Stomatal development events in maize
The development of maize stomatal complexes followed sequence events, which occurrence in the leaf basal meristem region. The series events begin with cell proliferation of meristematic cells which were found in the basal meristem region beside preligule band (PLB) (Fig. 3A). The remaining series events happened in the zone of stomata initial origin and the zone of stomatal differentiation, which are two consecutive zones above the meristem (Fig. 3A).
The development of stomatal in maize can be divided into six consecutive stages (Fig. 3B2-3B7)[26, 27]. It contained three asymmetrical divisions and one symmetrical division process. Initially, potential precursor cells proliferate in particular files at the distal end of the meristematic cell close to leaf base (Stage 1, Fig. 3B2). A stomatal initial from the first asymmetrical division produce the guard mother cell (GMC), which separated a small rectangular cell toward the leaf tip (Stage 2, Fig. 3B3). Subsequently, a pair of subsidiary cells were induced by the GMC asymmetrical divisions in the laterally adjacent subsidiary mother cells (SMC) (Stage 3 and 4, Fig. 3B4 and 3B5), finally the GMC then symmetrical division into two immature guard cells(GC) (Stage 5, Fig. 3B6). Four especially cells mature and expand to form a mature stomatal complex (Stage 6, Fig. 3B7).
Leaf tissue was extract from 10 days old maize seedlings, its leaf 2 was just emerging from the whorl (Fig. 2D). The coleoptille and leaf 1 were removed to expose developing tissue at the bases of leaf 2; There are three zones at basal leaf zones were excised for analysis based on the developmental stages they represented (Fig. 3A). Zone 1 is located at the base of about 80µm, it contains preligular band(PLB) structure, which are dividing cells with shape of primarily isodiametric. Zone 2 is between 100µm to 5500µm from the PLB, contains differentiation cells, which are varying size and shape cells. It contains the pathway of stomata development. The length of total cell at asymmetric entry division stage is about 350µm, and the SMC formation stage is about 400µm, the SMC asymmetric division is about 1600µm and the GMC symmetric division stage is 2700µm. Zone 3 is about 5500 µm away from the ligule, contains extensive differentiation cells which expand to mature stomata (Supplementary Fig. 1).
Exogenously application of 2,4-D (0.4mg/ml) repress the cell division in leaf meristem zone, decrease number of GMCs and make ligule disappear at leaf base.
The application of 2,4-D in this work influence the growth of maize seedling leaves. It mainly make the ligule disappear and a remarkable decrease in GMC formation. In the protodermal areas at leaf base, GMC number and the number of epidermal cells were decreased and the length and width of epidermal cell was increase comparable to the mock treatment at the asymmetric enter division stage. Some young subsidiary cells were induced by GMC, which is more closed to the leaf base than the control leaves (Fig. 4B3). Collectively, these data indicated that 2,4-D suppressed the development of ligule and decreased the number of cell entry into the first asymmetric division and suppressed the cell symmetric division.
There is a uniquely linear band, located at leave base, which will develop to ligule and auricle, named preligule band (PLB). The cells in PLB are smaller and run perpendicular to the proximal-distal axis of the developing leaf (Fig. 3B1 and 4A1). Cells periclinal divisions in PLB will develop to ligule, which are parallel to the leaf surface[28, 29]. On the developing ligule stage, the PLB was disappeared at 48 hours after 2,4-D treatment. And the smaller cells in PLB zone, which was characteristic of PLB cells were reduced. The cells number in this zone were also decreased significantly (Fig. 4B1).
At the cell proliferation stage, the cells number per area was less than mock treatment group (Fig. 4B2). And the width and length of cells in this zone was larger than mock treatment group. The average length of cells is 26.77µm after 0.4mg/ml 2,4-D treatment, is longer than mock significantly, which value was 17.47µm (Fig. 5A). And the average width of cells in 2,4-D treatment group (13.91µm) is also significantly larger than mock(12.19µm) (Fig. 5B).
At asymmetric entry division stage, the number of GMCs in 2,4-D treatment group was less than mock, and the symmetric division was also reduced compare to mock. The number of guard mother cells (GMC) was reduced. The density of GMCs was 5.6 per 160 ×160µm2 area leaf after 0.4mg/ml 2,4-D treatment and it was 16.6 at the mock treatment (Fig. 4B3, Fig. 5C). This result indicated that the first asymmetric division for GMCs was suppressed by 2,4-D. The events of periclinal division and perpendicular division were also reduced, it means that the division event of asymmetric and symmetric were suppressed by 2,4-D. The cells number on 2,4-D treatment group was less than mock group, it may be induced by decreased cell division. This may lead the length and width larger than mock and the cell number was less than mock. Additionally, there are some GMCs induce the SMCs at this stage, the young stomata complex formation more closed to the leaf base.
At SMC formation stage, there are some GMCs induced the two SMCs, and produce unnormal stomata complex which include two young subsidiary cells(YSCs) in 2,4-D treatment group (Fig. 4B4). And the stomata complex in mock group is include mature GMC and flanked by two nascent SCs (Fig. 4A4).
At SMC asymmetric division stage, the number of cells and stomata in 2,4-D treatment group was similar as mock treatment group (Fig. 4B5). It indicated that the 2,4-D did not influence the development of stomata on this stage, maybe because the initiate process of stomata at this stage had finished before 2,4-D treatment. It indicated that initial process of stomata may be influenced by 2,4-D, but when the initial process had been finished, the GMCs will develop into the mature stomata complex even under the condition of 2,4-D treatment.
The minimum concentration of 2,4-D which influence the formation of subsidiary cell in maize is 4x10 − 3 mg/ml.
At the asymmetric entry division stage, there are many young subsidiary cells were induced by GMCs in the 2,4-D treatment group at a concentration of 4x10− 3 mg/ml (Fig. 6C2). And there are many GMCs induced the subsidiary cells at 4x10− 2 mg/ml and 4x10− 1 mg/ml (Fig. 6D2, Fig. 6E2). The young GMCs at the asymmetric entry division stage didn’t induce the subsidiary cells under 2,4-D treatment conditions at a concentration of 4x10− 4 mg/ml (Fig. 6B2), just like the young GMCs in mock treatment group. This result indicated the minimum treatment concentration that affects formation of subsidiary cell is 4x10− 3 mg/ml for 2,4-D treatment.
At cell proliferation stage, there are many GMCs which are produced by asymmetric division under 2,4-D treatment conditions at a concentration of 0.4mg/ml (Fig. 6E1). For other concentration from 4x10− 2mg/ml to 4x10− 4mg/ml, the cells in stomatal lineage proliferated just like the cells in stomatal lineage in the mock treatment group (Fig. 6B1-6D1), it indicated that when the 2,4-D concentration is higher than 0.4mg/ml, the asymmetric division is closer to the leaf base.