Rapid, Reversible Alterations in Spinach Thylakoid Appression Upon Changes in Light Intensity
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Blackwell Science, LtdOxford, UK PCEPlant, Cell and Environment0016-8025Blackwell Science Ltd 2002 253March 2002 823 Rapid changes in thylakoid stacking P. R. Rozak et al. 10.1046/j.0016-8025.2001.00823.x Original ArticleBEES SGML Plant, Cell and Environment (2002) 25, 421–429 Rapid, reversible alterations in spinach thylakoid appression upon changes in light intensity P. R. ROZAK,1 R. M. SEISER,3,* W. F. WACHOLTZ2 & R. R. WISE1 Departments of 1Biology and 2Chemistry, University of Wisconsin-Oshkosh, Oshkosh, WI 54901-8640, USA and 3Department of Chemistry, Lawrence University, Appleton WI 54912, USA ABSTRACT more thylakoids per individual granum than chloroplasts in shade leaves (Anderson, Goodchild & Boardman 1973; Changes in light quantity and quality cause structural Boardman et al. 1975; Lichtenthaler et al. 1981). These dif- changes within the thylakoid membrane; long-term ferences can be seen between plants native to high- or low- responses have been described for so-called ‘sun’ and light habitats (Björkman & Holmgren 1963) as well as ‘shade’ leaves. Many leaves, however, experience changes in leaves at the interior or exterior of the canopy in an indi- irradiance on a time scale of minutes due to self-shading vidual plant (Lichtenthaler 1985). and sun flecks. In this study, mature, attached spinach In addition to long-term differences between sun and leaves were grown at 300 µmol photons m−2 s−1 then rapidly shade environments, many plants experience rapidly chang- switched to a different light treatment. The treatment irra- ing light conditions in the form of sun flecks, during which diances were 10, 800 or 1500 µmol m−2 s−1 for 10 min, or 10 irradiances can vary from less than 10 to over 1000 µmol or 20 min of self-shading (about 10 µmol m−2 s−1). Image photons m−2 s−1 in a matter of seconds (Pearcy 1990). These analysis of transmission electron micrographs revealed that rapid changes in irradiance can lead to photo-inhibition a 10 min switch to a lower light intensity increased grana (Powles & Björkman 1981; Raven 1989). In response, chlo- size and number per chloroplast profile by 10–20%. roplasts are capable of rapid biochemical and bioenergetic Returning the leaves to 300 µmol m−2 s−1 for 10 min reversed adjustments via mechanisms such as state transitions (Ben- the phenomenon. Chlorophyll fluorescence measurements nett 1991). State transitions allow chloroplasts to balance of detached, intact leaves at 77 K were suggestive of a tran- relative turnover rates of the two photosystems due to the sition from state 2 to state 1 upon shading. Diurnal ultra- mobility of the antenna proteins surrounding photosystem structural measurements of granal size and number did not II (PSII) (Allen 1992) and the extreme lateral heterogene- reveal a significant net change in ultrastructure over the ity of the protein complexes in the thylakoid membrane time scale of hours. It is concluded that spinach chloroplasts (Andersson & Anderson 1980). Under state 2 conditions can alter the degree of thylakoid appression in response to (moderate, high, or 650 nm light) a portion of the light har- irradiance changes on a time scale of minutes. These ultra- vesting chlcrophyll binding proteins of PSII (LHCII) is structural responses are caused by biochemical and bio- phosphorylated and the resulting LHCII-PO is forced to physical adjustments within the thylakoid membrane that 4 migrate from the appressed region to the nearest unap- serve to maximize photosynthesis and minimize photo- pressed region. Under state 1 conditions (darkness, shade, inhibition under rapidly fluctuating light environments. or 710 nm light) LHCII-PO4 is dephosphorylated, and LHCII returns to an appressed region (Staehelin 1976; Key-words: chloroplast ultrastructure; sun flecks; thylakoid Telfer et al. 1983). Measurements of 77 K chlorophyll fluo- appression; thylakoid stacking. rescence in both isolated thylakoids and intact leaves dem- onstrate that a portion of the LHCII antennae proteins serving PSII under state 1 conditions becomes energetically INTRODUCTION disconnected from PSII under state 2 conditions (Bennett, Leaves that develop under high light are physiologically Steinback & Arntzen 1980; Dau & Hansin 1988; Bennett and anatomically distinct from those that develop in the 1991). Because the mechanism maintaining thylakoid shade. In comparison to ‘shade’ leaves, ‘sun’ leaves are appression is based on a mutual attraction between LHCIIs thicker, smaller, contain more protein and chlorophyll, and in adjacent thylakoids (Ryrie, Anderson & Goodchild 1980; have higher light-saturated photosynthetic rates (Board- Sculley et al. 1980; Chow, Miller & Anderson 1991), any man 1977; Cui, Vogelmann & Smith 1991). Chloroplast change in the amount of LHCII associated with PSII would ultrastructure is also responsive to growth light intensity, in be expected to: (1) alter the amount of energy being fun- that chloroplasts from sun leaves have more grana and neled into that photosystem; (2) influence the extent of thy- lakoid appression; and (3) change the size of the individual Correspondence: Dr Robert R. Wise. Fax: +1 920 424 3404; grana stacks. E-mail: [email protected] Given that: chloroplast ultrastructure in sun and shade *Present address: Department of Cell Biology, Duke University Medical plants responds in a predictable fashion to long-term dif- Center, Durham, NC 27710, USA. ferences in growth light intensity, leaves are frequently © 2002 Blackwell Science Ltd 421 422 P. R. Rozak et al. subjected to rapid changes in irradiance, and short-term given above for growth-chamber-grown plants. Plants were biochemical adjustments that influence thylakoid stacking sampled on 8 September 1999, at 0530 h (26 min before civil are possible, we sought to determine whether chloroplast twilight), 1252 h (solar transit), and 2040 h (53 min after ultrastructural adjustments to varying light intensities are civil twilight). The times for these three events were taken solely a long-term growth response or if they may be from the US Naval Observatory Astronomical Applications attributable to the short-term bioenergetic status of the Department web page (http://aa.usno.navy.mil/AA/data/). chloroplast itself. The fundamental question may be posed, Diurnal solar irradiance was measured every 2 min (data ‘how dynamic is thylakoid stacking?’ An answer to this not shown) and logged using the data log function on a self- question was sought by subjecting spinach leaves to a vari- recording infrared gas analyzer (PP Systems, Haverhill, ety of irradiance treatments that mimicked sunflecks in the MA, USA). field. The size and number of grana in two-dimensional chloroplast sections from these leaves were assessed by Electron microscopy transmission electron microscopy (TEM) and image analysis. At the termination of the light treatment the leaf tissue was excised and immediately cut into 0·5 mm2 segments in a pool of 2·5% glutaraldehyde in a sodium cacodylate buffer MATERIALS AND METHODS (0·1 mol m−3, pH 7·2) and kept under the treatment light conditions for 10 min. Fixation continued under dim light Plant material for 60 min, then the samples were rinsed four times in the Spinach (Spinacia oleracea L. cv Bloomsdale Longstanding; same buffer for 15 min each. Post-fixation was in 1% OsO4 Excel Seeds, Minneapolis, MN, USA) seeds were sown in a in the cacodylate buffer for 60 min followed by two 5 min commercial potting soil in 10 cm × 10 cm pots. Plants were water rinses. Dehydration was in a graded ethanol series. raised in a Percival model 35LLVLX growth chamber (Per- All fixation and dehydration steps were at room tempera- cival Scientific, Boone, IA, USA) under a photoperiod of ture. Tissue was embedded in Spurr’s (1969) resin and poly- 12 h light : 12 h dark (300 µmol m−2 s−1 PAR) supplied by merized at 60 °C for 8 h. Thin (∼70 nm) sections were cut 40 W GE Soft White fluorescent bulbs at 23 °C. All irradi- using a diamond knife on a Leica Ultracut UCT ultrami- ances were measured with a Li-Cor LI 189 light meter crotome and contrasted in 0·02 N calcined lead (Haniachi et equipped with a LI 190SA quantum sensor (Li-Cor Inc., Lin- al. 1986) for 2 min, prior to viewing with a Zeiss EM10CA coln, NE, USA). Relative humidity averaged 80%. Plants transmission electron microscope at 60 kV. All chloroplast were watered daily and fertilized every other day (Miracle- profiles were photographed at 10 000 × magnification. Mag- Gro nutrient solution, 15-30-10 N-P-K, half strength, Scott’s nification was calibrated by photographing a test standard Co., Marysville, OH, USA). Fully mature leaves after 6 (replica of a 73 152 lines per cm block, Ladd Red. Ind, Bur- weeks of growth were chosen for experimentation. lington, VT, USA) at the same magnification. Only fully dif- ferentiated chloroplasts from the palisade mesophyll layer were chosen for analysis in order to reduce variability due Experimental light treatments to tissue effects (cf. Terashima & Inoue 1985). Negatives of All light treatments were performed on intact, attached selected chloroplasts were printed on Kodak Polycontrast leaves. Nine separate experiments were performed. Fully photographic paper. Clear acetate film was laid over pho- dark-adapted, growth-chamber-grown leaf tissue was col- tographs and grana were traced by hand with a permanent lected after 11·5 h of darkness just prior to lights on and marker. The tracings were digitized to TIFF format files designated as ‘dark-adapted control’. The light-adapted using a flatbed scanner. control was 2 h of 300 µmol m−2 s−1, i.e. 2 h after lights on. Five additional experiments were performed by switching Sampling for morphometric analysis light-adapted control plants to either more or less light (see Table 2). Self-shading was effected by detaching a spinach Three epoxy blocks from each of the nine light treatments leaf from a separate plant and laying it on top of a treat- were randomly selected for sectioning.