2.3 Spatial distribution of water protons
MR imaging was used to measure the spatial distribution of water in imbibition control seed and H2SO4 seed (Fig. 4A 4B). The images presented are limited to one 2D median or near-median slice in the axial planes and one superficial slice in the coronal plane. The slices were taken as a series of sections from single seed at a given time (Gruwel, M. L. etc.,2002 ; Gruwel, M. L. etc.,2004).
Figure 4A1 (0 h) shows that the initial water content in the control seed is mainly distributed in the hypocotyl. AfterA2(4 h) of soaking, water enters radicle through micropyle. After A3(13 h), A4(20 h), A5(28 h), A6(38 h), A7(48 h) and A8(60 h) of imbibition, moisture enters the cotyledons from the hilum and continues to enter radicle and hypocotyl through micropyle. After soaking for A9(72 h), A10(82 h), A11(97 h), A12(120 h), A13(138 h), A14(161 h) and A15(185 h), the red water signal in the cotyledons, radicles and hypocotyls is more pronounced, and obvious red water signals begin to appear on both sides of the endosperm. After imbibition for A16(42d), A17(46d), A18(51d), A19(57d), A20(63d) and A21(71d), the red water signals on the cotyledons, radicles, hypocotyls, and outside endosperms are more apparent, but there is no water signal on the inside of the endosperm.
Figure 4B1 (0 h) shows that there was no obvious water signal in the H2SO4 treated seeds. After 3B2 (2 h), 3B3 (4 h), 3B4 (6 h), 3B5 (8 h), 3B6 (14 h), 3B7 (23 h) and 3B8 (30 h) of soaking, water enters the radicle through the micropyle. After imbibition for B9 (38 h), B10 (48 h), B11 (58 h), B12 (70 h), B13(82 h), B14 (92 h), B15 (107 h), B16 (130 h), B17(148 h), B18 (171 h) and B19 (195 h), water enters cotyledons through the hilum, and penetrates into endosperm on both sides through micropyle and hilum. After soaking for B20(21d), B21(31d), B22(35d), B23(42d), B24(46d) and B25(51d), water enters into the whole seed coat and the inside of the endosperm, and the red water signal of the whole embryo is more pronounced. After B26(57d), B27(63d) and B28(71d) of soaking, the entire seed coat, endosperm, embryo cavity and embryo all showed strong red water signals.
The SNR data of control seeds during imbibition and cold stratification process (Fig. 5A). The imbibition phase I (0-185 h), the SNR value of hilum showed three times ups and downs, and the amplitude of second rise and fall was larger; the SNR value of micropyle showed three times ups and downs, then continue to rise; the SNR value of seed coat experienced three sharp rises and falls, and then continued to rise; the SNR value of seed coat experienced two times small amplitude of rises and falls, and then continued to rise. The imbibition phase II (185 h-744 h), the SNR values of hilum, micropyle, seed coat and endosperm showed continuous decline to saturation. The saturation stage (744 h- 1512 h), the SNR value of hilum showed falling after rising, and almost flat after saturation; the SNR value of micropyle showed rising after rising and falling, and slowly decrease after saturation; the SNR value of seed coat showed falling after rising, and slowly decrease after saturation ;the SNR value of seed coat showed two times sharp rises and falls, and then rise rapidly after saturation (1512 h-1704 h).
The SNR data of H2SO4 treated seeds during imbibition and cold stratification process (Fig. 5B). The imbibition phase I (0-185 h), the SNR value of hilum rises and falls, then rises again; the SNR value of micropyle rose again after two times of rises and falls, and the range of first time rises and falls was sharply obvious; the SNR value of seed coat has gone through three times of sharp rises and falls; the SNR value of endosperm showed three smaller amplitudes rises and falls. The imbibition phase II (185 h-744 h), the SNR value of hilum and micropyle continued to decrease to saturation; the SNR value of seed coat falls to saturation after sharply falls and rises; the SNR value of endosperm smoothly rises and falls to saturation. The saturation stage (744 h- 1104 h), the SNR values of hilum, micropyle, seed coat and endosperm showed continues to rise and then decline. The germination phase (1104 h- 1704 h), the SNR value of hilum and endosperm rises slowly; the SNR value of seed coat and micropyle showed the same trend, which increased rapidly to 1224 h (51d), then decreased rapidly to 1512 h (63d), and then increased. We found that the SNR value of micropyle and seed coat was the highest at 1224 h (51d), which was probably the key point of seed dormancy release.
2.4 Tissue observation in PSD
We using PSD to detect the changes of morphological properties of endosperm cells in control seed and H2SO4 treated seeds. The elongated thick-walled cells are closely arranged in the elongation zone of the cotyledons (Fig. 6A1). The long strips of tetragonal parenchyma cells are closely arranged in the procambium of cotyledon (Fig. 6A2). The bud tip of the germ consists of irregular thick-walled cells (Fig. 6A3). The endosperm consists of a stack of tetragonal parenchyma cells (Fig. 6A4). The elongated thick-walled cells are closely arranged in the elongation of the radicles (Fig. 6A5). The root end of the radicles is composed of irregular thick-walled cells (Fig. 6A6). The long strips of tetragonal parenchyma cells are closely arranged in the cell division area (Fig. 6A7).
The procambium of cotyledon and the elongation of the radicles will eventually develop into vascular bundles. The morphological properties of endosperm cells in control seed have no obvious change in cold stratification (Fig. 6B). After 35 days of cold stratification, the morphological properties of endosperm cells in H2SO4 treated seeds changes from an elongated state to a small polygonal shape, and the arrangement becomes tight (Fig. 6C5). After 55 days of cold stratification, the morphological properties of endosperm cells become polygonal, containing small round particles and closely packed (Fig. 6C9)(Sechet and Frey et al., 2016). After 60 days of cold stratification, the red granules in the outer parenchyma cells of the endosperm have been degraded and the radicle has broken through the endosperm cap (Fig. 6C10). It can be seen that H2SO4 treatment accelerated the degradation of endosperm contents and shortened the time of seed dormancy release.
2.5 Tissue observation in TEM
The endosperm cells of control seed contained a large number of circular, elliptical, and polygonal high electron dense substances, which are surrounded by lipids (Fig. 7A1). After 10 days of cold stratification, the endosperm cells still contained more lipid particles and a large amount of high electron dense substances (Fig. 7A2). On the 20th days of cold stratification, the lipids began to decompose, but the high electron dense substance did not change (Fig. 7A3). On the 30th days of cold stratification, the lipid in the endosperm cell continued to decomposed and a large amount of high electron density substance still remained (Fig. 7A4). On the 40th day of cold stratification, the lipids gradually ablated and decomposed into small vacuoles, and there is no change in the high electron density substance (Fig. 7A5). On the 50th day of cold stratification, the lipids gradually ablated and decomposed into big vacuoles, but the high electron dense substance showed no change (Fig. 7A6). On the 55th day of cold stratification, the lipids gradually ablated and decomposed into vacuoles, and a few irregular shaped high electron dense materials began to degrade (Fig. 7A7)(Bewley, 1997). On the 60th day of cold stratification, most of the lipid droplets have broken down, and the round and elliptical high electron dense matter begins to degrade in small portion (Fig. 7A8). In conclusion, during the whole cold stratification process, the high electron dense substance in the cells of the control seed degraded very slowly, but the lipids were decomposed almost completely.
Part of the round high-electron dense substance in the endosperm cells after soaking for 15 min in H2SO4 showed obvious holes and cracks, and containing a lot of lipid droplets (Fig. 7B1). After 10 days of cold stratification, the pores and cracks of the high electron dense substance become larger and more pronounced, and the irregularly high electron dense substance begins to degrade, and the amount of lipid droplets are still existed (Fig. 7B2). After 20 days and 30 days of cold stratification, the irregularly high electron dense substances continue to degrade (Fig. 7B3,4). After 40 days of cold stratification, the irregular high-electron dense substances degraded significantly, and round and elliptical high-electron dense substances begin to degrade initially (Fig. 7B5). After 50 days of cold stratification, the round high electron dense substance is obviously degraded (Fig. 7B6). After 55 days of cold stratification, the round high electron dense substance is highly degraded (Fig. 7B7). After 60 days of cold stratification, the high electron density substance degraded into white vacuoles which contained the residual black fragments that are not thoroughly degraded, and still contained a large amount of lipid droplets (Fig. 7B8). The most obvious change in the cold stratification process of H2SO4 treated seeds is that there are obvious holes and cracks in the high electron density substance in the cells, and the degradation and ablation speed are accelerated(Leubner-Metzger, 2003). Obvious pores and cracks undoubtedly greatly contribute to the improvement of endosperm permeability (Finch Savage, etc.,2006).