2.1 Structural of the Sinojackia xylocarpa Hu drupe and seed
The drupe of Sinojackia xylocarpa Hu is oblong shape, with a dense pale brown lenticels surface, a pointed conical tip, a blunt circular fruit handle, and a thick cork. From the structural point of view (Fig. 2Ⅰ), the drupe can be divided into exocarp, mesocarp, endocarp, outer seed coat, inner seed coat, endosperm[7]. The seed of Sinojackia xylocarpa Hu has a hard shell, thick membranous coat and brown color, contains 1-3 pieces of fine-column seed embryos. The seed tip is long conical, and the seed tail is pointed conical. A thin layer of the inner seed coat, light brown, tightly wrapped in the endosperm. From the structural point of view (Fig. 2Ⅱ), the seed can be divided into outer seed coat, inner seed coat, endosperm, cotyledon, hypocotyl, radical.
2.2 Tissue observation in scanning electron microscopy (SEM)
Observed by SEM, the tip of the drupe is raised (Fig. 3A). The fruit handle has an obvious hole, and the hole is associated with water entry into drupe (Fig. 3B)[8]. The surface of the exocarp is an irregular scaly stratum corneum composed of densely packed thick-walled cells. There are a small number of minute cracks in the recessed portion (Fig. 3C)[9]. The mesocarp is grayish-white, and the surface is mostly linear, semi-circular and torn, composed of large parenchyma cells (Fig. 3D)[10]. The endocarp consists of honeycomb-shaped parenchyma cells. The cells are large and relatively obvious. They are loosely arranged, with pores connected in the middle, with lateral vascular bundle distribution, low degree of lignification, and a highly elongated palisade tissue layer. The cells are arranged loosely and have distinct cell gaps (Fig. 3E).
The shell is elongated, gray, boney, like a jujube nucleus, with a highly corked surface, and uneven, irregular longitudinal edges, which are difficult to separate from the endocarp. The surface cells are papillary bulges, arranged neatly and tightly. The outer cells are fibrous thickened. The inner cells develop into thick-walled tissues, and the shell is obviously thickened from the outer to inner cell walls (Fig. 3F)[11]. The seed coat is thick and membranous, with a tan color, densely arranged cells, uneven surface, and tightly close to the shell. The membranous seed coat has a strip-like fibrous tissue, which interlaced and arranged in a woven pattern and has a fine structure, with fine cracks and small holes on the surface of the seed coat (Fig. 3G)[12]. The outer surface of the endosperm has an irregular quadrilateral shape, which is closely arranged, the inner surface of the endosperm is uneven, and the cells are highly dense (Fig. 3H).
2.3 Spatial distribution of water protons
MRI was used to measure the spatial distribution of water in imbibition Sinojackia xylocarpa Hu drupe, seed and embryo (Fig. 4Ⅰ, 4Ⅱ, 4Ⅲ)[13]. MR images were obtained in axial orientations, coronal, which are parallel to the embryonic axis. The images presented in Fig. 4Ⅰ, 4Ⅱ, 4Ⅲ are limited to one 2D median slice in the coronal. The slices were taken as a series of sections from a single sample at a given time.
2.3.1 Spatial distribution of water protons in Sinojackia xylocarpa Hu drupe
Fig.4ⅠA shows MR images of dry Sinojackia xylocarpa Hu drupe. The initial moisture content of the mature dry drupe was 22.46% and can readily see in the coronal plane. The water distribution is inhomogeneous, and mainly distributed in the vascular bundle and embryonic axis. After 4h of soaking, an intense water signal was observed in the fruit handle. Water moves initially through the fruit handle hole, then along the vascular bundle and endocarp enters the drupe. In images obtained after 13h,20h,28h,38h,48h,60h and 72h of imbibition, the water gradually penetrates into the mesocarp and exocarp[14]. An intense water signal was observed in the fruit tip after 82h of steeping. Then it was continued to imbibition for 97h,120h,138h,161h and 185h, moisture migrated from the fruit handle to the tip, while the water continued to permeate and move along the vascular bundle to the mesocarp and exocarp on both sides. During the next time of imbibition 21d, the unwetted area of the peel and the seed gets smaller and disappears completely after 31d. But the seed coat always has no red water signal. (Because of the use of AB glue to fix the drupe, the water can’t enter the small part of the right side of the peel.)
Fig.4ⅡA(0h) shows MR images of dry Sinojackia xylocarpa Hu embryo were taken from the MR images of the drupe and amplified. From the longitudinal section of 0h, the initial moisture of the embryo is mainly distributed along the center of the embryonic axis. When inflated for 7B(4h), the radicle and hypocotyl showed a red water signal. When the swelling was 7C(13h), the cotyledon showed a red water signal. After7D(20h), 7E(28h), 7F(38h), 7G(48h) swelling, the whole embryo showed obvious red water signals. After 7H(60h), 7I(72h), the water enters from the radicle and the small hole in the lower right end of the endosperm. After swelling for7J(82h), 7K(97h), 7L(120h), 7M(138h), 7N(161h), 7O(185h), the whole embryo has a significant red water signal, and the volume of the embryo is gradually increased largely. After inflating 7P(21d) and 7Q(31d), the embryo was enlarged to the entire embryo cavity, and the channel of moisture entering the lower right side of the endosperm is visible.
Fig.4ⅢA(0h) shows MR images of dry Sinojackia xylocarpa Hu seed. From the coronal plane, the initial moisture of the dry seed is mainly distributed in the embryo, sporadically distributed in the seed tip and tail, which was 7.67%. After 9B(4h),9C(13h), 9D(20h), 9E (28h), 9F(38h)and 9G(48h)of soaking, it can be seen that water entered from the seed tail and the seed tip, and the red water signal area of embryo of the seed cavity increased significantly. Water entered from seed tip penetrated into cotyledon, while water entered from seed tail, then moved to the hypocotyl from both the right lower pore of the endosperm and the radicle. After 9H(60h), 9I(72h), 9J(82h) and 9K (97h) of imbibition, the red water signal of the influent channel of the seed tail and the seed tip were obvious. After swelling for9L(120h), 9M(138h), and 9N(161h), the water gradually fills the seed cavity, and the gap between the seed coat and the endosperm. After 9O(185h) of inflating, the whole seed cavity, and the gap between the seed coat and the endosperm was completely filled with water. The seeds swelled obviously and filled the entire seed cavity. After 9P (21 d) and 9Q (31 d) of imbibition, the seed tail, seed tip, water intake channel, and the whole seed cavity all showed an extremely strong red water signal. But the seed shell always has no water signal. However, the seed shell does not have any water signal during the whole imbibition process.
Figure 5ⅠA shows the change of SNR in different areas of the drupe during 31d imbibition time. Changes in the intensity of the MRI SNR data generally show three distinct stages of the imbibition: an imbibition phase I, an imbibition phase II and a saturation phase. Five areas in the MR images were integrated separately in Figure 5ⅠA: the fruit handle region (black solid square data set), the exocarp region (red solid circle data set), the mesocarp region (blue solid triangle data set), the endocarp region (green solid inverted triangle data set), and the tip region (purple solid rhombus data set). The SNR intensity of the five parts of the drupe was compared as follows: exocarp >fruit handle hole > endocarp> fruit tip> mesocarp. Finally, the SNR values of the four areas are all greater than 0.5dB.
Figure 5Ⅰ A1: the imbibition phase I (0-60h), the SNR value of exocarp showed a sharp rise after a brief rise, then rises quickly, the SNR value of mesocarp and endocarp generally showed a slow upward trend; the imbibition phase II (60h-185h), the SNR values of exocarp experienced two rises and falls, the SNR values of mesocarp and endocarp continued to rise slowly; the saturation stage (185h- 504h), quickly the SNR values of exocarp dropped to saturation and slowly increased after saturation, the endocarp SNR value rises slightly and remains saturated, the exocarp SNR value continued to rise to saturation, and slowly decrease after saturation (504h-744h).
Figure 5Ⅰ A2: the imbibition phase I (0-48h), the SNR value of fruit handle and tip rising sharply; the imbibition phase II (48h-185h), after a sharp decline, the SNR values of fruit handle rises sharply to saturation, the SNR values of tip rise after falling, then fall to saturation, the saturation stage (185h- 744h), after the fruit handle and tip SNR values reach saturation, they begin to decrease.
Figure 5Ⅱ B shows the change of SNR data in different areas of the seed during 31d imbibition time. Five areas in the MR images were integrated separately in Figure10A. Figure10A: the seed tail region (black solid square data set), the embryo cavity tail region (red solid circle data set), the shell region (blue solid triangle data set), the embryo cavity tip region (green solid inverted triangle data set), the seed tip region (purple solid rhombus data set).The SNR intensities of the five regions during imbibition are compared as follows: embryo cavity tail > seed tail > embryo cavity tip > seed tip. The SNR data of seed coat was always low, basically maintained at around 0.1dB, showed that the seed coat has almost no water absorption.
Figure 5Ⅱ B 1:the imbibition phase I (0-97h), the seed tail SNR value increased uniformly, the seed tip SNR value increased rapidly; the imbibition phase II (97h-185h), the SNR values of both increased uniformly; the saturation stage (185h- 504h), the SNR values of the seed tail and seed tip both quickly rise to saturation, and slowly decrease after saturation (504h-744h).
Figure 5Ⅱ B 2: the imbibition phase I (0-60h), the embryo cavity tail SNR value starts to fall and rise, then fall and rise rapidly, the embryo cavity tip SNR value begins to rise and fall, then rise; the imbibition phase II (60h-185h); After falling, the SNR values of embryo cavity tail and tip rises again, then falls again and rises to saturation; the saturation stage (185h- 504h) the embryo cavity tail SNR value rises slowly, the embryo cavity tail SNR value keeps falling. After the SNR values of embryo cavity tail and tip reached saturation, they continuously rising.
Figure 5 Ⅲ C shows the change of SNR in different areas of the embryo in the drupe during 31d imbibition time. Four regions of the embryo MR images are integrated into Figure 4Ⅱ C respectively: the radicle region (black solid square data set), the cotyledon region (red solid circle data set), the endosperm region (blue solid triangle data set), the shell region (green solid inverted triangle data set).The SNR intensities of the four regions during imbibition are compared as follows: radicle > cotyledon > endosperm > seed coat. All inflation time displayed that the SNR values of the radicle and cotyledon areas are always above 0.6dB, and the SNR values of the endosperm and seed coat areas are always below 0.4dB.
Figure 5 Ⅲ C 1:the imbibition phase I (0-97h), the SNR values of radicle and cotyledon rise sharply and then drop rapidly, and then rise sharply; the imbibition phase II (97h-185h), the SNR values of radicle and cotyledon decreased and then rose, the saturation stage (185h- 504h), the SNR values of radicle and cotyledon continued to rise to saturation, and quickly decrease after saturation (504h-744h).
Figure 5 Ⅲ C 2: the imbibition phase I (0-48h), the SNR values of endosperm and shell are on the rise; the imbibition phase II (48h-161h), the SNR values of endosperm and shell are decreased, rise, fall and rise again; the saturation stage (161h- 504h), the SNR values of endosperm keep falling, the SNR values of shell decline slowly, after the endosperm and shell SNR values reach saturation, they begin to increase. (504h-744h). The SNR values endosperm and shell are always low, the SNR data of the endosperm is maintained between 0.15 dB and 0.4dB, and the SNR data of the shell is always below 0.2dB.
Figure 5 Ⅳ D shows the change of SNR in different areas of the embryo in the seed during 31d imbibition time. Four areas in the MR images were integrated separately in Fig.4 Ⅳ D respectively: the radicle region (black solid square data set), the hypocotyl region (red solid circle data set), the cotyledon region (blue solid triangle data set), the endosperm region (green solid inverted triangle data set). The SNR intensities of the four regions during imbibition are compared as follows: hypocotyl > cotyledon > radicle > endosperm.
Fig.5 Ⅳ D 1:the imbibition phase I (0-48h), the radicle SNR value rises and falls, then rises, the cotyledon SNR value rises after falling, the endosperm SNR value drops rapidly after a rapid rise; the imbibition phase II (48h-185h), after three rises and then dropped mode, the radicle SNR value reached to saturation, the cotyledon SNR value rises after two rising and then falling mode to saturation, the endosperm SNR value rises to saturation after a sharp drop; the saturation stage (185h- 504h), the SNR values of the radicle, cotyledon and endosperm both continue(s) to drop, and quickly increase after saturation (504h-744h).
Fig.5 Ⅳ D 2: the imbibition phase I (0-48h), the hypocotyl SNR value rises after continuous decline, the cotyledon SNR value rises after falling, the endosperm SNR value drops rapidly after a rapid rise; the imbibition phase II (48h-185h); the hypocotyl and cotyledon SNR value after two rising and then falling mode to saturation, the endosperm SNR value rises to saturation after a sharp drop; the saturation stage (185h- 504h), the SNR values of the hypocotyls, cotyledon, and endosperm both continue(s) to drop, and quickly increase after saturation (504h-744h). In different periods of inflation, the SNR data changes of the hypocotyl, cotyledon and radicle are basically the same. The SNR data of the endosperm never exceeds 1.1dB.
2.4 Tissue observation by paraffin sections
Paraffin sections were used to observe the morphological structure of the cells in the peel, shell, and endosperm. The tip consists of epidermis, cork layer and cortex. The epidermis is regularly distributed with lenticels and highly lignified. The parenchyma cells of different sizes in the cortex are closely packed. The tip consists of epidermis, cork layer and cortex. The epidermis is regularly distributed with lenticels and highly lignified. The parenchyma cells of different sizes in the cortex are closely packed(Fig.6A). The fruit handle is made up of closely packed thin-walled cells containing green cellulose cell walls(Fig.6B). The epidermis of the exocarp is distributed with lenticels and keratinized(Fig.6C). The mesocarp is composed of parenchyma cells of different sizes and shapes. The vascular bundle composed of numerous small cells extends into the parenchyma cells of the endocarp(Fig.6D). The shell consists of closely arranged skeletal stone cells and round thick-walled cells of varying sizes, presenting a highly corked red color(Fig.6F).
The endosperm is made up of closely packed and different sizes and shapes thin-walled cells containing green cellulose cell walls. The endosperm cells contain darkly colored round particles, and the number of particles in a single cell is different(Fig.6G). According to the determined experiment by further oil red O fat stain, iodine-potassium iodide starch stain, periodic acid-Schiff polysaccharide stain, we found that the starch content and the fat content were high, but less sugar. The fat has high hydrophobicity, and the accumulation of a large amount in the cells does not increase the osmotic potential of the cells, which hinders the free exchange of water inside and outside the embryo, thereby hindering the material metabolism of the endosperm. In addition, dense endosperm cells create severe mechanical barriers. It can be seen that the permeability problem and mechanical obstacle of the endosperm may be an important reason for the embryo fails to germination.
2.5 Tissue observation in transmission electron microscopy
The hydration of Sinojackia xylocarpa Hu seeds may be related to changes in the content of endosperm cells. In order to detect the endosperm cell contents, the ultrastructural characteristics of endosperm cell contents of dry Sinojackia xylocarpa Hu seeds were compared by transmission electron microscopy (TEM)[15]. Electron microscope observation shows that the inner wall cells of the endosperm at the radicle position contained a large number of high electron-dense substances and lipid droplets, and their shape and size are different. The volume of a high electron-dense substance is larger than that of lipid droplets, and the lipid droplets are densely arranged. The high electron density substances in the outer wall cells of endosperm at radicle position are smaller, the lipid droplets are arranged tightly, and high electron-dense substances are also present between the lipid droplets.
Compared with the inner wall cells of the endosperm at the radicle position, the high electron-dense substance of the inner wall of the endosperm in the middle position is larger, accounting for 1/2 of the cell volume, and the lipid droplets are densely arranged. The high-electron dense substance in the outer wall cells of the endosperm in the middle part is small, and the volume is relatively small, the lipid droplets vary in size, arranged neatly, and high electron-dense substances are also present between the lipid droplets.
It can be seen that the endosperm contains a large number of lipid droplets and high electron-dense substances, which may have certain mechanical obstacles to the germination of the embryo.