Abstract
Ultrastructural investigations of cells and organelles by transmission electron microscopy (TEM) usually lead to two-dimensional information of cell structures without supplying exact quantitative data due to the limited number of investigated ultrathin sections. This can lead to misinterpretation of observed structures especially in context of their three-dimensional (3D) assembly. 3D investigations and quantitative morphometric analysis are therefore essential to get detailed information about the arrangement and the amount of subcellular structures inside a cell or organelle, respectively, especially when the plant sample was exposed to environmental stress. In the present research, serial sectioned chloroplasts, mitochondria, and peroxisomes from first year spruce needles (Picea abies (L.) Karst.) were 3D reconstructed and digitally measured using a computer-supported image analysis system in order to obtain a detailed quantitative characterization of complete cell organelles including precise morphological data of drought-induced fine structural changes. In control plants, chloroplast volume was composed of 56% stroma, 15% starch, 27% thylakoids, and 2% plastoglobules. In drought-stressed chloroplasts, the relative volume of both the thylakoids and the plastoglobules significantly increased to 37% and 12%, respectively. Chloroplasts of stressed plants differed from control plants not only in the mean thylakoid and plastoglobules content but also in the complete lack of starch grains. Mitochondria occurred in variable forms in both control and stressed samples. In stressed plants, mitochondria showed a significant smaller mean volume which was only 81% when compared with the control organelles. Peroxisomes were inconspicuous in both samples and their volume did not differ between control and drought-stressed samples. The present study shows that specific subcellular structures are subject to significant quantitative changes during drought stress of spruce needles giving a detailed insight in adaptation processes of the investigated cell organelles.
Similar content being viewed by others
References
Armstrong AF, Logan DC, Tobin AK, O'Toole P, Atkin OK (2006) Heterogeneity of plant mitochondrial responses underpinning respiratory acclimation to the cold in Arabidopsis thaliana leaves. Plant Cell Environ 29:940–949
Austin JR, Frost E, Vidi PA, Kessler F, Staehelin LA (2006) Plastoglobules are lipoprotein subcompartments of the chloroplast that are permanently coupled to thylakoid membranes and contain biosynthetic enzymes. Plant Cell 18:1693–1703
Bäck J, Neuvonen S, Huttunen S (1994) Pine needle growth and fine structure after prolonged acid rain treatment in the subarctic. Plant Cell Environ 17:1009–1021
Bigras FJ (2005) Photosynthetic response of white spruce families to drought stress. New Forest 29:135–148
Bortz J, Lienert GA, Bohenke K (2000) Verteilungsfreie Methoden in der Biostatistik. Springer, Berlin
Bourett TM, Czymmek KJ, Howard RJ (1999) Ultrastructure of chloroplast protuberances in rice leaves preserved by high-pressure freezing. Planta 208:472–479
Brix H (1979) Effects of plant water stress on photosynthesis and survival of four conifers. Can J For Res 9:160–165
Charon J, Launay J, Carde JP (1987) Spatial organization and volume density of leucoplast in pine secretory cells. Protoplasma 138:45–53
Deo PM, Biswal UC, Biswal B (2006) Water stress-sensitized photoinhibition in senescing cotyledons of clusterbean: changes in thylakoid structures and inactivation of photosystem 2. Photosynthetica 44:187–192
Donohoe BS, Mogelsvang S, Staehelin LA (2006) Electron tomography of ER, Golgi and related membrane systems. Methods 39:154–162
Duan B, Lu Y, Yin C, Junttila O, Li C (2005) Physiological responses to drought and shade in two contrasting Picea asperata populations. Physiol Plant 124:476–484
Escalona JM, Flexas J, Medrano H (1999) Stomatal and non-stomatal limitations of photosynthesis under water stress in field-grown grapevines. Aust J Plant Physiol 26:421–433
Fink S (1988) Histological and cytological changes caused by air pollutants and other abiotic factors. In: Schulte-Hostede S, Darral NM, Blank LW, Wellburn AR (eds) Air pollution and plant metabolism. Elsevier, Amsterdam, pp 36–54
Fink S (1991) Structural changes in conifer needles due to Mg and K deficiency. Nutr Cycling Agroecosyst 27:23–27
Forschner W, Schmitt V, Wild A (1989) Investigations on the starch content and ultrastructure of spruce needles relative to the occurrence of Novel forest decline. Bot Acta 102:208–221
Günthardt-Goerg MS, Vollenweider P (2007) Linking stress with macroscopic and microscopic leaf response in trees: new diagnostic perspectives. Environ Poll 147:467–488
Hanson MR, Sattarzadeh A (2008) Dynamic morphology of plastids and stromules in angiosperm plants. Plant Cell Environ 31:646–657
Hernández JA, Diaz-Vivancos P, Rubio M, Olmos E, Ros-Barceló A, Martínez-Gómez P (2006) Long-term PPV infection produces an oxidative stress in a susceptible apricot cultivar but not in a resistant cultivar. Phys Plant 126:140–152
Holopainen T, Anttonen S, Palomäki V, Kainulainen P, Holopainen JK (1996) Needle ultrastructure and starch content in Scots pine and Norway spruce after ozone fumigation. Can J Bot 74:67–76
Holzinger A, Buchner O, Lütz C, Hanson MR (2007a) Temperature-sensitive formation of chloroplast protrusions and stromules in mesophyll cells of Arabidopsis thaliana. Protoplasma 230:23–30
Holzinger A, Wasteneys GO, Lütz C (2007b) Investigating cytoskeletal function in chloroplast protrusion formation in the arctic-alpine plant Oxyria digyna. Plant Biol 9:400–410
Holzinger A, Kwok EY, Hanson MR (2008) Effects of arc3, arc5 and arc6 mutations on plastid morphology and stromule formation in green and nongreen tissues of Arabidopsis thaliana. Photochem Photobiol 84:1324–1335
Kierzkowski D, Samardakiewicz S, Robakowski P (2007) Variation in ultrastructure of chloroplasts in needles of silver fir (Abies alba Mill.) saplings growing under the canopies of diverse tree species. Pol J Ecol 55:821–825
Kivimäenpää M, Sutinen S (2007) Microscopic structure of Scots pine (Pinus sylvestris (L.)) needles during ageing and autumnal senescence. Trees 21:645–659
Kivimäenpää M, Sutinen S, Karlsson PE, Selldén G (2003) Cell structural changes in the needles of Norway spruce exposed to long-term ozone and drought. Ann Bot 92:779–793
Kivimäenpää M, Jönsson AM, Stjernquist I, Selldén G, Sutinen S (2004) The use of light and electron microscopy to assess the impact of ozone on Norway spruce needles. Environ Pollut 127:441–453
Kivimäenpää M, Selldén G, Sutinen S (2005) Ozone-induced changes in the chloroplast structure of conifer needles, and their use in ozone diagnostics. Environ Pollut 137:466–475
Lo YS, Hsiao LJ, Jane WN, Charng YC, Dai H, Chiang KS (2004) GFP-targeted mitochondria show heterogeneity of size, morphology, and dynamics in transgenic Nicotiana tabacum L. plants in vivo. Int J Plant Sci 165:949–955
Logan DC (2006a) Plant mitochondrial dynamics. Biochim Biophys Acta 1763:430–441
Logan DC (2006b) The mitochondrial compartment. J Exp Bot 57:1225–1243
Lütz C, Engel L (2007) Changes in chloroplast ultrastructure in some high-alpine plants: adaptation to metabolic demands and climate? Protoplasma 231:183–192
Lütz C, Moser W (1977) On the cytology of high alpine plants. The ultrastructure of Ranunculus glacialis. Flora 166:21–34
McIntosh R, Nicastro D, Mastronarde D (2005) New views of cells in 3D: an introduction to electron tomography. Trends Cell Biol 15:43–51
Menzel D (1994) An interconnected plastidom in Acetabularia: implications for the mechanism of chloroplast motility. Protoplasma 179:166–171
Munné-Bosch S, Jubany-Marí T, Alegre L (2001) Drought-induced senescence is characterized by a loss of antioxidant defences in chloroplasts. Plant Cell Environ 24:1319–1327
Mustárdy L, Buttle K, Steinbach G, Garab G (2008) The three-dimensional network of the thylakoid membranes in plants: quasihelical model of the granum–stroma assembly. Plant Cell 20:2552–2557
Natesan SKA, Sullivan JA, Gray JC (2005) Stromules: a characteristic cell-specific feature of plastid morphology. J Exp Bot 56:787–797
Ögren E, Öquist G (1985) Effects of drought on photosynthesis, chlorophyll fluorescence and photoinhibition susceptibility in intact willow leaves. Planta 166:380–388
Olmos E, Sanchez-Blanco MJ, Ferrendez T, Alarcon JJ (2007) Subcellular effects of drought stress in Rosmarinus officinalis. Plant Biol 9:77–84
Pechová R, Kutík J, Holá D, Kočová M, Haisel D, Vičánková A (2003) The ultrastructure of chloroplasts, content of photosynthetic pigments, and photochemical activity of maize (Zea mays L.) as influenced by different concentrations of the herbicide amitrole. Photosynthetica 4:127–136
Peltier JP, Marigo G (1999) Drought adaptation in Fraxinus excelsior L.: physiological basis of the elastic adjustment. J Plant Physiol 154:529–535
Pfeiffer S, Krupinska K (2005) New insights in thylakoid membrane organization. Plant Cell Physiol 46:1443–1451
Rantanen L, Palomäki V, Harrison AF, Lucas PW, Mansfield TA (1994) Interactions between combined exposure to SO2 and NO2 and nutrient status of trees: effects on nutrient content and uptake, growth, needle ultrastructure and pigments. New Phytol 128:689–701
Rozak PR, Seiser RM, Wacholtz WF, Wise RR (2002) Rapid, reversible alterations in spinach thylakoid appression upon changes in light intensity. Plant Cell Environ 25:421–429
Ruetze M, Schmitt U (1988) Histologie der Alterung von Fichtennadeln (Picea abies (L.) Karst.). Angew Bot 62:9–20
Schiffgens-Gruber A, Lütz C (1992) Ultrastructure of mesophyll cell chloroplasts of spruce needles exposed to O3, SO2 and NO2 alone and in combination. Environ Exp Bot 32:243–254
Shimoni E, Rav-Hon O, Ohad I, Brumfeld V, Reich Z (2005) Three-dimensional organization of higher-plant chloroplast thylakoid membranes revealed by electron tomography. Plant Cell 17:2580–2586
Siefermann-Harms D, Boxler-Baldoma C, Von Wilpert K, Heumann HG (2004) The rapid yellowing of spruce at a mountain site in the Central Black Forest (Germany). Combined effects of Mg deficiency and ozone on biochemical, physiological and structural properties of the chloroplasts. J Plant Physiol 161:423–437
Sutinen S (1987) Cytology of Norway spruce needles. II. Changes in yellowing spruces from the Taunus mountains, West Germany. Eur J For Path 17:74–85
Sutinen S, Skärby L, Wallin G, Sellden G (1990) Long-term exposure of Norway spruce, Picea abies (L.) Karst. to ozone in opentop chambers. New Phytol 115:345–355
Thomas H (1997) Chlorophyll a: a symptom and a regulator of plastid development. New Phytol 136:163–181
Whatley JM, Hawes LR, Horne JC, Kerr JDA (1982) The establishment of the plastid thylakoid system. New Phytol 90:619–629
Wheeler WS, Fagerberg WR (2000) Exposure to low levels of photosynthetically active radiation induces rapid increases in palisade cell chloroplast volume and thylakoid surface area in sunflower (Helianthus annuus L.). Protoplasma 212:38–45
Wulff A, Ahonen J, Kärenlampi L (1996) Cell ultrastructural evidence of accelerated ageing of Norway spruce needles in industrial areas. New Phytol 133:553–561
Yang Y, Liu Q, Han C, Qiao YZ, Yao XQ, Yin HJ (2007) Influence of water stress and low irradiance on morphological and physiological characteristics of Picea asperata seedlings. Photosynthetica 45:613–619
Yordanov I, Velikova V, Tsonev T (2000) Plant responses to drought, acclimation, and stress tolerance. Photosynthetica 38:171–186
Yoshinaga K, Arimura SI, Niwa Y, Tsutsumi N, Uchimiya H, Kawai-Yamada M (2005) Mitochondrial behaviour in the early stages of ROS stress leading to cell death in Arabidopsis thaliana. Ann Bot 96:337–342
Ytterberg AJ, Peltier JB, Van Wijk KJ (2006) Protein profiling of plastoglobules in chloroplasts and chromoplasts. A surprising site for differential accumulation of metabolic enzymes. Plant Physiol 140:984–997
Zechmann B, Müller M, Zellnig G (2003) Cytological modifications in zucchini yellow mosaic virus (ZYMV)-infected Styrian pumpkin plants. Arch Virol 148:1119–1133
Zechmann B, Mauch F, Sticher L, Müller M (2008) Subcellular immunocytochemical analysis detects the highest concentrations of glutathione in mitochondria and not in plastids. J Exp Bot 59:4017–4027
Zellnig G, Perktold A (2003) Diurnal variation of chloroplast fine structures of spinach. Acta Biol Slov 46:43–47
Zellnig G, Zechmann B, Perktold A (2004) Morphological and quantitative data of plastids and mitochondria within drought stressed spinach leaves. Protoplasma 223:221–227
Acknowledgements
We thank Ing. Gerhard Graggaber for skillful technical assistance. This work was supported by the Austrian Science Fund (P13614-BIO and P-15374 to G.Z.).
The authors declare that they have no conflict of interest.
Author information
Authors and Affiliations
Corresponding author
Additional information
Dedicated to Professor Cornelius Lütz on occasion of his 65th birthday
Rights and permissions
About this article
Cite this article
Zellnig, G., Perktold, A. & Zechmann, B. Fine structural quantification of drought-stressed Picea abies (L.) organelles based on 3D reconstructions. Protoplasma 243, 129–136 (2010). https://doi.org/10.1007/s00709-009-0058-3
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00709-009-0058-3