Anatomy and cell wall ultrastructure of sunflower stalk rind

The anatomy and ultrastructure of sunflower stalk rind are closely related to its conversion and utilization. We studied systematically the anatomy and ultrastructure of the stalk rind using light, scanning electron, transmission electron and fluorescence microscopy. The results showed that the stalk rind consisted of phloem fibers (PF), xylem fibers (XF), vessel elements (V), ground parenchyma cells (GPC), axial parenchyma cells (APC), xylem ray parenchyma cells (XRPC), and pith ray parenchyma cells (PRPC). These cell walls were divided into the middle lamella, primary wall, and secondary wall (S). It was found that the S of PF, XF and V was further divided into three layers (S1–S3), while the S of APC, GPC, XRPC and PRPC showed a non-layered cell wall organization or differentiated two (S1, S2) to seven layers (S1–S7). Our research revealed the plasmodesmata characteristics in the pit membranes (PMs) between parenchyma cells (interGPCs, inter-XRPCs, and inter-PRPCs). The morphology of the plasmodesmata varied with the types of parenchyma cells. The thickness and diameter of PMs between the cells (inter-Vs, V–XF, V–APC, and V–XRPC) were greater than that of PMs between parenchyma cells. The cell corners among parenchyma cells were intercellular space. The lignification degree of vessels was higher than that of parenchyma cells and fibers. The results will provide useful insights into the biological structure, conversion and utilization of sunflower stalk rind.


Introduction
The ultrastructure of plant cell walls is important not only for plant biology, but also for the conversion and utilization of mature plants [1]. Considerable progress has been made in understanding the basic structure and function of wood cell walls, but the variation in cell wall structures in grasses remains poorly understood because of the high complexity and dynamic characteristics of the cell wall [2,3]. One of the key steps in lignocellulosic conversion is cell wall deconstruction. The anatomy and cell wall ultrastructure of lignocellulosic materials are closely related to their conversion and utilization [4].
Plant cell walls are typically described as complex multi-layered structures consisting of an ordered array of cellulose microfibrils embedded in a matrix of non-cellulosic polysaccharides and lignin [5]. The ultrastructure of plant cell walls includes a complex hierarchical structure and heterogeneous distribution of chemical components between different layers [6]. Plant cell walls are usually divided into the middle lamella (ML), primary wall (P), and secondary wall (S). The S is the thickest and accounts for the largest proportion of the cell wall volume [7]. Lignin is the primary component of plant cell wall. Generally, the concentration of lignin in cell corners (CCs) or compound middle lamella (CML) is higher than that in the S. It is worth noting that, as an important morphological region of the cell wall, there are few reports on the cell pits and intercell pit membranes (PMs). In the lignocellulosic biorefining process, the characteristics of cell wall layers and PMs affect the transverse transport of the solution, and then affect the efficiency of biomass conversion [8,9]. Sunflower (Helianthus annuus L.) is one of the main oil crops. With the increasing demand for vegetable oil, the planting area of sunflowers is increasing year by year. The global sunflower planting area exceeded 26.5 million hectares in 2017, and approximately 80-186 million tons of residual stalk was generated [10]. The stalk consists of the rind and pith, in which the weight percentage of rind is approximately 90% [11]. The stalk rind can be used for papermaking [12], producing composite materials [13], chemicals [14], and energy fuels [15]. Sun et al. studied the cell composition and arrangement of the base, middle, and top of sunflower stalks, and analyzed the effects of each portion microstructure on mechanical properties and water diffusion [16]. Mehdikhani et al. studied the length and width of stalk rind fiber, and discussed its performance in papermaking [10]. Currently, the anatomy of sunflower stalk rind is not detailed, and the ultrastructure of its fibers, vessel elements (V), and parenchyma cells has not been reported. Hence, we analyzed the anatomy of stalk rind using light microscopy (LM), the cell pits characteristics using scanning electron microscopy (SEM), the ultrastructure of cell wall layers and PMs using transmission electron microscopy (TEM), and lignin distribution using fluorescence microscopy (FM) and scanning electron microscopy with energy dispersive X-ray analysis (SEM-EDXA), and measured cell wall layer thickness, the PMs thickness and diameter using a combination of TEM images and ImageJ software. This study aimed to reveal the anatomy and cell wall ultrastructure of sunflower stalk rind and to provide the basis for its conversion and utilization.

Materials
Sunflower stalk was collected from Wuchuan County, Inner Mongolia, China. After sunflower maturation, stalks with a height of approximately 2 m and diameter of approximately 20 mm were selected. The middle section of the stalk was taken, and the pith was removed manually. The stalk rind was air-dried and stored for further use.

LM
The stalk rind was cut into strips (0.5-1 cm height) and embedded with polyethylene glycol 2000 [17]. Transverse and radial sections (30 μm thickness) were prepared using a sliding microtome (TU-213, Yamato Kohki Industrial, Saitama, Japan), then stained with Safranin O and mounted in glycerol. Anatomy of the stalk rind was observed using a light microscope (BH2, OLYMPUS, Japan).

SEM
The radial sections (30 μm thickness) of the stalk rind were selected. Before imaging, the sections were sputter-coated with 8 nm of gold using an E-1010 sputter coater (Hitachi, Hitachinaka, Japan) for 120 s. The cell pit characteristics were observed using an environmental scanning electron microscope (Quanta 200, FEI Co., OR, USA) at an acceleration voltage of 20 kV.

TEM
The stalk rind was cut into sticks (1 × 1 × 1 mm 3 ). The samples were embedded in Spurr's epoxy resin [18]. Transverse ultra-thin sections (90 nm) were cut with a diamond knife using an ultramicrotome (EM-UC6, Leica, Wetzlar, Germany). The sections were stained with 1% w/v KMnO 4 (prepared in 0.1% w/v sodium citrate) for 2 min at 25 °C. The cell wall layering structure and PMs characteristics were observed under a transmission electron microscope (JEM-1400, JEOL, Tokyo, Japan) at an accelerating voltage of 80 kV.

FM
Transverse sections (15 μm thickness) were dehydrated in a graded series of ethanol solutions, and then were mounted in 70% glycerol and covered with a coverslip. Sections were observed with a fluorescence microscope (BX51, Olympus, Tokyo, Japan) using mercury lamp for illumination and using light at 330-385 nm for imaging lignin autofluorescence.

SEM-EDXA
Transverse sections (15 μm thickness) were extracted with a benzene-ethanol mixture (2:1, v/v) for 24 h. The sections were then reacted with 1% w/v KMnO 4 for 5 min at 25 °C. A gold film (8 nm thickness) was coated on the sections with an E-1010 sputter coater (Hitachi). Finally, the distribution of lignin in the cell walls was analyzed by measuring the Mn-Kα X-ray counts using a Quanta 200 environmental scanning electron microscope (FEI Co.) equipped with an energy dispersive X-ray analyzer (Inca X-act, Oxford, England). The accelerating voltage and probe current used were 20 kV and 80 μA, respectively. The point measurement of Mn-Kα X-rays was performed using at least 10 cells per region.

Ultrastructure measurements
ImageJ software (available at http:// rsb. info. nih. gov/ ij) was used to measure the cell wall layer thickness, PMs diameter (D PM ), and PM thickness (T PM ) based on TEM images. The cell wall layer thickness was measured for at least 20 cells per cell type. The D PM and T PM measurements were recorded for more than 20 PMs per pit-pair type.

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 (S 1 ), a middle (S 2 ), and an inner layer (S 3 ) (Fig. 2a). The S layering structure of the XF resembled that of the PF, and was also divided into S 1 , S 2 , and S 3 (Fig. 2b). The layering structure of the S of stalk rind fibers was similar to that of wood fibers [21], except that the S 3 of stalk rind fibers was very thin, which was occasionally difficult to distinguish from the S 2 . The S of the V was also differentiated into S 1 , S 2 , and S 3 , in which the S 1 was clearly distinguished from the S 2 , and the S 3 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 (S 1 -S 3 ) or seven layers (S 1 -S 7 ) (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 (S 1 , S 2 ) or three layers (S 1 -S 3 ), 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 (S 1 -S 3 ) or four layers (S 1 -S 4 ) (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 nonlayered 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 KMnO 4 . KMnO 4 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 S 2 layer of vessel was higher than that of fiber and parenchyma cells, and the S 2 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 > PR PC. The thickness of S 2 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 S 1 layer, with an average thickness of 0.20-0.57 μm. The thickness of CML layer and S 3 layer was the smallest, with an average Fig. 3 The thickness of each layer in sunflower stalk rind cell walls thickness of 0.06-0.17 μm and 0.07-0.23 μm, respectively. The average thickness of S 2 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 S 2 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 intervessel 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 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].  (3.17-5.15 μm) between the cells (inter-Vs, V-XF, V-APC, and V-XRPC) were all larger than the T PM (0.14-0.23 μm) and the D PM (1.32-1.63 μm) between parenchyma cells (inter-GPCs, inter-XRPs, and inter-PRPCs). The D PM was related to the aperture of simple pit [33], and the D PM 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 > PRP C > PF > XF > GPC. The SEM-EDXA measurements were consistent with the FM and TEM observations. the thickness of each layer were different between cell types. The S of PF, XF, and V were divided into three layers (S 1 -S 3 ). The S of APC, GPC, XRPC, and PRPC showed a non-layered cell wall organization or differentiated two (S 1 , S 2 ) to seven layers (S 1 -S 7 ), with three layers being the most common.
The order of average thickness of each cell wall was PF > XF > V > GPC > APC > XRPC > PRPC. The CCs among parenchyma cells were intercellular space. (3) The type, distribution of pits and the ultrastructure of PMs were diverse. The pits of V and PF were mostly bordered, while pits of XRPC, APC, GPC, and XF were simple. The pits on parenchyma and fiber cell walls were scattered randomly, and the pits on the V cell walls were distributed in an alternate pattern. The PMs between parenchyma cells were perforated by plasmodesmata, and the morphology