Identification of the initial water-site and movement in Gleditsia sinensis

1 seeds and its relation to seed coat structure 2 Mingwei Zhu, Song Dai, Qiuyue Ma and Shuxian Li* 3 1 The Key Lab of Cultivar Innovation and Germplasm Improvement of Salicaceae, College of Forestry, Nanjing 4 Forestry University, Nanjing 210037, China; zmw1994@njfu.edu.cn (M.Z.) 5 2 Institute of Forestry Scientific Research and Technology Extension, Tongren Academy of Sciences, Tongren 6 55430, China; 717977709@qq.com (S.D.) 7 3 Institute of Leisure Agriculture in Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China; 8 yue.870808@163.com (Q.M.) 9 * Correspondence: shuxianli@njfu.com.cn; Tel.: +86-025-8542-7403 10 The mechanism of water imbibition in Gleditsia sinensis seed 11

Studying water uptake and distribution during the rapid initial uptake stage is essential to Fig. 1 Water absorption curves for intact and manually cracked Gleditsia sinensis seeds with duration of soaking.

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Error bars represent the standard deviation.

Effect of hot water treatment on seed coat impermeability 90
The impact of water temperature on seed coat impermeability is summarized in Table 1. The 91 percentage of swollen seeds in the control group was 3.8%, which indicated that the hard seed coat 92 strongly inhibited water absorption. The hot water treatments increased the permeability of the 93 seed coat. With increase in water temperature, the percentage of swollen seeds increased 94 significantly. The swollen percentage in the 70, 80, and 90 °C treatments increased significantly to 95 38.6%, 82.8%, and 91.4%, respectively, but the difference between the 80 and 90 °C treatments 96 was not significant. The viability of the control seeds was 91.3%, which was higher than the 97 viabilities of 89.3% and 87.3% when the seeds were immersed in water at 70 and 80 °C , 98 respectively, but the differences in viabilities among these treatments and the control were 99 nonsignificant. The viability of seeds soaked in 90 °C water plummeted to 57.0%. Thus, high 100 water temperatures aided imbibition but an excessively high water temperature caused a degree of 101 damage to the seeds. In this experiment, water with an initial temperature of 80 °C was optimal to 102 break the seed coat impermeability of G. sinensis seeds. 103 Table 1 Effects of hot water treatments to break the seed coat impermeability on percentage of 104 swollen seeds and viability of Gleditsia sinensis seeds 105 107

Ultramorphological characteristics of the seed coat 108
Stereomicroscopic observation revealed that the G. sinensis seed shape is oblong with a distinct 109 dorsoventral asymmetry. The hilar region is flat on one side of the seed and consists of the 110 micropyle, hilum, and lens, which are linearly aligned ( Fig. 2A). The entire seed, especially the 111 micropyle and hilum, are covered with a thick layer of waxy substances (Fig. 2B), which can 112 prevent water absorption of the seeds. Observation of the longitudinal section revealed that the 113 embryo was encased by a thick nucellar-endosperm casing (Fig. 2C). The colloidal endosperm is 114 distributed unevenly in the seed, with higher concentrations in the dorsoventral regions and lower 115 concentrations in the two lateral sides, the radicle, and the chalazal region ( Fig. 2D-F).

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SEM images of the seed coat surface revealed many intersecting vertical and horizontal cracks, 122 including in the hilar region ( Fig. 3A-C). After breaking seed coat impermeability, an area of the 123 seed coat was detached at the lens (Fig. 3D). The seed coat cracks of permeable seeds were deeper 124 than those of the control, and internal tissues were observed through the cracks (Fig. 3C, D). No 125 other visible differences to the seed coat were observed between impermeable (control) and 126 permeable seeds.  layer of cells became more loosened than in the control, especially those away from the 146 D C micropylar side (Fig. 4C, F). A linear narrow channel which connected the lower side of the cavity 147 and the inside of the seed coat was found (indicated by green arrow, Fig. 4G). The cuticle layer 148 ruptured near the hilar region and the layer of palisade cells in the lens was raised (Fig. 4H).

Tracking the imbibition pathway by TTC staining 156
Dye-tracking tests, such as TZ staining, can be used to locate the site of water entry into a seed 157 [11]. The principle of TZ staining is that the dehydrogenases responsible for the staining reduction 158 are only active in the living cells of imbibed seeds. When a colorless TTC solution is imbibed by 159 the seed, it will accept hydrogen from the dehydrogenases, producing a red, stable, and 160 non-diffusible substance. The non-imbibed tissues cannot stain red, even though the cells are 161 living, thus the color change in a viable seed distinguishes the tissues that have been imbibed. 162 As indicated by the control permeable seed (Fig. 5A), the embryo color before dying was 163 yellow. After 6 h of incubation in TTC solution, red staining was first observed at the tip of the 164 radicle (Fig. 5B). After staining for 12 h, the entire radicle and the right side of the adaxial 165 cotyledon (which is under the lens) were stained red, whereas the left side was not stained red ( Fig.  166 5C). Subsequently, red staining expanded along each lateral side to the chalazal of the cotyledons 167 ( Fig. 5D), and the red-stained area of the cotyledon on the hilar side (right side) was significantly 168 larger than that on the other side. At 48 h, the entire lateral region of the cotyledon was stained and 169 H G the stain area gradually penetrated to the central axis of the cotyledon, but the majority of the 170 central cells of the cotyledon were unstained (Fig. 5E). After sufficient imbibition, the radicle 171 elongated slightly and the entire embryo was stained red (Fig. 5F). These results indicated that the 172 initial site of water absorption was the tip of the radicle. The direction of water migration in the 173 seeds was from the radicle towards the cotyledon apices along the lateral sides of the cotyledons. 174 Subsequently, water entered the central cells of the cotyledons. After 12 h, the first area of the 175 cotyledon to be stained was on the left side, but the red-stained area on the right side of the 176 cotyledon was more extensive than that on the left side after staining for 24-48 h. This trend can 177 be explained by the presence of a vascular bundle on the side of the hilar region. 178   the seed volume increased significantly, so 13-20 layers of the seeds at each stage were scanned 186 by MRI. And in this paper, for each stage we only showed two layers (coronal and sagittal plane) 187 of MRI images (Fig. 6). In the mature seed (0 h), the seed was presented as black (Fig. 6A, a) 188 which inferred that the water content or the hydrogen proton count was extremely low. After 4 h 189 imbibition, a bright spot at the cavity region under the micropyle appeared (Fig. 6b), which 190 suggested that the initial water absorption site was the micropyle. After imbibition for 8 h, the seed 191 coat of the hilar region was imbibed noticeably (Fig. 6C) and the bright area of the right side 192 (indicated by a yellow arrow) was larger and brighter than that on the left side. According to the 193 anatomy of the seed coat, we know the brighter parts are the narrow space between seed coat and 194 endosperm. Also, at this time the intersection of the radicle tip, the endosperm, and the cotyledon 195 became brighter (indicated by a red arrow). At 12 h, the intensity of water signal of the entire 196 radicle increased significantly. Meanwhile, the signal at the intersection of the endosperm and the 197 cotyledon was enhanced further. Therefore, for G. sinensis seeds, after water entered the seeds 198 through the micropyle, two water movement paths were found: migration along the space between 199 the seed coat and the endosperm to the chalazal (Path Ⅰ); migration from the embryonic axis to the 200 cotyledons (Path Ⅱ) with considerable swelling of the seeds. Up to 20 h, from Fig. 6E, we found in 201 the right side of the intersection between the endosperm and the cotyledons, the speed of water 202 movement was still significantly quicker than that of the other side since a bright signal was 203 observed in a larger area at the chalazal in the right side. From this map we also found that the 204 embryonic axis exhibited very strong signals, but water migration through Path Ⅱ was far slower 205 than that through Path Ⅰ because most part of the cotyledon was black. At 24 h, the signals at the 206 intersection between endosperm and the cotyledons of both sides had arrived to the chalazal. 207 When the seed was imbibed for 48h, the plumule had been elongated as shown in the picture (Fig.  208   6g). In Fig. 6G, we can see the vascular clearly in the seed coat (indicated by a blue arrow) for the 209 exhibited bright signal. Before 48 h, the vascular should have exhibited a bright signal but we 210 were unable to confirm this phenomenon during scanning. After imbibition for 96 h (Fig. 6H, h), 211 water continued to enter into the cotyledons, and the cotyledons were brighter than before, but the 212 seed coat was still black.

Water absorption by blocking different regions of the seed 218
The water absorption rate in treatment I (seed coat surface was blocked and submergence in water 219 for 144 h) was 8.6%, which was considerably lower than that of the control (Fig. 7). Thus, 220 Vaseline™ effectively prevented seeds from absorbing water. Therefore, it was feasible to 221 determine water absorption in different regions of the G. sinensis seed coat by applying 222 Vaseline™ to block water uptake in specific regions. 223 The water uptake rate in treatment V (no blocking) was significantly higher than that of the 224 three blocking treatments throughout the imbibition period. At 8 h, seed water absorption in 225 treatment V attained 19.8%, which was significantly higher than that of treatment II (10.5%). The 226 increase in water absorption in treatments III and IV (2.1% and 1.5%, respectively) was 227 significantly lower than that of treatment II. These results indicated that the hilar region was the 228 initial site for water absorption. At up to 32 h, seeds of treatment V maintained the most water 229 absorbed of 130.4%. Absorption in treatment II also increased rapidly to 70.7%. The absorption 230 rates of treatments III and IV were 18.8% and 5.8%, respectively, which differed significantly. 231 Thus, the rate of water absorption in treatment III increased more rapidly than that of treatment IV. 232 Therefore, after the initial main phase of water absorption in the hilar region, the medial region of 233 the seed began to uptake water, whereas little water was absorbed by the distal region. By 84 h, 234 the seeds in treatment V attained a plateau phase and the amount of water absorbed was 179.3%. that hot water treatment increased germination in Astragalus hamosus, and that a water temperature exceeding 80 °C was lethal to seeds [16]. In the present experiment, the optimal 255 treatment to break the seed coat impermeability of G. sinensis seeds was soaking in water at an 256 initial temperature of 80 °C for 5 min. It was also concluded that seed viability was sensitive to 257 water temperature. Kang proposed that 100 °C hot water treatment was the optimal temperature to 258 break hardseededness and the seed still showed a high germination rate [17]. However, in the 259 present study, 90 °C treatment reduced seed viability to 57.0%. 260

Seed coat structure and its relation to water absorption 261
Stereomicroscopic observation revealed that the entire seed coat of G. sinensis was covered with 262 waxy substances that may prevent water from entering the seeds. The hilum region of mature G. 263 sinensis seeds was coated with a thicker layer of wax and the micropyle was sealed. These 264 characteristics may be responsible for the impermeability of the seed coat. After hot water 265 treatment, SEM images showed that the wax in the hilum region was reduced or disappeared. The 266 micropyle was opened and cracks in the seed coat were wider and deeper (Fig. 4E), and the cuticle 267 in the hilar region was ruptured. These structural changes may improve the permeability for seed 268 imbibition. An interesting phenomenon observed in the present study was that a large piece of the 269 seed coat at the lens had detached and the layer of palisade cells at the lens was raised (Fig. 4F). 270 Although the degree of damage to the lens was more severe than that to the micropyle, MRI maps 271 showed that the lens of G. sinensis seeds was not the first site to imbibe water. Thus, we could not 272 identify the water-gap from morphological changes by itself in the seed coat after physical 273 dormancy was broken. 274 Structural studies of impermeable seeds are important to better understand the causes and 275 suitable dormancy release methods when subjected to treatments for dormancy release [18,19].
Previous research has shown that water impermeability is due to the palisade layer [20]. 277 According to Geisler et al. [21], resistance to water absorption by Leguminosae seeds is primarily 278 due to water-impermeable substances in the palisade cells. Baskin [22] stated that the heavily 279 lignified cell walls in the palisade layer may also render the seed impermeable to water. In some 280 species, the palisade layer of the seed coat contains a light line, which is comprised of a high 281 concentration of callose, a water-repellent substance that also contributes to seed coat 282 impermeability [21-23]. Therefore, the presence of the palisade layer and the light line may be 283 associated with the hardseededness and impermeability of the seed coat of G. sinensis. 284 In the present study, a vascular bundle, a distinctive structure in Leguminosae seeds, was 285 observed in G. sinensis seeds that connected the hilum to the lens. de Paula et al. [24] concluded 286 that the vascular bundle was associated with movement of water in Senna macranthera seeds. A 287 vascular bundle was also reported in seeds of Lupinus luteus [8] and S. multijuga [19]. From the 288 maps of anatomy, it can be speculated that some other structures are also beneficial for water 289 migration within permeable seed. For example the sclerenchymatous cells becomes loose in the 290 left side of the seed around the micropyle. Between the lower side of the micropyle cavity and the 291 inner seed coat, a linear channel exists, which may be helpful for water enter into radicle. 292

Location of water entry and the pathway of water movement in the seeds after reduction of 293 seed coat hardness 294
Dye-tracking experiments like the TZ test have been used to identify the migration of water in the 295 seeds of some plants. In this study we found that the radicle was the first region to stain red. The 296 radicle is adjacent to the micropyle, therefore we suspected that the initial imbibition site of G. 297 sinensis seeds was the micropyle. However, owing to methodological limitations, water movement in the seed coat and endosperm cannot be assessed directly. A fundamental basis of the TZ test is 299 that only living cells can stain red. The seed coat and colloidal substances of the endosperm 300 cannot stain, thus water movement in the seed coat and endosperm could not be detected. In 301 addition, this method is labor-intensive, time-consuming, destructive, and requires monitoring 302 several seeds to allow for variation among seeds. 303 The MRI technique, which is a non-destructive, real-time visualization technology, effectively 304 overcomes the aforementioned shortcomings by acquiring spatial and proton mobility information 305 in biological systems [25]. The technology has been widely used to study seed imbibition [8, 306 26-28]. In the present research, the MRI maps acquired at 4 h imbibition ( Fig. 6- in Delonix regia. These results indicated that the primary site of water entry into seeds after 313 permeabilization of the seed coat varied among species. 314 The MRI maps also enabled us to determine water movement in G. sinensis seeds. When water 315 entered into the seed, the signal strength was enhanced gradually. The scanner maps at 8 h 316 indicated that water entered into the seed from the micropyle, was stored in the cavity, then 317 migrated through the narrow channel indicated by the green arrow in Fig 4G, and finally split  318 along two paths where some water migrated along the Path Ⅰ to the chalazal region and the other 319 water reached the embryonic axis and spread into the cotyledons through Path Ⅱ. of the seed (Fig. 5, 6D). These findings clearly certified that the vascular bundle is an important 322 structure for water movement. In addition to the seed coat and embryo, G. sinensis seeds contain a 323 colloidal endosperm which is composed predominantly of galactomannan [31]. The MRI maps at 324 24 h revealed that the bright signal of 1 H protons in the endosperm had reached the chalazal region, 325 but nearly half of the cotyledons did not exhibit water signal. Thus, the endosperm may prevent 326 water from entering the seed and hinder the early imbibition process. 327 The quantitative results of the blocking experiment confirmed that the hilar region played an 328 important role in imbibition by water absorption during the initial 8 h. Based on the results in 329 treatment III and Ⅳ, the medial region and the bottom of the chalazal region also showed 330 capability for imbibition at 144 h, however, water absorption was substantially less than that of the 331 hilum region. In G. sinensis seeds, the micropyle, hilum, and lens are in overly close proximity to 332 differentiate with the naked eye and reliably block them individually. From the presently applied 333 methods we can conclude that initial imbibition occurred in the hilar region, but we were unable to 334 quantify which structure was the primary water-gap for water absorption. 335 Three methods of identification of the route of water entry into seeds of G. sinensis seeds were 336 used in this study, however, none of these studies clearly documented water movement in the seed 337 coat, and thus further study is required. 338

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In our study we found that 80 °C water was optimal to break hardseededness of G. sinensis, which 340 is an effective and convenient method. SEM images revealed that the palisade layer and light line 341 in the seed coat can hinder water entry into the seed. When hardseededness was broken, the micropyle was the initial site for water imbibition. Water entered the seed through the micropylar