Ultrastructure of the Tobacco Leaf Epidermis
Total Page：16
File Type：pdf, Size：1020Kb
ULTRASTRUCTURE OF THE TOBACCO LEAF EPIDERMIS J. Peacock’, L. van Rensburg’, C.F. van der Merwe’ and W. Pretorius3 1. Research Institute for Reclamation Ecology, Potchefstroom University for Christian Higher Education, Private Bag X6001, Potchefstroom, 2520, South Africa. 2. Unit for Electron Microscopy, University of Pretoria, Pretoria, 0002, South Africa. 3. Laboratory for Electron Microscopy, Potchefstroom University for Christian Higher Education, Potchefstroom, 2520, South Africa. Our studies are concerned with epidermal water relations and water loss from leaves of plants which are of agronomic significance. A correlative study with TEM and FE-SEM of the cuticle and cell wall of Nicotiana tabacum L. was performed as their integrated structure-function relations are of importance in the transport of water and nutrients. The cell surface is at the heart of many key events in plant growth and development [ 11. Together the cuticle and cell wall control the exchange of matter between leaf and atmosphere. The FE-SEM study and the TEM ontogenetic study revealed new information on the epidermis ultrastructure bringing new hypothesis to light on epidermal transport. Two fixation schedules were carried out for TEM and all material was dehydrated in an ethanol series and embedded in Spur-r or Quetol 65 1. (a) Primary fixation with aqueous 1% 0~0~ vapour for 2 hours followed by post-fixation with aqueous 1% 0~04 for 30 min. (b) Primary fixation with 2.5% GA or 1% FA for 1 hour followed by post-fixation with aqueous 1% 0~04 for 30 min. For FE-SEM samples were plunge frozen in liquid propane at -180°C. The tissue was fractured in liquid nitrogen and freeze dried. A number of samples were fixed and critical point dried. The epidermal wall was isolated enzymatically through incubation with pectinase buffered at pH 4 and cellulase buffered at pH 5 with sodium acetate for 15min. An amorphous procuticle (Fig 1) is replaced early in organ differentiation by a cuticle proper (CP) which is of lamellate ultrastructure (Fig 2). Cutin-rich cystoliths (Fig 3 and 4) accumulate under the CP starting the construction of the cuticular layer (CL). The globules fuse laterally to form a continuous matrix that is permeated with electron-dense fibrillar cell wall (CW) material that is held apart by the growing cutin globules (Fig 5). The cutin cystoliths are structurally bound to the cuticle as they are still discernible after cellulase and pectinase incubation (Fig 6). It appears as if the cystolith is open on one side forming a tear drop shape possibly depositing cutin. The mature tobacco leaf epidermis exhibit a typical bilayered cuticular membrane (CM), composed of a thin outer CP and a thicker CL [2]. The cuticle proper consists of electron-opaque lamellae alternating with electron- lucent lamellae, while a reticulate pattern is discernible in the cuticular layer. The FE-SEM study revealed the same gross morphology of the epidermis as that seen in the TEM work consisting of the CM and CW (Fig 7). However, the CM could not be distinguished into the CP and CL as was the case with TEM (Fig 7). The CM appeared to have a distinct platelike structure (Fig 7). A possible explanation for the platelike structure is that it may be the consequence of freezing at -18OOC where the cuticle may lose its elasticity. A hypothesis to explain the fracture plane is that there could be a resemblance in the spatial size of the platelike structure and the fibrils in the CL seen with TEM. The cellulose microfibrils of the CW is revealed in Fig 8 after the cuticle is pulled away like a lid. The general structure of the primary cell wall has been envisioned for many years to be composed of cellulose microfibrils in an amorphous matrix of pectins, hemicellulose and glycoproteins. Cellulose microfibrils are embedded in a matrix, but as the matrix is not crystalline it has not been possible to see the structure by using conventional methods. The distribution and function of pectin is no longer compatible with the idea that it is simply a gel [ 11. It appears that the organization of pectic substances is a major control element in defining the sieving properties of the wall [2]. The FE-SEM and TEM ontogenetic study have revealed new information but it is clear that the epidennal wall still has many secrets that elude us. Many questions concerning the relationship between cuticular composition, structure and fkction remains unanswered. Fig I. An electron-dense procuticle (C) and cell wall (CW). Bar = IOOnm. Fig 2. The procuticle is transformed into a lamellate cuticle proper (CP) with the first lamellae (arrows) discernible. Bar = IOOnm. Fig 3. Arrows indicate the cutin cystoliths which accumulate under the CM in the CW. A lamellate cuticle proper is discernible. Bar = IOOnm. Fig 4. Cutin cystoliths are discernible with FE-SEM. Bar = IOOnm. Fig 5. Lateraffy fused globules forming a continuous matrix permeated with electron-dense tibrils (arrows). Bar = 1OOnm. Fig 6. Cutin cystoliths (arrows) discernible after cellulase and pectinase incubation. Bar = 1OOnm. Fig 7. Epidexmal wall with the platelike (arrows) CM and CW. Bar = 100~1. Fig 8. The cell wall (CW) reveal cellulose microfibrils a&r the CM is pulled away like a lid. Bar = 1OOnm. References 1. Roberts K., Current Opinion in Cell Biology, 2 (1990) 920. 2. Kriiger H. ef al., Annalsof Botany, 77 (1996) 11. 3. Baron-EpelO ef al., Planta 175 (1988) 389. .