123

124 2.3 Anatomy and morphology of T. domingensis

125 We analyzed the anatomy of vegetative organs (leaves, rhizomes, and roots)
126 searching for tissues related to both enhanced gas diffusion (aerenchyma) and reduced
127 gas diffusion (meristems and suberized tissues). The base of the leaves in Typha species
128 show an intercalary meristem (Kaul 1974), and the roots of T. domingensis show an
129 external cortex lacking intercellular spaces (Corrêa et al., 2017). The anatomical traits
130 evaluated in roots were: area of the root section, area occupied by aerenchyma
131 chambers, percentage of aerenchyma chambers in the root (calculated as the ratio of the
132 aerenchyma chambers area to the root area). Additionally, the anatomical traits
133 evaluated in rhizomes were: area of the rhizome section, area occupied by aerenchyma

4
134 chambers, percentage of aerenchyma chambers in the rhizome (calculated as the ratio of
135 the aerenchyma chambers area to the rhizome area). Lastly, the anatomical traits
136 evaluated in leaves were: area of the leaf section, area occupied by aerenchyma
137 chambers, percentage of aerenchyma chambers in the leaf (calculated as the ratio of the
138 aerenchyma chambers to the leaf area).
139 For anatomical analysis, plant parts were fixed in FAA 70% solution
140 (formaldehyde, acetic acid, and 70% ethanol at 0.5:0.5:9 proportions) for 48 hours and
141 then stored in 70% ethanol until further analysis (Johansen 1940). Furthermore, the
142 samples were dried with increasing ethanol concentrations (70, 80, 90, and 100%) at 2-
143 hour intervals and embedded in Historesin according to the manufacturer’s instructions
144 (Leica Microsystems, Wetzlar, Germany). Transversal sections were obtained using a
145 semi-automated rotary microtome Yidi YD-335 (Jinhua Yidi Medical Appliance CO.,
146 LTD, Zhejiang, China). Then, the sections were stained with toluidine blue 1% (w v-1)
147 and mounted on slides with Entellan (Merck, Darmstadt, Germany). The slides were
148 photographed using a microscope attached to an image capturing system (CX31,
149 Olympus, Tokyo, Japan), and quantitative anatomical analysis was performed using
150 UTHSCSA ImageTool software.
151 Fluorescence microscopy was performed to identify suberized or lignified
152 tissues that may form barriers for gas diffusion. Cross and longitudinal sections of
153 transition regions between the leaf and rhizome and the rhizome and root were placed in
154 a solution containing distilled water and 0.1% berberine hemisulphate (w v-1) for 1 hour
155 and then washed in distilled water. Thereafter, sections were kept in 0.5% aniline blue
156 (w v-1) solution for 30 minutes and then washed twice with distilled water. The sections
157 were mounted in a solution of 0.1% FeCl3 (w v-1) in 50% glycerol (w v-1) (Brundrett et
158 al., 1988). Then, the slides were analyzed with a fluorescence microscope (BX60,
159 Olympus) equipped with a cooled monochrome camera (Olympus). Images were
160 captured with ultraviolet excitation/emission wavelengths of 358–461 nm (Brundrett et
161 al., 1988).
162 In addition to the anatomical analysis, T. domingensis plants were analyzed to
163 measure the total air volume that filled the aerenchyma spaces in different organs. The
164 volumes of roots, rhizomes, and leaves from ten plants were measured via the water
165 displacement method using a measuring cylinder (data not shown). Based on the
166 volume of each organ and their percentage of aerenchyma, we calculated the volume of
167 air space in each plant part (the organ volume multiplied by the aerenchyma

5
168 proportion). These air-filled spaces inside each organ are important to calculate the
169 amount of gas necessary to provide enough pressure for gas movement across the
170 barriers found between plant organs (leaf-rhizome and rhizome-root interfaces) and
171 from the root to the soil (mainly exodermis and epidermis).
172 The anatomical analysis was used to identify the resistances to gas diffusion
173 along the entire plant structure. Incorporating some adaptations and considerations, we
174 assumed a model similar to that used for CO2 diffusion in the leaf, according to
175 Terashima et al., (2011). In our model, the following assumptions regarding resistance
176 to gas diffusion were necessary: (a) the intercellular spaces and aerenchyma cause low
177 resistance; (b) tissues with primary cell walls and without lignin or suberin deposition
178 (epidermis, meristems, and parenchyma) present the greatest resistances on their plasma
179 membranes, thin walls, and cytosol; (c) meristems and epidermis generate high
180 resistance because of the absence of intercellular spaces; (d) lignified or suberized
181 tissues cause the highest resistances because of their thick walls and the deposition of
182 these substances on their secondary cell walls.
183

184 2.4 Determination of the pressure limit necessary for ROL

185 To determine the pressure limit required for noticeable ROL, plants were
186 individually placed in plastic pots containing 3.5 L of deionized water and a sealed
187 rubber hose that was attached to T. domingensis leaves and to an air compressor. These
188 leaves were cut on the apical third, so that the pressurized air enters the leaf and passes
189 through the leaf-rhizome and rhizome-root interfaces and from the root to the solution.
190 Moreover, a multiparameter probe (YSI, 5565 MPS, Yellow Springs, Ohio, USA;
191 version 1.12) that detects dissolved O2 in the water was used to determine ROL. The
192 pressure was gently increased until the limit required for ROL detection was found, and
193 it was then registered. The experiment was replicated ten times, and the mean ±
194 standard deviation was calculated for the experimental plants.
195

196 2.5 The influence of plant parts on ROL

197 One of the hypotheses that may explain ROL in macrophytes is that O2 enters
198 the plant via stomata or broken stems or leaves and then finds its way through leaves,
199 towards the rhizome, then to the roots, and finally, into the soil (Colmer 2003).
200 Therefore, leaves and stems (shoots) may be crucial for the mechanism by which O2
201 enters the plant, if this is the only way it happens. We examined T. domingensis plants

6
202 in three distinct conditions to test this hypothesis: intact plants, leafless plants (all leaves
203 were carefully removed leaving only rhizomes and roots), and plants with leaves cut in
204 half (through the middle). Leafless plants were kept underwater the entire time (no
205 contact with air). The dissolved O2 was measured using the multiparameter probe
206 containing an O2 electrode (previously mentioned). Moreover, all data sampling was
207 performed in the morning (between 7 and 9 a.m.), and the electrode was kept resting in
208 the solution for three minutes to stabilize it before every measurement. The
209 experimental design was completely randomized with three treatments and ten
210 replicates. Measurements were taken for ten days, and the average was calculated for
211 each replicate.
212

213 2.6 CAT activity in T. domingensis roots and its role in ROL
214

215 The CAT activity on Typha species is remarkably high (Corrêa et al., 2015), and
216 that O2 is a product of this enzyme’s reaction, whose substrate (H2O2) is increased under
217 flooding conditions (Voesenek and Bailey-Serres 2015). Thus, we performed
218 experiments containing substances that modify CAT activity to determine the role of
219 CAT as a source of O2 for ROL. For this, we used both intact and leafless plants
220 (similar method to that previously described) and subjected them to three treatments: (1)
221 SNP (Êxodo Científica, São Paulo, Brazil), described by Fu et al., (2016) as a CAT
222 activator; (2) AT (Sigma Aldrich, St. Louis, MO, USA), described by Aver`yanov et al.,
223 (2015) as a CAT inhibitor; and (3) control treatment (distilled water), without
224 modifying CAT activity. All plants were placed in plastic pots containing 3.5 L of
225 distilled water and 0.1 mM SNP, 0.1 mM AT, or only distilled water (for the control
226 plants). In addition, the experiment was conducted using a 2×3 factorial design with ten
227 replicates, and the entire experiment was repeated three times.
228 The O2 dissolved in the solution was measured using the multiparameter probe
229 previously mentioned. Measurements were taken before and 12 hours after the addition
230 of CAT activity modifiers. Then, their difference was calculated and expressed as ΔO2.
231 The roots were collected 2 hours after the application of CAT activity modifiers,
232 placed in liquid nitrogen, and stored at −80 °C until they were analyzed. According to
233 Biemelt et al., (1998), CAT was extracted as follows: 0.2 g of fresh root mass was
234 ground in liquid nitrogen and homogenized in 1.5 mL of an extraction buffer that
235 contained 1.47 mL of potassium phosphate buffer 0.1 M (pH 7.0), 15 µL of EDTA 0.1

7
236 M (pH 7.0), 6 µL of DTT 0.5 M, 12 µL of PMSF 0.1 M, ascorbic acid 0.001 M, and 22
237 mg of polyvinylpolypyrrolidone. The extract was centrifuged at 12,000 g for 30 minutes
238 at 4 °C, and the supernatant was collected and stored at −20 °C until further analyzed.
239 Moreover, CAT activity was evaluated according to Havir and McHale (1987) as
240 follows: 10 µL aliquots of the enzyme extract were added to 170 µL of an incubation
241 medium that contained 90 µL of potassium phosphate 200 mM (pH 7.0), 71 µL of
242 water, and 9 µL of H2O2 250 mM, and then incubated at 28 °C. Enzyme activity was
243 determined by the decrease in absorbance at 240 nm measured every 15 seconds for 3
244 minutes, monitoring the consumption of H2O2. The molar extinction coefficient used
245 was 36 mM-1 cm-1. The specific activity of CAT was calculated on the basis of the total
246 amount of proteins in the samples determined according to Bradford (1976). We
247 calculated the enzymatic activity in triplicate, and the mean was calculated for each
248 replicate.
249 H2O2 was determined according to Velikova et al., (2000) as follows: 200 mg of
250 fresh roots were ground in liquid nitrogen and 20% polyvinylpolypyrrolidone (w m-1)
251 and further homogenized in 1.5 mL of trichloroacetic acid 0,1% (w v-1). The
252 homogenate was centrifuged at 12,000 g for 15 minutes at 4 °C. The H2O2 content was
253 determined measuring the absorbance at 390 nm in a reaction medium that contained
254 500 µL of extract, 500 µL of potassium phosphate buffer 10 mM (pH 7.0), and 1 mL of
255 potassium iodide 1 M. We calculated the H2O2 content in the roots in duplicate, and the
256 data were averaged to one replicate.
257

258 2.7 Statistical analysis

259 The data were submitted to one-way or two-way ANOVA using the SISVAR
260 5.0 software (Ferreira 2011). Prior to parametric analysis, the data were tested for a
261 normal distribution by using the Shapiro-Wilk test and means compared by the post-hoc
262 Scott-Knott test; p< 0.05 was considered statistically significant’.

263 3 Results
264 3.1 Anatomy of T. domingensis
265 The leaves of T. domingensis comprised one-layered epidermis without
266 intercellular spaces. Internally, 3–5 layers of palisade parenchyma were followed by 3–
267 6 layers of large ground parenchyma cells, both having particularly small intercellular
268 spaces (Figure 1). These three tissues showed limited gas diffusion capacity, and this

8
269 pattern could be found on both adaxial and abaxial leaf sides. The large aerenchyma
270 chambers were found on the central part of the leaf (Figure 1A). Furthermore, the leaf-
271 rhizome interface included an intercalary meristem that maintains the mitotic capability
272 of the leaf base and promotes continuous growth (Figure 1C and D). However, this
273 meristem and the region where cells are still differentiating lacked intercellular spaces
274 limiting gas diffusion from the leaf aerenchyma to the rhizome (Figure 1 C and D).
275 Similarly, the rhizome of T. domingensis comprised one-layered epidermis
276 without intercellular spaces (Figure 2 A and B). Internally, three or four layers of
277 exodermis were found, having thick walls and lacking intercellular spaces. The
278 aerenchyma was found on the innermost part of the rhizome cortex; this area showed
279 small vascular bundles and parenchyma. The innermost part of the rhizome comprised
280 an atactostelic cylinder with ground parenchyma and large, scattered vascular bundles;
281 this part showed few intercellular spaces (Figure 2A).
282 T. domingensis roots comprised one-layered epidermis without intercellular
283 spaces. Internally, three layers of parenchyma cells with thick walls formed the
284 exodermis (Figure 2 C and D). The middle cortex was composed of large aerenchyma
285 chambers, while the innermost part of the cortex contained parenchyma cells with few
286 intercellular spaces. Moreover, the vascular cylinder (with xylem and phloem) was
287 found at the center of the roots. Intercellular spaces were nearly absent in this part of the
288 root (Figure 2B). The root-rhizome interface was composed of root external tissues as
289 well as rhizome external parts, namely the exodermis and epidermis of both, but lacked
290 direct connections between the two organs along the root axis (Figure 2E). This
291 interface showed scarce intercellular spaces (Figure 2E), and fluorescence microscopy
292 revealed significant deposition of lignin and suberin on the exodermis of both the
293 rhizome and root (Figure 2F).
294 The leaf of T. domingensis showed the highest aerenchyma percentage among
295 the vegetative organs, followed by the root and then the rhizome. In contrast, the low
296 percentage of aerenchyma found in the rhizome indicates that few parts are filled with
297 air, although this organ has a large area section with 6.1 mm2. In fact, the largest air
298 spaces were found on the leaves of T. domingensis and the smallest on the rhizomes
299 (Table 1). The leaf pressure threshold required to promote ROL was 0.08 ± 0.01 MPa.
300 We found miniscule intercellular spaces and deposition of compounds restrictive
301 to air diffusion in the leaf-rhizome and rhizome-root interfaces. Furthermore, the
302 deposition of lignin and suberin was remarkably abundant in cell walls of the root

9
303 exodermis and in the external layers of the rhizome. The connection between the root
304 and rhizome showed large areas with lignified and suberized tissues and lacked
305 intercellular spaces (Figure 2 E and F). Additionally, the leaf base showed scarce
306 intercellular spaces because of the intercalary meristem found in this region (Figure 1C
307 and D).
308

309 3.2 The influence of plant parts on ROL

310 All T. domingensis plants on experimental conditions regarding leaves (intact,
311 leafless, or cut leaves) exhibited ROL; however, the differences among them were not
312 statistically significant (P = 0.08) (Table 2).
313 3.3 CAT activity on T. domingensis roots and its role on ROL
314 No interaction was found between the effect of the plant parts and enzyme
315 modifiers (P = 0.98) and no effect of intact or leafless plants was found (p = 0.05) but
316 significant effect was found to the CAT modifiers (p < 0.01). Both intact and leafless
317 plants showed similar rates of ROL (Table 3). The highest and lowest means of ROL
318 were found in plants treated with sodium nitroprusside (SNP) and 3-amino-1,2,4-
319 triazole (AT), respectively, whereas control plants means showed intermediate values
320 (Table 3).
321 CAT activity on intact plants was modified after the application of SNP and AT;
322 interestingly, AT exposure resulted in higher CAT activity than SNP exposure. In
323 addition, intact plants always showed higher CAT activity than leafless plants (Table 4).
324 The H2O2 concentration on T. domingensis roots was not significantly different between
325 leafless and intact plants. However, plants subjected to AT showed highest H2O2 levels
326 in their roots (Table 4).

327 4 Discussion
328 4.1 Does ROL depend on macrophyte shoots?
329 The models that have been proposed to explain ROL argue that the O2 enters the
330 plant via broken shoots, then diffuses throughout the rhizome and roots, and finally is
331 released into the soil (Armstrong and Armstrong 1988; Armstrong 1980; Konnerup et
332 al., 2011). According to Colmer (2003), the O2 enters the plants via stomata or broken
333 shoots, and then diffuses from the shoot to roots, where ROL occurs. However, our
334 results indicated that the origin of the O2 may not rely exclusively on the shoot air
335 uptake, because even leafless plants exhibited ROL. In addition, the pressure threshold

10
336 to promote noticeable ROL on T. domingensis (0.08 MPa) was considerably higher than
337 the estimated values of internal pressure in other macrophytes; for instance, Konnerup
338 et al., (2011) found an average internal pressure of 0.0004 MPa in Cyperus L. species. It
339 seems unlikely that the high pressure on the leaf mesophyll necessary to promote ROL
340 could be reached only by air uptake of leaf stomata.
341 In this study, we measured ROL during 10 days for each experiment, which
342 certainly exceeds the time needed to deplete the aerenchyma-stored gas. In fact, the
343 amount of O2 released in two consecutive days (1.15 ±0.3 mg L-1, Table 2) was too high
344 to be sourced only from aerenchyma-stored gas. In this experiment, the van’t Hoff
345 equation could be used to estimate the amount of O2 in T. domingensis roots: n = PV /
346 RT; where P is the pressure for ROL in standard atmospheres (atm), V is the volume of
347 aerenchyma gas in liters (L), R is the gas constant, and T is the temperature in kelvin
348 degrees (K). In our experimental conditions, we could consider the values for the
349 pressure for ROL (measured), root aerenchyma volume (Table 1), average temperature,
350 and gas constant to be 0.7 atm (0.07 MPa), 0.00056 L (where 0.0001288, or 23%, is the
351 estimated O2), 293.15 K (20 ºC), and 0.082, respectively. Therefore, n = (0.7 × 0.00012)
352 / (0.082 × 293.1), which is approximately equal to 0.0000375 M of O2 –– that is to say,
353 about 1.2 mg of O2 were present in the roots. Notably, this amount of O2 is released on a
354 daily basis (1.15 ± 0.3 mg L-1), which means the entirety of O2 present in the roots could
355 be exhausted in a single day. Consequently, the total amount of O2 stored in the roots of
356 T. domingensis is insufficient to provide constant O2 release for 10 days, implying that
357 particularly in leafless plants, O2 in the root aerenchyma must be replenished by a
358 mechanism different from those previously proposed (shoot uptake and transport).
359 The ROL was not affected by the removal of the shoot, measured as the
360 difference of dissolved O2 in solution between consecutive days (Table 3). In addition,
361 differences could not be found between intact and leafless plants (Table 2 and 3). The
362 analysis of these results showed that ROL occurs independently of the shoot integrity.
363 Conclusively, our results showed that ROL is independent of macrophyte shoots
364 and is maintained even in the absence of this part of the plant. Moreover, it suggests that
365 a mechanism different from air diffusion from shoots is necessary to replenish the O2
366 released from ROL.
367

368 4.2 Anatomical barriers for O2 diffusion throughout the shoot-root-soil continuum

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369 Most studies concerning O2 diffusion through the plant and its role in ROL
370 disregard many anatomical traits that could significantly limit the plant’s internal air
371 flow. Different plant tissues show variable permeability to O2 depending on the
372 abundance of intercellular spaces. The resistance to gas diffusion is low in intercellular
373 spaces but is greatly increased in the presence of cell membranes and cells’ liquid
374 compartments (Terashima et al., 2011). Thus, tissues that typically have a low
375 percentage of intercellular spaces show a high resistance to O2 diffusion.
376 Anatomical barriers present in specific tissues of T. domingensis have been
377 previously reported; however, their role in O2 diffusion has not been discussed or
378 considered when suggesting the models to explain ROL. For instance, Kaul (1974)
379 identified the intercalary meristem on the leaf base. Furthermore, the root exodermis of
380 several aquatic plants limits the O2 diffusion from the roots to allow its use in root
381 respiration (Armstrong and Armstrong 1988; Armstrong et al., 1992; Brix and Schierup
382 1990; Corrêa et al., 2017). According to Pi et al., (2009), gas diffusion naturally occurs
383 under low resistance conditions within one of the plant’s organs containing abundant
384 intercellular spaces. The high percentage of aerenchyma present and the volume of air
385 contained in it provided the necessary conditions for gas distribution in the roots and
386 leaves of T. domingensis. However, the aerenchyma percentage found in the rhizome is
387 significantly low when compared to that of roots and leaves, and thus, this organ has a
388 limited gas diffusion capacity.
389 The multiseriate exodermis in the root and the suberin deposition in the root-
390 rhizome interface further impede O2 diffusion. The hydrophobic property of suberin
391 (Song et al., 2011) is another factor that limits (or even blocks) the gas diffusion
392 through the root-rhizome interface as well as the O2 released from the root external
393 cortex and epidermis.
394 In addition to those anatomical barriers hindering O2 diffusion throughout the
395 plant organs, leaf tissues also present difficulties for the air uptake reaching the
396 aerenchyma and for the gas diffusion between consecutive chambers. For instance, the
397 stomatal resistance is very well known (Terashima et al., 2011), which is largely
398 reduced when stomatal pores open. This is the way air enters leaves providing carbon
399 dioxide (CO2) for photosynthesis. However, in T. domingensis leaves, stomata are
400 located just above the palisade parenchyma that lacks (or has unnoticeable) intercellular
401 spaces (Figure 1), which constitutes a second resistance barrier for air diffusing into
402 aerenchyma. Successively, ground parenchyma creates a third resistance barrier. The

12
403 sum of these resistances comprises a strong barrier for air to fill the aerenchyma
404 chambers sufficiently to reach the high pressure required for ROL (0.08 MPa).
405 Figure 3 indicates the significant resistance locations on the structure of T.
406 domingensis. Considering the main tissues and organs interfaces, the total resistance for
407 O2 diffusion through the plant body could be defined as follows: RTO2 = Rl + Rt + Rlri +
408 Rriro + Ree; where RTO2 = total resistance to O2 diffusion, Rl = leaf resistance, Rt =
409 trabeculae resistance, Rlri = resistance of the leaf-rhizome interface, Rriro = resistance of
410 the rhizome-root interface, and Ree = resistance of the roots exodermis and epidermis
411 (Figure 3). Consequently, the accumulated resistances may severely limit the process in
412 the current model for O2 diffusion (Colmer 2003). Thus, the anatomy of aquatic
413 macrophytes (such as T. domingensis) is adapted to store O2 in the aerenchyma and to
414 diffuse it within an organ; nonetheless, the diffusion between organs is limited by
415 barriers in their interfaces.
416 4.3 CAT activity is an O2 source for ROL
417 The CAT enzyme consumes H2O2 to produce O2 and H2O; its activity is
418 associated to scavenging of H2O2 (a ROS) to protect plant cells against oxidative stress
419 (Møller 2001). Flooding causes O2 deprivation that promotes formation of ROS, and
420 thus, aerenchyma tissue has evolved to store O2 to avoid this effect (Voesenek and
421 Bailey-Serres 2015). Therefore, aquatic macrophytes experience constant O2 deficiency
422 promoting the increase of H2O2 in roots, which is detoxified by CAT activity. This
423 provides a continuous source of H2O2 for CAT. Consequently, this enzyme is able to
424 operate steadily in the roots of aquatic macrophytes, such as T. domingensis.
425 In aquatic macrophytes, CAT activity is increased by several environmental
426 factors such as heavy metals (Pereira et al., 2014), population density (Corrêa et al.,
427 2015), UV radiation (Xu et al., 2014), and O2 availability (Xu et al., 2011). Thus, this
428 enzyme is exceptionally responsive to environmental conditions. The CAT modifiers
429 used in this study were efficient for altering this enzyme’s activity, and these changes
430 were sufficient to shift the levels of ROL in the plants (Table 3). The role of this
431 enzyme in ROL was convincingly evidenced by the 81.9% increase and 49.5%
432 reduction of ROL obtained from treatments with a CAT activator and inhibitor,
433 respectively. In fact, changes in CAT activity were proportionally related to ROL.
434 These results support the effects of CAT modifiers and the enzyme’s use of
435 H2O2 as substrate. CAT activity was reduced by its inhibitor (AT), and during its effect,
436 H2O2 was accumulated in T. domingensis tissues. After that, the excess H2O2 in the

13
437 roots increased CAT activity. Therefore, the plants subjected to the inhibitor showed the
438 highest CAT activity because the enzyme’s substrate accumulated, which corresponds
439 to the Michaelis-Menten kinetics that state the enzyme activity rates depend on the
440 concentration of substrate (Reuveni et al., 2014).
441 Another issue was whether CAT would be able to provide sufficient O2 to match
442 the levels of ROL we measured (1.15 mg L-1). The average CAT activity for control
443 plants was 0.08 µmol H2O2 min-1 µg-1 protein (data not shown). The method to assess
444 CAT activity derives it from H2O2 consumption; however, analysis of the equilibrated
445 equation showed that for every mol of this substrate, 0.5 mol of O2 would be produced
446 (Møller, 2001). Thus, 0.04 µmol O2 min-1 µg-1 protein is produced from CAT activity in
447 T. domingensis roots. Furthermore, 12 g of fresh mass was the average mass of the root
448 system of the T. domingensis experimental plants measured at a similar growth stage
449 and size (data not shown). The protein proportion found on T. domingensis roots was
450 0.08 mg protein g-1 fresh mass. Therefore, the total protein in the root system of these
451 plants was 0.96 mg (960 ug-1). Furthermore, the O2 production capacity of the T.
452 domingensis root system was 38.4 µmol O2 min-1, which is equivalent to 1.23 mg O2
453 min-1 of enzyme activity. Thus, this enzyme has an exceptional capacity to produce O2,
454 and a single minute of its operation could provide sufficient O2 to maintain the ROL
455 measured from T. domingensis roots. Additionally, its enzyme activity rate was similar
456 to those previously reported for Typha species and for other aquatic macrophyte species
457 as well (Corrêa et al., 2015; Yang and Ye 2015). This shows that for aquatic
458 macrophyte roots, CAT could be a reliable source of O2 to supply the aerenchyma and
459 the root’s aerobic metabolism. In addition, CAT could support ROL rates found in
460 aquatic macrophyte roots.
461

462 4.4 Complementary model to explain the aerenchyma O2 origin and ROL
463 The O2 supplied via transport from the shoots may be insufficient for ROL
464 because of anatomical barriers. Complementary, CAT acting as an O2 source in the
465 roots is a plausible way to replenish the gas lost in ROL. Moreover, O2 produced
466 directly in the roots avoids all anatomical barriers except for the root exodermis (Ree
467 resistance), which substantially facilitates O2 reaching the soil. Consequently, we
468 propose a complementary model shown in Figure 4.
469 5 Conclusion

14
470 In summary, anatomical barriers comprised of tissues lacking intercellular
471 spaces constrain the O2 pathway from the shoot to the roots in aquatic macrophytes. The
472 root CAT has sufficient activity and substrate to provide enough O2 to fill aerenchyma
473 chambers and to support ROL. Moreover, the O2 released by aquatic macrophytes in
474 wetlands partially originates from CAT activity. T. domingensis roots produced
475 sufficient O2 for ROL independently of the aerial plant part.
476

477 Disclosures:
478 The authors declare no conflicts of interest.
479

480 Acknowledgments and Funding Source:

481 The authors thank CNPq [Conselho Nacional de Desenvolvimento Científico e
482 Tecnológico (National Counsel of Technological and Scientific Development)], CAPES
483 [Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (Coordination for the
484 Improvement of Higher Education Personnel)], and FAPEMIG [Fundação de Amparo à
485 Pesquisa do estado de Minas Gerais (Minas Gerais State Research Foundation)] for
486 funding and research grants awarded to complete the present study.
487

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657 Zhang, L., Yang, Q., Wang, S., Li, W., Jiang, S., Liu, Y., 2017. Influence of silicon treatment on
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660

661

662

663

664

665

666

667

668

669

670

671

672

673

674

675

676

677

678

679

18
680 FIGURE LIST:
681 Figure 1. Anatomical structure of Typha domingensis leaves (A and B) and leaf-rhizome connection (C
682 and D). ade= adaxial epidermis, abe= abaxial epidermis, pp= palisade parenchyma, ae= aerenchyma, is=
683 intercellular space, gp= ground parenchyma, vb= vascular bundle, fb= fibers, rhi= rhizome, le= leaf,
684 asterisk= leaf intercalary meristem. Bars= 300 µm (A), 50 µm (B) and 200 µm (C and D).
685
686 Figure 2. Anatomical structure of the rhizome (A and B), root (C and D) and the rhizome-root interface
687 (E and F) of Typha domingensis. The bright areas on the F image show lignin/suberin deposition on
688 fluorescence microscopy and the staining with cyanide blue. ep= epidermis, ex= exodermis, ae=
689 aerenchyma, is= intercellular space, gp= ground parenchyma, vb= vascular bundle, vc= vascular cylinder,
690 fb= fibers, rhi= rhizome, ro= root, arrow= rhizome-root interface. Bars= 300 µm (A, B C and E), 100 µm
691 (D) and 200 µm (F).
692
693 Figure 3. Scheme of the path to O2 diffusion from the atmosphere to soil throughout the plant body,
694 when considering anatomical resitances. RTO2 = total resistance to O2 diffusion; Rl = leaf resistance, Rt =
695 trabeculae resistance; Rlri = resistance of the leaf-rhizome interface; Rriro = resistance of the rhizome-root
696 interface; Ree = resistance exodermis and epidermis of the roots.
697
698 Figure 4. A complementary proposal for the origin of O2 on the aerenchyma and to permit ROL in
699 aquatic macrophytes such as T. domingensis.
700

701

702

703

704

705

706

707

708

709

710

711

712

713

714

19
715 TABLE LIST:
716 TABLE 1. Aerenchyma and internal air volume characterization of the vegetative organs of T.
717 domingensis Pers. Data are shown as mean ± standard deviation.
Organ Organ section Aerenchyma area Aerenchyma Air volume
(mm2) (mm2) (%) (mL)

Root 1.35±0.50 0.51±0.38 37.77±13.75b 0.56±0.16b

Rhizome 6.1±0.89 0.49±0.14 8.03±2.56c 0.33±0.17b

Leaf 8.18±6.03 4.29±3.48 52.44±12.86a 2.41±0.98a

718 Means followed by the same letters in columns do not differ according to the Scott-Knott test at p < 0.05.
719

720

721

722

723

724

725

726

727

728

729

730

731

732

733

734

735

736

737

738

739

740

741

742

20
743 TABLE 2. Root oxygen loss (ROL) expressed as the mean difference of the dissolved oxygen between
744 two consecutive days on the solution containing intact, leafless and T. domingensis plants with cut
745 leaves. Data is shown as mean ± standard deviation.
ROL

(mg L-1)

Intact Plants 0.43± 0.4a

Leafless Plants 0.64± 0.4a

Cut leaves 0.49± 0.4a

746 Means followed by the same letters in columns do not differ according to the Scott-Knott test at p < 0.05.
747

748

749

750

751

752

753

754

755

756

757

758

759

760

761

762

763

764

765

766

767

768

769

770

21
771 TABLE 3. Root oxygen loss (ROL) expressed as the mean difference of the dissolved oxygen between
772 two consecutive days after the application of treatments. The ROL was calculated in the solution
773 containing intact and leafless T. domingensis plants subjected to AT (CAT inhibitor) and SNP (CAT
774 activator). Data are shown as mean ± standard deviation.
Treatment ROL (mgO2 L-1)

Intact plants 1.00 ± 0.3a

Leafless 1.33 ± 0.4a

Treatment ROL (mgO2 L-1)

Control 1.05 ± 0.1b

AT (CAT inhibitor) 0.53 ± 0.2c

SNP (CAT activator) 1.91 ± 0.2a

775
776 Means followed by the same letters in columns do not differ according to the Scott-Knott test at p < 0.05.
777

778

779

780

781

782

783

784

785

786

787

788

789

790

791

792

793

794

795

796

797

22
798 TABLE 4. CAT activity and H2O2 concentration in roots of T. domingensis evaluated from intact or
799 leafless plants passed 2 h after the application of AT (CAT inhibitor) and SNP (CAT activator). Data is
800 shown as mean ± standard deviation.
CAT activity on roots (µmol H2O2 min-1 ug-1 protein)
Control AT SNP
Intact Plants 0.0315±0.002aB 0.1041±0.02aA 0.0481±0.03aB
Leafless 0.0046±0.003bA 0.013±0.001bA 0.0108±0.004bA
Root H2O2 concentration (mmol H2O2 mg-1 FM)
Control AT SNP
Intact Plants 0.255±0.13aA 0.390±0.21aA 0.040±0.11aA
Leafless 0.053±0.12aB 0.530±0.50aA 0.118±0.16aB
801
802 Means followed by the same lowercase letters in columns and uppercase letters in rows do not differ
803 according to the Scott-Knott test at p < 0.05.
804

23