Three-Dimensional Analysis of the Mesophyll Plastids Using Ultra High Voltage Electron Microscopy

초고압전자현미경에 의한 엽육세포 색소체 미세구조의 3차원적 분석

  • Kim, In-Sun (Biology Department, College of Natural Sciences, Keimyung University) ;
  • Park, Sang-Chan (College of Life Sciences and Biotechnology, Division of Life Sciences, Korea University) ;
  • Han, Sung-Sik (College of Life Sciences and Biotechnology, Division of Life Sciences, Korea University) ;
  • Kim, Eun-Soo (Department of Biological Sciences, College of Science, Kunkuk University)
  • 김인선 (계명대학교 자연과학대학 생물학과) ;
  • 박상찬 (고려대학교 생명과학대학 생명과학부) ;
  • 한성식 (고려대학교 생명과학대학 생명과학부) ;
  • 김은수 (건국대학교 이과대학 생명과학과)
  • Published : 2006.09.30

Abstract

Image processing by ultra high voltage electron microscopy (UHVEM) and tomography has offered major contributions to research in the field of cellular ultrastructure. Furthermore, such advancements also have enabled the improved analysis of three-dimensional cellular structures in botany. In the present study. using UHVEM and tomography, we attempted to reconstruct the three-dimensional images of plastid inclusions that probably differentiate during photosynthesis. The foliar tissues were studied Primarily with the TEM and further examined with UHVEM. The spatial relationship between tubular elements and the thylakoidal membrane and/or starch grains within plastids mainly have been investigated in CAM-performing Sedum as well as in $C_4$ Salsola species. The inclusion bodies were found to occur only in early development in the former, while they were found only in mesophyll cells in the latter. The specimens were tilted every two degrees to obtain two-dimensional images with UHVEM and subsequently comparison has been made between the two types. Digital image processing was performed on the elements of the inclusion body using tilting, tomography, and IMOD program to generate and reconstruct three-dimensional images on the cellular level. In Sedum plastids, the inclusion bodies consisted of tubular elements exhibiting about 20 nm distance between elements. However, in Salsola, plastid inclusion bodies demonstrated quite different element structure, displaying pattern, and origin relative to those of the Sedum. The inclusion bodies had an integrative relationship with the starch grains in both species.

발달중인 Sedum 및 Salsola의 엽육조직을 chemical fixation과 high pressure freezing (HPF) 등으로 고정한 후, 초박 및 후박절편으로 제작 carbon coating하여 TEM 및 UHVEM으로 연속절편에 의한 2-D영상과 tilt image data를 수집하였다. 이후 초미세 구조들에 대하여 tilting 및 tomography 기법, 그리고 디지털화한 image의 3-D 입체구조 재구현에 필수적인 IMOD 프로_그램을 적용한 image 처리과정을 거쳐 UHVEM data에서 색소체내 초미세구조의 정보를 추출하여 세포수준에서의 3-D image를 분석하였다. 색소체 기질에서 녹말입자 및 틸라코이드에 인접하여 형성되는 CAM및 $C_4$ 식물 색소체 결정체들은 어떤 막으로도 둘러싸이지 않는 구조로서, Sedum rotundifolium 색소체내 결정체는 수 ${\mu}m$에 이르는 커다란 크기로 형성된다. 결정체 내에는 약 20nm격자거리로 이루어진 기원을 알 수 없는 수백-수천 개의 미세소관성 요소들이 평행 또는 격자상태로 정교한 구조를 이루며, 티라코이드 및 녹말입자와 인접하여 발달하였다. $C_4$ 광합성 수행 Salsola komarovii의 경우, 결정체는 엽육세포 색소체에서만 발달하며 결정체 구성 기본요소들이 비교적 규칙적인 격자거리를 이루며 수십 개 배열하는 구조를 형성하였다. 특히, tilted image및 3-D 입체구조 연구에서 결정체 형성에는 이들과 인접하여 발달하는 틸라코이드막이 관여함을 알아 낼 수 있었다. 이는 엽육세포 색소체에는 결정체들이 식물이 수행하는 광합성 유형에 따라 각기 다른 구성요소로 형성되어 Sedum의 경우와 같이 발달 중인 엽육조직에서 분화하거나 Salsola에서와 같이 세포 유형에 따라 상이하게 발달하는 것으로 추정되었다.

Keywords

References

  1. Frank J, Wagenknecht T, McEwen BF, Marko M, Hsieh C, Mannella CA: Three-dimensional imaging of biological complexity. J Struct Biol 138 : 85-91, 2002 https://doi.org/10.1016/S1047-8477(02)00019-9
  2. Giddings TH: Freeze-substitution protocols for improved visualization of membranes in high-pressure frozen samples. J Microsc 212 : 53-61, 2003 https://doi.org/10.1046/j.1365-2818.2003.01228.x
  3. Gilbert PFC: Reconstruction of a 3-dimensional structures from projections and its application to electron microscopy II. Direct methods. Proc R Soc Lond B Biol Sci 182 : 89-102, 2002
  4. Gilky JC, Staehelin LA: Advances in ultra-rapid freezing for the preservation of cellular ultrastructure. J Electron Microsc Tech 3 : 177-210, 1986 https://doi.org/10.1002/jemt.1060030206
  5. Gunning BES, Steer MW: Plant Cell Biology: Structure and Function. Jones and Bartlett Publishers, Boston, pp. 1-60, 1996
  6. Katsuna N, Kumagai F, Sato MH, Hasezawa S: Three-dimensional reconstruction of tubular structure of vacuolar membrane throughout mitosis in living tobacco cells. Plant Cell Physiol 44 : 1045-1054, 2003 https://doi.org/10.1093/pcp/pcg124
  7. Kim I: Chloroplast microtubules in young leaves of Sedum rotundifolium. J Plant Biol 40 : 115-119, 1997 https://doi.org/10.1007/BF03030243
  8. Kim I: High Voltage Electron Microscopy of Structural Patterns of Plastid Crystalline Bodies in Sedum rotundifolium. Kor J Electron Microsc 36 : 73-82, 2006a
  9. Kim I: Changes in plastid ultrastructure during Sedum rotundifolium leaf development. J Plant Biol 49, 2006b. (in press) https://doi.org/10.1007/BF03178815
  10. Kim I, Fisher DG: Structural aspects of the leaves of seven species of Portulaca leaves growing in Hawaii. Can J Bot 68 : 1803-1811, 1990 https://doi.org/10.1139/b90-233
  11. Koster AJ, Grimm R, Typke D, Heherl R, Stoscheck A, Walz J, Baumeister W: Perspectives of molecular and cellular electron microscopy. J Struct Biol 120 : 276-308, 1997 https://doi.org/10.1006/jsbi.1997.3933
  12. Kremer JR, Mastronarde DN, McIntosh JR: Computer visualization of the three-dimensional image data using IMOD. J Struct Biol 116 : 71-76, 1996 https://doi.org/10.1006/jsbi.1996.0013
  13. Lee YN: Flora of Korea. Kyo-Hak Publishing Co., Seoul, p. 271, 1996
  14. Mastronarde DN: Dual axis tomography: An approach with alignment methods that preserve resolution. J Struct Biol 120 : 343-352, 1997 https://doi.org/10.1006/jsbi.1997.3919
  15. McDonald MS: Photobiology of Higher Plant. Wiley, Chichester, pp. 148-198, 2003
  16. Otegui MS, Mastronarde DN, Kang BH, Bednarek SY, Staehelin LA: Three-dimensional analysis of syncytial-type cell plates during endosperm cellularization visualized by high resolution electron microscopy. Plant Cell 13 : 2033-2051, 2001 https://doi.org/10.1105/tpc.13.9.2033
  17. Otegui MS, Staehelin LA: 3D tomographic analysis of postmeiotic cytokinesis during pollen development in Arabidopsis thaliana. Planta 218 : 501-515, 2004 https://doi.org/10.1007/s00425-003-1125-1
  18. Pyankov V, Ziegler H, Kuzmin A, Edwards G: Origin and evolution of $C_4$ photosynthesis in the tribe Salsoleae (Chenopodiaceae) based on anatomical and biochemical types in leaves and cotyledons. Plant Syst Evol 230 : 43-74, 2001 https://doi.org/10.1007/s006060170004
  19. Reiner H: The EM program package : A platform for image processing in biological electron microscopy. J Struct Biol 116 : 30-34, 1996 https://doi.org/10.1006/jsbi.1996.0006
  20. Ryberg H, Ryberg M, Sundqvist C: Plastid ultrastructure and development. In: Sundqvist C, Ryberg M, eds, Pigment- Protein Complexes in Plastids: Synthesis and Assembly, pp. 25-62, Academic Press, San Diego, 1993
  21. Sage RF, Monson RK: $C_4$ Plant Biology. Academic Press, San Diego. pp. 49-209, 1999
  22. Saito C, Ueda T, Abe H, Wada Y, Kuroiwa T, Hisada A, Furuya M, Nakank A: A complex and mobile structure forms a distinct subregion within the continuous vacuolar membrane in young cotyledons of Arabidopsis. Plant J 29 : 245-255, 2002 https://doi.org/10.1046/j.0960-7412.2001.01189.x
  23. Salemma R, Brandao I: Development of microtubules in chloroplasts of two halophytes forced to follow Crassulacean acid metabolism. J Ultrastruct Res 62 : 132-136, 1978 https://doi.org/10.1016/S0022-5320(78)90026-6
  24. Santos I, Salemma R: Chloroplast microtubules in some CAM plants. Bol Soc Brot Ser 2, 53 : 1115-1122, 1981
  25. Santos I, Salemma R: Stereological study of the variation of chloroplast tubules and volume in the CAM plant Sedum telephium. Z Pflanzenphysiol 113 : 29-37, 1983 https://doi.org/10.1016/S0044-328X(83)80016-6
  26. Segui-Shimarro JM, Austin JR, White EA, Staehelin LA: Electron tomographic analysis of somatic cell plate formation in meristematic cells of Arabidopsis preserved by high-pressure freezing. Plant Cell 16 : 836-856, 2004 https://doi.org/10.1105/tpc.017749
  27. Smith JD, Todd P, Staehelin LA: Modulation of statolith mass and growing in white clover (Trifolium repens) grown in 1-g, microgravity and on the clinostat. Plant J 12 : 1361-1373, 1997 https://doi.org/10.1046/j.1365-313x.1997.12061361.x
  28. Verma DPS, Hong Z: The ins and outs in membrane dynamics: tubulation and vesiculation. Trends Pl Sci 10 : 159-165, 2005 https://doi.org/10.1016/j.tplants.2005.02.004
  29. Voznesenskaya EV, Franceschi V, Artyusheva EG, Black CC, Pyankov VI, Edwards E: Development of the$C_4$ photosynthetic apparatus in cotyledons and leaves of Salsola richteri (Chenopodiaceae). Int J Plant Sci 164 : 471-487, 2003 https://doi.org/10.1086/374828
  30. Waters M, Pyke K: Plastid development and differentiation. In: Moller SG, ed, Plastids, pp. 30-59, Blackwell Publishing, Oxford, 2005