Morphologies of the epicuticular surfaces
Figure 1 showed the typical ESEM images of the epicuticular surfaces of rice and P. tenuiflora leaves. The epicuticular surface of rice leaves (Fig. 1a and b) contained epidermis cell (EC), stomatal guard cell (GC), stomatal subsidiary cell (SbC), trichome (TC), and wart-like protuberance (silica cell, SC). Crystalline wax covered over the epicuticular surfaces. Wax crystals appeared as randomly distributed crystals over the epicuticular surfaces (Fig. 1b). The wax crystals showed no specific orientation, and their planes were standing with acute angles to the epicuticular surface. The random orientation of small-sized crystalline waxes formed the micro-networks. The heights of platelet wax crystals were less than 0.2 μm. There was no noticeable difference of crystalline wax layers on EC, GC, SbC, and SCs, but no on TC.
Figure 1c and d showed the similar wax crystals on the epicuticular surface of the P. tenuiflora leaves. It seemed that there were two different sized crystals in the waxy networks (Fig. 1d). Smaller crystals formed more dense networks within the networks formed by bigger crystals. There was no distinction of waxy morphology between on EC, GC, and SbC. There were no SC and TC on the P. tenuiflora leaves. The density of the wax crystal networks in P. tenuiflora was higher than that in rice.
NaHCO3 stress induced changes of rice epicuticular surfaces
As NaHCO3 stress increased for the rice samples, the epicuticular morphologies changed (Fig. 2). Figure 2b showed that wart-like protuberance silica cells merged and enlarged to be the big protuberances (MSC). The distribution density of wax crystal networks deceased and disappeared on the apex surfaces of the MSC. At 100 mM NaHCO3 with 7 days exposure, leaked columns (C) and/or swollen corn-shapes (S-Cs) appeared on the surface (Fig. 2c). Interestingly, wax crystals remained on the surfaces of the S-Cs. Diameter of the leaked columns was 2 ~ 5 μm, while the size of the swollen corn was bigger than 10 μm. Cracked side view of the S-Cs revealed the cubic crystals as marked arrow in Fig. 2d, indicating NaCl crystals. There were also the solidified particles underneath of the cell wall as marked number 6 on Fig. 2d.
EDX element analysis of the epicuticular surface of rice leaves
EDX microanalysis spectroscopies were obtained from the different spots over the epicuticular surfaces as marked numbers in Fig. 2. For the controlled rice leave surfaces, there were no significant difference among the EDX spectroscopies obtained from SbC, EC and SC. C and O elements (Fig. 3a and b) were dominated. Traces of other elements including gold were also detected. Relatively high gold peak came from the gold coating. The level of silicon accumulation was low on both epidermal region and silica cells. For the NaHCO3 exposed rice, there were significant changes in Na and Cl counts at the points on the merged and enlarged silica cells (MSC) (Fig. 3c), the swollen corn shapes (S-C) (Fig. 3d) and the leaked columns (C) (Fig. 3e). Weight %s of Na and Cl on MSC were counted 12.5 % and 0.3 %, respectively. EDX spectra from the localized swollen corn surfaces showed that concentrations of Na and Cl were 20 ~ 30 time higher than those form the normal controlled surfaces. The particles underneath cell wall also showed high counts of Na and Cl (Fig. 3f). The cubic crystals on cross-section surface of the swollen corn appeared, indicating NaCl crystal. At the higher saline stress, condensed Na and Cl were leaked trough the ruptured surfaces to form the NaCl columns. There was more excessive Cl−than Na+ on the swollen corns, while excessive Na+ than Cl− on the leaked columns.
We have scanned over the surfaces to visualize the morphology depended Na+ distribution by using ESEM. Figure 4 showed Na and K contour maps over the surfaces after 7 and 9 days exposure to100 mM NaHCO3. Compartmented Na+ was found underneath the epicuticular surfaces, but no K+. The surface morphologies over the high Na+ accumulations were different from those over the control surfaces. It seemed that the degree of Na+ was associated with the morphological changes of the epicuticular surface.
Absorption comparison of cytosolic Na+ and K+ in rice and p. tenuiflora
Plant usually balances at low cytosolic [Na+], and a cytosolic [K+]/[Na+] >1 [12]. Figure 5 showed that Na+ influx from the high external [NaHCO3] altered the [K+]/[Na+] in the rice. Na+ distribution ratio of shoot to root for rice also increased significantly from 0 mM to 150 mM NaHCO3 stress, appearing as [Na+]shoot/[Na+]root > 1 (Fig. 5a). It seems the absorbed Na+ ions from root were transported to the shoot. Consequently, the [K+]/[Na+] ratios in rice shoots decreased gradually lower than 1 (Fig. 5b). Transported Na+ ions were accumulated to be toxic effects in rice shoot. At the 200 mM NaHCO3 stress, [Na+]shoot dropped dramatically (# marked in Fig. 5a and b). This [Na+] decrement may be caused from a dye-functioned rice (yellowish colored shoot) due to high toxicity. Localized NaCl swollen corn shapes and columns formed by rupturing and/or leaking highly accumulated NaCl were correlated to decreased cytosolic [Na+] at extremely high NaHCO3 stress.
For P. tenuiflora, Na+ concentrations of both root ([Na+]root) and shoot ([Na+]shoot) were always balanced well at a very low level. This stable [Na+]root/[Na+]shoot indicated that the external saline stress barely affected P. tenuiflora, which was different from those for rice. Figure 5b showed that ratio of [K+]/[Na+] decreased for both P. tenuiflora and rice, but for P. tenuiflora, [K+] was twice higher than [Na+] as NaHCO3 stress increased, while [K+] became almost 5 times lower than [Na+] for rice.
Surface morphology and EDX profiles for P. tenuiflora
Figure 6 showed the epicuticular morphologies of P. tenuiflora with EXD characterization. Interestingly, epicuticular surface morphology of P. tenuiflora had no remarkable changes with experiencing the NaHCO3 stress (Fig. 6). Morphology of waxy crystal network on the P. tenuiflora epidermis surfaces was similar as that on the controlled rice leave surfaces. Even at high NaHCO3 stress, 150 mM for 21 days exposure, the wax and surface morphologies had no remarkable changes, EDX profiles also showed no remarkable changes of the element concentrations either, including Na+ and K+.