Formation Mechanism and Anatomical Structure of the Knee Roots of Taxodium Ascendens

22 Aims: Flooding seriously limits the growth and distribution of plants. Taxodium ascendens is a typical 23 tree species with high flood tolerance, and it can generate knee roots in the wetlands. This study was 24 conducted to understand the formation mechanism of the knee roots. 25 Methods: The number and size of knee roots and soil flooding conditions were investigated in this 26 study. Furthermore, physiology, biochemical responses, and the anatomical structure of knee roots and 27 underground roots were measured at different developmental stages. 28 Results: The results show that the formation of knee roots was significantly affected by the soil water table ( P < 0.05). Moreover, knee root formation was affected by ethylene and indole-3-acetic acid 30 (IAA) concentrations in the roots. The 1-aminocyclopropane-1-carboxylic acid (ACC) content and 31 ACC synthase activity were significantly lower in the knee roots than in the underground roots. The 32 ethylene release rate was significantly higher in the knee roots than in the underground roots ( P < 33 0.05), and IAA content first increased and then decreased with knee root development. The cells of the 34 periderm at the apex of the knee roots were dead and had a large number of intercellular spaces, which 35 was beneficial for the growth of T. ascendens . Conclusions: Seasonal flooding induced the production of endogenous hormones, resulting in the 37 formation of knee roots, which improved root respiration and ventilation. The results obtained can gain a basis for the formation mechanism of knee roots and provide scientific evidence for the afforestation and management under wetland conditions.


Introduction 44
Nowadays, flood damage is receiving a lot of attention, as flooding and waterlogging phenomena 45 tend to be closely associated with global climate changes (Hirabayashi et al. 2013, Zhou et al. 2020. 46 Flooding and submergence are major abiotic stresses that determine species distribution, growth, and 47 knee roots and without knee roots was randomly conducted in 15 plots (1 m × 1 m) within a range of 2 120 m from T. ascendens. The underground roots (depth, 0-50 cm) were excavated, cleaned, dried, and 121 weighed. 122

Sampling of knee roots and assay of physiological indicators 123
In October 2016, the middle water table sites were chosen to investigate the development stages 124 of the knee roots. The knee roots were divided into three stages on the basis of size and age: young-125 aged stage (Fig. 1A), middle-aged stage (Fig. 1B), and old-aged stage (Fig. 1C). The knee roots at the 126 young-aged stage were less than 5 cm in height and less than 5 years of age. The knee roots at the 127 middle-aged stage were 5 to 10 cm in height and 5 to 10 years of age. The knee roots at the old-aged 128 stage were more than 10 cm in height and more than 10 years of age. The age of the knee roots was 129 determined using the annual rings in the transverse section. The knee root samples were divided into 130 two parts: the swollen part (upside) and non-swollen part (underside). Underground roots 131 approximately 10 mm in diameter were collected as the control. The root tissues were randomly 132 selected for physiological assay. 133

Ethylene release rate of the roots 134
Fresh root cambium tissues (0.6 g) were sealed in a closed bottle and incubated for 4 h at 30 °C. 135 Then, gas samples (0.5 mL) were collected from the incubator to determine the ethylene concentration 136 by using a gas chromatograph equipped with a flame ionization detector (FID) and electron capture 137 detector (ECD) (Agilent 7890A; Agilent Technologies Inc. USA). The external standard method was 138 used to calculate the ethylene concentration of the samples. 139

ACC content 140
The ACC content was determined according to Hoffman et al. (1983). The root cambium tissue 141 samples were cut into small pieces and mixed completely. Then, the samples (1.0 g) were 142 homogenized in 8 mL of ethanol (95%) and centrifuged (8000 × g) at 4 °C for 15 min; the 143 supernatant was transferred to a plastic bottle, 6 mL of ethanol (80%) was added to the residue and 144 shaken for 30 min, the supernatant after centrifugation was transferred to the previous bottle, and the 145 bottle was dried in a water bath at 40 °C. The dried residue was dissolved with 5 mL of distilled water 146 and then centrifuged (8000 × g) at 4 °C for 10 min. The supernatant was used as the ACC fluid; 1 mL 147 of the fluid and 40 µL of HgCl2 (25 mmol·L -1 ) were added into test tubes (20 mL) closed with rubber 148 stoppers. Then, 1 mL of NaOCI-NaOH (v:v = 2:1) was injected into the test tubes with a syringe. The 149 tubes were shaken for 10 min, and gas samples (0.5 mL) were collected from the tubes to determine 150 the ethylene concentration via gas chromatography. The ACC concentration of fresh root samples was 151 calculated using the following formula: 152 where C is the ethylene concentration measured using gas chromatography (nL·L -1 ); VL is the volume 154 of sample bottles without solution (mL); V is the volume of the extracting solution (mL); R is the 155 transfer coefficient from ACC to ethylene; V1 is the volume of the extracting solution used for 156 measurement (mL); V2 is the gas sample volume for gas chromatography (mL); W is the fresh weight 157 of the root sample (g); and 22.4 is 1 molar gas constant under normal atmospheric conditions (L·mol -1 ). 158

ACC synthase activity 159
The ACC synthase activity was determined according to Mehta et al. (1988). Briefly, 1 g of root 160 cambium tissues were ground in a mortar with the extraction buffer (2 mL) and a small amount of 161 quartz sand at 4 °C and centrifuged at 10000 × g·for 20 min. The extraction buffer solution contained 162 400 mmol·L -1 potassium phosphate buffer solution (pH 8.5), 1 mmol·L -1 ethylene diamine tetraacetic 163 acid, 0.5% (v:v) β-mercaptoethanol, and 10 μmol·L -1 pyridoxal phosphate (PLP). The supernatant was 164 used to determine ACC enzyme activity; 0.4 mL of the enzyme extract and 1.6 mL of the buffer 165 solution (containing 50 μmol·L -1 SAM, 10 μmol·L -1 PLP, and 50 mmol·L -1 Hepes-KOH, pH 8.5) were 166 added to the test tubes and incubated at 32 °C for 1 h. Then, 0.1 mL of mercuric chloride (500 167 mmol·L -1 ) was added to stop the reaction, the test tubes were closed and incubated in ice water for 5 168 min, and 0.2 mL of 5% NaOCI-NaOH (v:v = 2:1) was injected into the test tubes with a syringe. The 169 test tubes were shaken for 10 min, and 0.5 mL gas sample was collected to determine the ethylene 170 content by using gas chromatography. 171

IAA content 183
The IAA content was determined according to Chen and Zhao (2008), and Yuan et al. (2008). Corp.). A Symmery C18 column (Waters Corp.) (4.6 × 250 mm, 5 μm) and a detection wavelength of 193 254 nm were used. A sample (50 μl) was automatically injected at a flow rate of 0.5 ml min -1 . 194 Quantification was made by comparing the peak areas with the known amounts of IAA (Sigma). 195

Anatomical analysis of knee roots and underground roots 196
Three samples containing bark and currently produced xylem from the apex of knee roots at the 197 middle-aged stage and mid-sized underground roots were obtained (Fig. 2). Small pieces (10 × 10 × 198 10 mm) of these root materials were fixed in FAA solution (formalin:acetic acid:ethanol:water,

Statistical analysis 205
The data were calculated and plotted using Microsoft Office Excel 2016, and all data were 206 subjected to analysis of variance by using IBM SPSS Statistics 19.0. To determine the effects of the 207 knee roots on the growth of T. ascendens, correlation analysis was performed using IBM SPSS 208 Statistics 19.0. The data were presented as mean ± standard deviation (M ± SD) values, and 209 differences in the data were evaluated using Duncan's test at a significance level of 0.05. 210

Relationships between the morphological characteristics of knee roots and underground water 212 table 213
The tree height, DBH, underground roots, and knee roots of T. ascendens were investigated, and 214 the results showed that the formation and distribution of knee roots were significantly affected by the 215 water table. The knee root density in the middle water table was 143.94% and 147.69% higher than 216 that in the high water table and low water table, respectively. The height and diameter of the knee 217 roots were also observed to be higher in the middle water table site (Table 1); thus, the middle water 218 table significantly increased knee root formation and growth (P < 0.05). 219

Effects of the knee roots on the growth of T. ascendens 220
The weight of underground roots with knee roots was significantly higher than that of 221 underground roots without knee roots (Fig. 3). Furthermore, the correlation between tree height and 222 DBH and knee roots in the middle water table was analyzed; the height of T. ascendens was positively 223 correlated with the number and size of knee roots (Table 2), and the DBH of T. ascendens was 224 significantly positively correlated with the number and size of knee roots. The number of underground 225 roots was positively correlated with the mean height and surface area of knee roots, and the weight of 226 underground roots was significantly correlated with the mean height and surface area of knee roots. 227

ACC content and ACC synthase and ACC oxidase enzyme activities 229
The ACC content in the knee roots at different development stages did not show significant 230 differences (P > 0.05): KY > KM > KO (Fig. 4A). However, the ACC content was significantly lower 231 in the knee roots than in the underground roots (P < 0.05). The ACC synthase activity showed the 232 same trend as ACC content and was not significantly different among the development stages of the 233 knee roots (Fig. 4B), and the ACC synthase activity was significantly lower in the knee roots than in 234 the underground roots (P < 0.05). The ACC oxidase activity in the knee roots at different development 235 stages was in the order of KY > KM > KO, and ACC oxidase reduced with the growth of knee roots 236 (Fig. 4C). The ACC oxidase activity was significantly higher in the knee roots than in the underground 237 roots, except in KO ( Fig. 4C; P < 0.05). 238

Endogenous hormone release rates 239
Different ethylene release rates were observed in the underground roots and knee roots (Fig. 5A). 240 The ethylene release rate at different stages was significantly higher in the knee roots than in the 241 underground roots (P < 0.05). In addition, significant differences were observed among the different 242 development stages of the knee roots (P < 0.05). The maximum ethylene release rate from the knee 243 roots was observed in KO, and the minimum, in KM. 244 The IAA content was in the order of UR > KM > KO > KY (Fig. 5B), with no statistically 245 significant differences between UR and KM (P > 0.05) and KO and KY (P > 0.05). 246

Anatomical structure of the knee roots and underground roots 247
The knee roots had 3-4 layers of rhytidome, which were formed by the integration of the 248 periderm and phloem, and some parts of the periderm had branches (Fig. 6). The periderm was 249 composed of many layers of cork cells, and the cork layer, cork-forming layer, and inner cork layer 250 were closely overlapped. The phloem, isolated from the periderm on the apex side of the knee roots, 251 was dead, the cells were broken, and the arrangement of cells was loose and porous. The phloem 252 parenchyma cells were close to the cambium and rectangular. The cell wall of the phloem fiber was 253 thickened and showed an increase in lignification. The phloem ray expanded obviously, and the 254 arrangement was loose. The knee roots were mainly composed of the secondary xylem; in the cross-255 section, xylem tracheids were arranged in order; the early tracheids were rectangular, square, or 256 polygonal; the late tracheids were obviously smaller than the early tracheids; and the cell wall was 257 thicker. The rhytidome layers of the underground roots were lesser than those of the knee roots (1-2 258 layers). The width of the phloem was smaller than that of the knee roots; the xylem tracheids were 259 arranged in order, and the shape was similar to that of the knee roots. 260

Discussion 261
In plants, flood tolerance is related to shifts in anatomical and morphological characteristics 262 Voesenek 1996, Hua et al. 2017). Under flooding conditions, the formation of knee roots is 263 a morphological adaptation of T. ascendens to environmental stress (Fig. 7). Taxodium ascendens with 264 knee roots had more underground roots and showed better tree growth, which suggests that the knee 265 roots are beneficial for the growth of T. ascendens. Our results suggest that the middle water table is 266 more suitable for the formation and growth of knee roots, which is consistent with the findings of 267 knee roots, knee roots at the young-aged stage showed the highest ACC oxidase activity, knee roots at 285 the old-aged stage may be less affected by flooding stress, and showed the lowest ACC oxidase 286 activity. When the underground roots of T. ascendens were exposed to flooding in the growing season, 287 the activity of ACC synthase was enhanced by anaerobic stress, which led to the accumulation of ACC. 288 When the water table receded, the upper surface of the roots distributed in the shallow soil received 289 oxygen, and the ACC was transported to better-aerated tissues. This resulted in ethylene synthesis by 290 ACC oxidase; ethylene promoted the uneven growth of morphologically upper and lower roots, 291 leading to the formation of knee roots. Thus, the knee roots had significantly higher ethylene content 292 than the underground roots (Fig. 5A), and the highest ethylene content was observed in the old-aged 293 stage of the knee roots. This is consistent with the results of Pesquet and Tuominen (2011), who 294 reported that the ethylene content was maximum before cell death and lignification. The knee roots 295 formed easily when the tree was exposed to anoxic conditions (reduction state) and aerobic conditions 296 (oxidation state) alternately. Thus, our results suggest that the high water table (the underground roots 297 experienced flooding from June to September, and the soil was in the reduction state during the 298 growing season) and low water table (no flooding throughout the year, and the soil was in oxidation 299 state) are not suitable for the formation of knee roots; only the middle water table (flooding period was 300 1 to 2 months from July to August, and the annual average water table was from -0.6 m to -1.2 m) is 301 suitable for the formation of knee roots. Moreover, ACC and some related enzymes were found in the 302 knee root tissues, suggesting that the formation and development of T. ascendens knee roots are 303 related to ethylene production and accumulation. 304 The phytohormone IAA is essential for root development and adventitious root formation (Visser 305 and Voesenek 2004, Kitomiy et al. 2008. IAA also plays an important role in the development and 306 activities of the cambium (Uggla et al. 1998, Bhalerao andFischer 2014). Our study suggests that, 307 with the development of the knee roots, the IAA content first increased and then decreased; IAA 308 content was lower in the knee roots at the young-aged and old-aged stages and higher in those at the 309 middle-aged stage. High IAA content is beneficial for cambium cell enlargement, expansion, and . In our study, the cells of the periderm at the apex of the knee roots were dead, arranged 320 loosely, and had a large number of intercellular spaces, which is conducive to gas exchange between 321 the knee roots and air. Because the knee roots were exposed to air to resist the adverse effects of 322 external environmental factors, the periderm of the knee roots was obviously thicker than that of the 323 underground roots. Plant stems can form irregular wide rays composed of abnormally large ray cells 324 after the application of ethephon (Pallardy 2011). Because the knee roots were stimulated by ethylene, 325 phloem rays at the apex of the knee roots expanded obviously when compared with the underground 326 roots. Thus, the flooding resistance of T. ascendens is related to the formation of knee roots and 327 enhancement of air permeability. Although this study showed that ACC, ethylene, and IAA affected 328 the formation of knee roots, the underlying molecular mechanism is still unclear. We recommend the 329 transcription factors and gene expression of T. ascendens roots should be explored to further 330 understand the mechanism of flooding resistance. 331

Conclusions 332
Our results suggest that the formation and distribution of knee roots are significantly affected by 333 the water table. The middle water table significantly enhanced the formation and distribution of knee 334 roots in T. ascendens. Furthermore, the knee roots were beneficial for the growth of T. ascendens, and 335 the height and the DBH of T. ascendens were positively correlated with the number and size of knee 336 roots. The ACC content and ACC synthase activities in the knee roots at different development stages 337 did not show any significant differences, whereas they were significantly lower in the knee roots than 338 in the underground roots. The ACC oxidase activities in the knee roots decreased as the knee roots 339 developed. Ethylene and IAA affected the formation of knee roots. The ethylene release rate was 340 significantly higher in the knee roots than in the underground roots, and the IAA content first 341 increased and then decreased with the development of the knee roots. The anatomical structure of the 342 knee roots showed that cells of the periderm at the apex of the knee roots were dead, arranged loosely, 343 and had a large number of intercellular spaces that improved internal gas diffusion. In conclusion, 344 seasonal flooding induced the production of endogenous hormones, resulting in the formation of knee 345 root, which improved root respiration and ventilation, thus improving the flooding tolerance of T. 346

Conflicts of Interest 348
The authors declare no conflict of interest.    Horizontal line = 100 μm. 1, Rhytidome at the apex of the knee roots (middle stage); 2, rhytidome of 544 mid-sized underground roots; 3, phloem at the apex of the knee roots (middle stage); 4, phloem of 545 mid-sized underground roots; 5, xylem at the apex of the knee roots (middle stage); 6, xylem of mid-546 sized underground roots; rh: rhytidome; pe: periderm; ph: phloem; pr: phloem ray; pf: phloem fiber; 547 pc: phloem parenchyma cell; wr: wood ray.    Weight of underground roots with or without knee roots Values followed by the same letter(s) are not signi cantly different at P < 0.05, according to Duncan's multiple range tests. Error bars are standard error of the mean; n = 3. ACC content and ACC synthase and ACC oxidase activities in different roots of Taxodium ascendens (KO, KM, and KY refer to the knee roots at different development stages: old-aged stage, middle-aged stage, and young-aged stage, respectively. UR means underground roots.). Values followed by the same letter(s) are not signi cantly different at P < 0.05, according to Duncan's multiple range tests. Error bars are standard error of the mean; n = 3.

Figure 5
Ethylene release rates from different root types of Taxodium ascendens Figure 6 Transverse anatomical structure of knee roots and underground roots Horizontal line = 100 μm. 1, Rhytidome at the apex of the knee roots (middle stage); 2, rhytidome of mid-sized underground roots; 3, phloem at the apex of the knee roots (middle stage); 4, phloem of mid-sized underground roots; 5, xylem at the apex of the knee roots (middle stage); 6, xylem of mid-sized underground roots; rh: rhytidome; pe: periderm; ph: phloem; pr: phloem ray; pf: phloem ber; pc: phloem parenchyma cell; wr: wood ray.

Figure 7
Knee roots of Taxodium ascendens in the wetland