Anatomy features
The LM images showed the transverse and radial anatomy of sunflower stalk rind (Fig. 1). The stalk rind consisted of the epidermis (Ep), vascular bundles, and parenchyma tissues. The Ep was composed of epidermal membrane and epidermal cells, was located in the outermost layer of the stalk rind. The vascular bundle tissues were arranged in a ring and consisted of phloem (Ph), vascular cambium (VC), and xylem (X). Ph consisted of phloem fibers (PF). X consisted of xylem fibers (XF), vessel elements (V), axial parenchyma cells (APC), and xylem ray parenchyma cells (XRPC). The cell composition and arrangement of X were similar to that of porous wood [19]. There were several types of parenchyma cells in the stalk rind, including ground parenchyma cells (GPC), APC, XRPC, and pith ray parenchyma cells (PRPC). The GPCs were distributed near the Ep, while the APCs and XRPCs were scattered around the V and XF. The number of PRPC differentiated cells was large, usually present in multiple rows.
Cell wall layering structure
Morphological characteristics of the cell wall
The TEM images showed the cell wall layering structure of sunflower stalk rind (Fig. 2). The cell walls of fibers, V, and parenchyma cells were divided into the ML, P, and S. The boundary between the ML and P was not clearly distinguishable because of its high density and extreme thinness. Therefore, both the ML and the contiguous P were referred to as the CML [20]. The S of the PF was divided into an outer (S1), a middle (S2), and an inner layer (S3) (Fig. 2a). The S layering structure of the XF resembled that of the PF, and was also divided into S1, S2, and S3 (Fig. 2b). The layering structure of the S of stalk rind fibers was similar to that of wood fibers [21], except that the S3 of stalk rind fibers was very thin, which was occasionally difficult to distinguish from the S2. The S of the V was also differentiated into S1, S2, and S3, in which the S1 was clearly distinguished from the S2, and the S3 was very thin (Fig. 2c).
The layering structure of the S of parenchyma cells in stalk rind varied with the types of parenchyma cells. The S of the APC had a non-layered cell wall organization, and these cells were mostly distributed close to the vessels (Fig. 2c). The APC in oak earlywood also showed the characteristics of non-layered cell wall organization [22]. The S of the XRPC was resolved into three (S1–S3) or seven layers (S1–S7) (light and dark alternation) (Fig. 2d). XRPCs with the S divided seven layers were usually distributed between XFs. The S of the PRPC was differentiated into two (S1, S2) or three layers (S1–S3), and PRPCs with the S divided two layers was generally distributed in the X (Fig. 2e). The S of ray parenchyma cells in Cornus alba similarly consisted of two well-defined layers [23]. The S of the GPC was divided into three (S1–S3) or four layers (S1–S4) (Fig. 2f). GPCs with the S divided four layers was distributed near the Ep. To summarize, the S of parenchyma cells of sunflower stalk rind showed a non-layered cell wall organization or could be divided into two, three, four and seven layers, with three layers being the most common. It was interesting to find that the CCs among parenchyma cells in stalk rind were all intercellular space, and the volume of intercellular space in the GPC and PRPC was usually larger than that of the APC and XRPC. The intercellular space was most characteristic of mature tissue. The parenchyma cells in sunflower stalk rind were mature and fully differentiated, which was the outstanding characteristic of annual grass plants different from wood.
When the TEM was used to analyze the lignin distribution in cell wall, the ultra-thin sections needed to be stained by KMnO4. KMnO4 having a special reactivity with lignin, reacted fast with the double bonds in lignin molecules by forming manganese dioxide, which deposited on the reaction sites [24]. According to the difference in the intensity of staining at the reaction site, the high and low lignin concentrations were determined [25]. As shown in Fig. 2, the staining intensity was highest in the CC of fibers, followed by the CML of each cell, and the S layer with a lower staining intensity. That was, the CC of fiber cells had the highest lignin concentration, followed by the CML of each cell, and the S layer had a lower lignin concentration. Overall, the staining intensity of S2 layer of vessel was higher than that of fiber and parenchyma cells, and the S2 layer was the main part of cell wall. Hence, the lignification degree of vessel in stalk rind was higher than that of fiber and parenchyma cells (Fig. 2c arrow). Plant vessels mainly transported water and inorganic salts, which were subjected to great pressure of transpiration. The high degree of lignification of the vessel cell wall can increase the pressure resistance, so as to protect it from collapse during transporting [26].
Quantification of cell wall layers
On comparing the thickness of cell wall layers of fibers, V and parenchyma cells in the stalk rind, only parenchyma cells with the S divided three layers were selected for measurement. Figure 3 shows the average thickness of each layer in stalk rind cell walls. The order of average cell wall thickness was PF > XF > V > GPC > APC > XRPC > PRPC. The thickness of S2 layer of fibers, vessels and parenchyma cells in stalk rind was the largest, with an average thickness of 0.55–2.43 μm, followed by S1 layer, with an average thickness of 0.20–0.57 μm. The thickness of CML layer and S3 layer was the smallest, with an average thickness of 0.06–0.17 μm and 0.07–0.23 μm, respectively. The average thickness of S2 layer of the PF was greater than that of the XF, which indicated that the XF had a lower cell wall thickness compared to the PF. The average thickness of the S2 layer of the GPC was higher than that of APC, XRPC, and PRPC, which was the same as the features of cell wall thickness of basic parenchyma cells and vascular parenchyma cells in bamboo [27].
Pit characteristics of cells
Type and distribution of pits
The pit is a hole or concave in the process of plant secondary wall thickening [5]. The pit is the most obvious structural feature on the cell wall, and is the main transverse channel for water or nutrient transport in plants [28]. The SEM images showed pits characteristics of sunflower stalk rind cells (Fig. 4). The pits of the PF were mostly bordered (Fig. 4a), while pits of the XF were simple (Fig. 4b). The pits of both PF and XF were randomly distributed on the cell wall, but the number of pits in the XF was more than that in the PF. Pits of the V were bordered, and were distributed in an alternate pattern (Fig. 4c). Pits of the XRPC, APC, and GPC were simple, and were scattered randomly throughout the cell wall. The distribution of pits in the XRPC and APC was sparse and the pit aperture was large, while that in the GPC was dense and the pit aperture was small (Fig. 4d–f). Combined with the analysis of the anatomy and cell wall layering structure, the stalk rind had the characteristics of many parenchyma cells, thin cell wall of parenchyma cells, the intercellular space in CC among parenchyma cells, and a large number of pits on cell wall, which were conducive to the penetration of liquid chemicals during the conversion and utilization [29].
Morphological characteristics of pit membranes
The PM was a very important structural part of the pits, which was a safety valve for water transport in plants [8]. Figure 5a–d shows the PMs between the cells (inter-Vs, V–XF, XF–APC, and XF–XRPC) in sunflower stalk rind, and Fig. 5e–g shows the PMs between parenchyma cells (inter-GPCs, inter-XRPCs, and inter-PRPCs). It was worth noting that the PMs between parenchyma cells was perforated by plasmodesmata (Fig. 5e–g inset), while the PMs between other cells was not perforated. It might be because on the one hand, plasmodesmata are generally not found in bordered pit membranes that were formed between treachery cells (i.e., tracheid and vessel). In angiosperms, plasmodesmata are absent or rare in bordered pit membranes of inter-tracheid and inter-vessel bordered pits. On the other hand, plasmodesmata are abundant in a simple pit of parenchyma cells [30]. All PMs had perforations. Perforations allowed for free passage of water and nutrients, while limiting the passage of pathogens between cells [31]. Furthermore, the morphology of plasmodesmata on the PMs between parenchyma cells in stalk rind was different, which might be related to the physiological function of parenchyma cells. The GPCs are located near the Ep, and their main function is to store starch granules. The XRPCs are close to the V and involve in the transport capacity of the V, transporting water, nutrition, etc. However, the PRPC connects outward to the cortex and inward to the pith, and has both functions of the GPC and XRPC [32].
Quantification of pit membranes
Figure 6 shows the measurement results of TPM and DPM of sunflower stalk rind cells. The order of the TPM was V–V > V–XF > V–APC > V–XRPC > GPC–GPC > PRPC–PRPC > XRPC–XRPC. The order of the DPM was V–XRPC > V–APC > V–XF > V–V > XRPC–XRPC > PRPC–PRPC > GPC–GPC. The TPM (0.33–0.43 μm) and the DPM (3.17–5.15 μm) between the cells (inter-Vs, V–XF, V–APC, and V–XRPC) were all larger than the TPM (0.14–0.23 μm) and the DPM (1.32–1.63 μm) between parenchyma cells (inter-GPCs, inter-XRPs, and inter-PRPCs). The DPM was related to the aperture of simple pit [33], and the DPM results of parenchyma cells were also consistent with the larger pit aperture in XRPC than that in GPC.
Lignin distribution in cell walls
Autofluorescence images can visually display lignin distribution in the cell walls [34]. Figure 7 shows the autofluorescence images of sunflower stalk rind cell walls. The CC of the PF and XF assumed the greatest autofluorescence, while the CML of fibers, V, and parenchyma cells displayed greater autofluorescence than that of the S layer. The autofluorescence intensity of the V cell walls was higher than that of parenchyma and fiber cell walls. It suggested that the lignin concentration in the CC and CML was higher than that in the S layer, and the vessels are more lignified than parenchyma cells and fibers. The distribution of lignin in stalk rind cell walls was similar to that of poplar and wheat straw [35].
The distribution of lignin was indirectly analyzed by Mn–Kα X-ray point scanning method [36]. Figure 8 shows the Mn–Kα X-ray count measurements of the regions in the stalk rind cell walls. The CC of the fibers had the highest Mn–Kα X-ray counts. The Mn–Kα X-ray counts of the CML in fibers, V, and parenchyma cells were higher than that of the S layer. The order of the Mn–Kα X-ray counts of the S layer was V > APC > XRPC > PRPC > PF > XF > GPC. The SEM–EDXA measurements were consistent with the FM and TEM observations.