Cell structural changes in the needles of Norway spruce exposed to long-term ozone and drought

Abstract

Effects of ozone and/or drought on Norway spruce needles were studied using light microscopy and electron microscopy. Saplings were exposed to ozone in open-top chambers during 1992-1995 and also to drought in the late summers of 1993-1995. Samples from current and previous year needles were collected five times during 1995. Ozone increased the numbers of peroxisomes and mitochondria, which suggests that defence mechanisms against oxidative stress were active. The results from peroxisomes suggest that the oxidative stress was more pronounced in the upper side of the needles, and those from mitochondria that defence was more active in the younger needle generation. Possibly due to the good nitrogen status and the active defence, no ozone-specific chloroplast alterations were seen. At the end of the season, older needles from ozone treatments had smaller central vacuoles compared with other needles. Cytoplasmic vacuoles around the nucleus were increased by ozone in the beginning of the experiment, and did not increase towards the end of the season as in the controls. These results from vacuoles may indicate that ozone affected the osmotic properties of the cells. Decreased number and underdevelopment of sclerenchyma cells and proliferation of tonoplast were related to nutrient imbalance, which was enhanced by drought. Larger vascular cylinders and more effective starch accumulation before and after the drought periods compensated for the reduced water status. Numbers of peroxisomes and mitochondria were increased in the drought-exposed needles before the onset of drought treatments of the study year, i.e. these changes were memory effects. Interactions between ozone and drought were few.

Fig. 1. Schematic illustrations of cross‐sections of the mesophyll tissue of two spruce needles. The central circle is the vascular cylinder. Larger squares on the abaxial side of one needle and adaxial side of another needle show the areas of mesophyll tissue photographed with the light microscope. Smaller squares show the areas of mesophyll tissue in the thin sections, and black ovals show the area from which photographs were taken using a transmission electron microscope.

Fig. 2. A, Typical cross‐sectional area of the mesophyll tissue of a Norway spruce needle analysed by light microscopy. Arrows indicate cells with cytoplasmic vacuoles encircling the nucleus (N). Black granules inside the vacuole (V) are phenolic compounds (tannins). Ic, intercellular space. Current year needle collected from CF/D on 4 Oct. Bar = 50 µm. B, Typical cytoplasmic area analysed by transmission electron microscopy. White asterisks indicate mitochondria and black asterisks peroxisomes. C, Chloroplast; St, starch grain; Pg, plastoglobuli; W, cell wall. The black material at the left edge of the micrograph is vacuolar tannin lining the tonoplast. Current year needle collected from NF+/D on 4 Oct. Bar = 1 µm. C, Underdeveloped (thin cell walls, arrow) and well‐developed (thick cell walls, *) sclerenchyma cells in healthy looking vascular cylinder. E, endodermis; P, phloem; X, xylem. Current year needle collected from NF+/D on 28 July. Bar = 50 µm. D, Current year needle from NF+/D showing increased cytoplasmic lipid aggregates (L), which were increased by ozone in the current year needles. The low amount of plastoglobuli was typical to current year needles. Bar = 1 µm.

Fig. 3. Average length of starch grains in the endodermis (with s.e.) measured in the current (C) and second (C+1) year needles of Norway spruce saplings from four treatments: charcoal‐filtered air, well‐watered (white), charcoal‐filtered air, drought‐stressed (black), non‐filtered air with added ozone, well‐watered (vertical lines), non‐filtered air with added ozone, drought‐stressed (horizontal lines). The samples were analysed before the periods of reduced water supply (28 July) and at the end of the experiment, after re‐watering (4 Oct.). For statistics, see text.

Fig. 4. A, Proliferation of the tonoplast (arrows) was typical in drought treatments (cf. B and D). Current year needle collected from CF/D on 4 Oct. Bar = 1 µm. B, Negatively stained, bent thylakoids (T) fill nearly all the chloroplast area in a mesophyll cell. Stroma (S) is electron‐dense. Mitochondria (white asterisks) and peroxisomes (black asterisks) can also be seen. Arrows point to tonoplast of the cytoplasmic vacuoles (or invaginations of the central vacuole). Current year needle collected from CF/W on 18 Aug. Bar = 1 µm. C, Non‐osmiophilic plastoglobuli (Pg) were seen in all treatments, although they were on average more common in the older needles. Previous year needle from NF+/D on 16 Aug. Bar = 1 µm. D, The amount of plastoglobuli (Pg) was higher in the previous than current year needle generation (cf. Fig. 2D). The tonoplast is indicated by an arrow. Previous year needle collected from CF/W on 4 Oct. Bar = 1 µm.

Fig. 5. Mean number of peroxisomes in 100 µm² cytoplasm (with s.e., upwards for triangles and downwards for squares) in the mesophyll cells of needles of Norway spruce saplings exposed to charcoal‐filtered air (squares) or ozone (non‐filtered air × 1·5 ambient ozone concentration; triangles) and kept well‐watered (closed symbols) or drought‐stressed (open symbols) during a 10‐week study period in 1995. Samples from 16 Aug. and 18 Sept. were from periods of reduced water supply. For clarity, the aggregated values from current and second year needles from cells from upper and lower sides of the needles are presented. Significant interactions of drought × time (P = 0·002) and of drought × ozone × time (P = 0·038) were not modified by needle age or needle side.