| Summary: | 碩士 === 國立嘉義大學 === 生物資源學系研究所 === 101 === Selaginella erythropus is a member of the ancient vascular plants, belonging to Selaginellaceae (Lycopodiophyta, a.k.a. Lycophyta). This plant occurs in deep-shade environment native to Amazon tropical rain forests in South American. Recently, a unique chloroplast, bizonoplast, was found in the dorsal epidermal cells of microphylls in S. erythropus. Bizonoplast is a giant chloroplast appearing as cup-shape with dimorphic ultrastructure separated into two zones: an upper zone of parallel layers of 2 - 4 stacked thylakoid membranes, and a lower zone with both the stromal thylakoids and grana structure similar to the chloroplast of most land plants. Current understanding of bizonoplasts is confined to the basis of its unusual ultrastructure; its developmental morphogenesis and the correlation between dimorphic structure and photosynthetic activity need more study. The aims of this study are to reveal the chloroplast diversity in different tissues of microphylls and stems and to explore the developmental process of bizonoplasts in S. erythropus with light microscopy (LM), transmission electron microscopy (TEM) and confocal scanning laser microscopy (CSLM). In addition, quantitative immunogold labeling technique was conducted to labeling four types of photosynthetic key proteins to understand the differences of photosynthetic features between the upper and lower zones of a bizonoplast.
The results show that chloroplasts occur in both microphylls (dorsal and ventral sides of epidermal cell, mesophyll, guard cell and trichome) and stems (cortex cell, trabecular endodermal cell and pericycle cell) of S. erythropus. Bizonoplast is extremely large (26.74 ± 3.48 μm) and cup-shaped, with only one filling in a dorsal epidermal cell. A three-dimensional diagram of this bizonoplast was constructed based on the observation of both transverse and paradermal sections. The chloroplasts occurring in other cell types are the normal type with typical ultrastructure of thylakoid membranes. Both dorsal epidermal cells (with bizonoplast) in a microphyll and pericycle cells (with normal chloroplast, 3.54 ± 1.09 μm) of a stele in a stem show monoplastidity. Four normal chloroplasts (8.35 ± 1.11 μm) are found in a guard cell. The chloroplast number per cell varies from 4 to 13 in the following cell types: the mesophyll cell (8.06 ± 1.46 μm) and the ventral epidermal cell (7.03 ± 1.04 μm) of microphylls, and the cortex cell (5.38 ± 0.24 μm) and the trabecular endodermal cell (5.38 ± 0.24 μm) in stems. In addition, chloroplasts (5.33 ± 0.55 μm) were observed only in large trichomes near the base of a microphyll, ranging from 0 to13 per trichome. However, the chloroplasts found in trichomes have poor to limited developed thylakoid membranes.
The formation pattern of bizonoplasts of S. erythropus was studied with the prostrate and erect shoots by examining their development over time. The results show that the early developmental stage of a bizonoplast is similar to a normal chloroplast. After exposing to sun light about 2 - 3 days, these normal-like chloroplasts in dorsal epidermal cells of the shoot apex in both prostrate and erect branches initiate to differentiate a few groups of parallel thylakoid membranes on the top of original chloroplasts, and gradually form the upper zone of bizonoplasts. Based on TEM study, the development of bizonoplasts can be defined by five stages: (1) an initial normal chloroplast: oval-shaped (1.5 μm × 3.2 μm) with 3 - 5 layers of thylakiod membranes; (2) an early normal chloroplast: round shape (diameter 4.9 μm), with 5 - 7 layers of thylakiod membranes in grana and a small number of starch grains; (3) an initial bizonoplast: oval to cup-shaped (8.9 μm × 11.1 μm), with upper zones initiated (1/5 of a bizonoplast), thick grana and many starch grains in lower zones ; (4) a mature bizonoplast: cup-shaped (19.48 μm ×15.68 μm), with well-developed upper zones (about 1/3 of a bizonoplast) and many dense-stacking grana in lower zones intermingled with stroma thylakoid membranes, and numerous starch grains mainly in the middle; (5) a senescent bizonoplast: oval-shape (5.5 μm × 7.9 μm), with loosely arranged thylakoid membranes in both upper and lower zones containing many plastoglobuli.
Four photosynthetic key proteins were quantitative and qualitative analysis by immunogold labeling technique between the upper and the lower zones of a bizonoplast: psaA for photosystem I (PSI), psbA for photosystem II (PSII), RbcL for rubisco and AtpA for ATPase. The results show that the upper zone is significantly enriched in PSI than the lower zone, while the lower zone is significantly enriched in PSII and rubisco than the upper zone. There is no significant difference in ATPase between two zones. Based on these results, the unique bizonoplasts with dimorphic ultrastructure may have eco-physiological implications to adapt to deep shade environment. (1) The bizonoplast has better efficiency of capturing photons by having both parallel and horizontally extended thylakoids in the upper zone (rich in PSI, P700). (2) The bizonoplast increases the absorption of high-wavelength photons: the upper zone with higher ratio of PSI helps to absorb the far-infrared light, and the lower zone with higher ratio of PSII (P680) helps to absorb the infrared light. (3) The arrangement of a bizonoplast may have self-photoprotective mechanism: the numerous groups of parallel thylakoids of upper zone (with less PSII) can intercept high-density photons of sun flecks. Due to these thylakoid membranes of the upper zone contain less PSII, and thus less D1 protein of PSII of this upper zone will get damage from sun flecks. This arrangement of groups of parallel thylakoid membranes of an upper zone may help the thylakoid membranes of a lower zone to avoid damaging from high-density photons.
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