Does availability of potassium affect cold hardening of Scots pine through polyamine metabolism?

The effect of different potassium availability on the polyamines and frost resistance of Scots pine seedlings ( Pinus sylvestris L.) during cold hardening was studied. Scots pine seedlings were grown applying different rates of potassium by using the relative addition rate technique followed by a 2- or 9-week hardening period with decreased light intensity, day length and temperature. After 2 weeks of treatment the seedlings were not hardened (LT₅₀, =−11°C) and showed no differences in frost resistance, although differences in the polyamine levels between the K levels were observed. After 9 weeks of hardening the seedlings at the low, medium and high K levels showed a mean frost resistance (LTs.₅₀) of −81, −63, and −47°C, respectively. A negative effect of K on the frost resistance of the needles was also found in adult trees in September. The results indicate that at the early stage of cold hardening, potassium or free polyamine levels do not affect the frost resistance of Scots pine needles. However, in hardened seedlings and adult trees potassium displays a negative and putrescine a positive correlation with frost resistance, whereas spermidine and spermine show no correlation.     

Slightly elevated ozone exposure causes cell structural changes in needles and roots of Scots pine

Scots pine (Pinus sylvestris L.) seedlings were fumigated with 1.2–1.5 x ambient ozone over 2 seasons in an open-air experiment. Fumigation started in the early spring and continued into late autumn during both years. Needle and root cell structures were analyzed in the summer, autumn and early winter following the second fumigation period. Under the light microscope an increase in the intercellular space and disintegrating cells in the mesophyll tissue near the stomata and stomatal cavities were observed in the ozone-exposed needles. Darkening of chloroplast stroma, increased plastoglobulus size and decreased chloroplast size were characteristic ultrastructural changes associated with ozone exposure. In addition, less dense grouping of the chloroplasts in the needles of elevated ozone-exposed seedlings as compared to the controls (background ozone) was observed in the early winter. Fewer starch grains and an increased accumulation of tannin-like substances were detected in both mycorrhizal and uninfected roots of ozone-exposed seedlings as compared to the control seedlings. For the first time, we were able to show that the ozone-induced darkening of needle chloroplast stroma is a reversible symptom. An increased frequency of frost injury symptoms indicated that the winter hardening process was disturbed in the needles of ozone-treated seedlings.     

Carbohydrate distribution and cellular injuries in acid rain and cold-treated spruce needles

Summary The cellular structures of acid rain-irrigated needles of several provenances of Norway spruce (Picea abies L. Karst) seedlings were studied after winter experimental freezing. Frost injuries and recovery were characterized by visual damage scoring and classification of mesophyll cell alterations, also using histochemical methods for carbohydrate fluorescent staining. The treatment with-30° C during the late dormancy period was sufficient to cause significant injuries and intracellular degradation in the tissues of the green needles. The most affected seedlings in terms of visual injury scoring were found among those treated with clean water or at pH 3, while freezing injury, defined as an occlusion of phenolic substances in the central vacuole of the mesophyll cells, was most abundant in the needles from spruces irrigated either with clean water or at pH 4 or pH 3. Electron microscopy revealed the details of the injury, e. g. thinning out of the cytoplasm and chloroplast stroma, darkening of the chloroplasts and eventually swelling of the chloroplasts and protoplast. PAS and ConA reactions in the needle tissue revealed intense starch accumulation in the mesophyll and transfusion tissues as early as in March, with a tendency to increase, especially in the untreated needles during the recovery period. Plasma membrane disturbances were indicated by histochemical identification of callose deposits in the mesophyll cell walls, these being most abundant in the acid rain-treated needles. All these findings suggest that freezing at –30° C was more deleterious to the seedlings pretreated with acid or clean water than to those not given additional irrigation.     

Effects of drought and waterlogging on ultrastructure of Scots pine and Norway spruce needles

Effects of water stress on needle ultrastructure of 2-year-old Scots pine (Pinus sylvestris L.) and 5-year-old Norway spruce [Picea abies (L.) Karst.] seedlings were studied in greenhouse experiments. Drought stress was induced by leaving seedlings without watering, and waterlogging stress was produced by submerging the seedling containers in water. Needle samples for ultrastructural analyses were collected several times during the experiments, and samples for nutrient analyses at the end of the experiments. In drought stress, plasmolysis of mesophyll and transfusion parenchyma tissues, aggregation of chloroplast stroma and its separation from thylakoids and decreased size and abundance of starch grains in needles of both species were observed. The concentration of lipid bodies around the chloroplasts were detected in pine needles. Calcium and water concentrations in spruce needles were lower by the end of the experiments compared to controls. In waterlogging treatment, swelling of phloem cells in pine needles and large starch grains, slight swelling of thylakoids and increased translucency of plastoglobuli in chloroplasts of both species studied were observed. The phosphorus concentration in pine needles was higher while phosphorus, calcium and magnesium concentrations in spruce needles were lower in the waterlogging treatments compared to controls. Typical symptoms induced by drought stress, e. g. aggregation of chloroplast stroma and its separation from thylakoids, were detected, but, in waterlogging stress, ultrastructural symptoms appeared to be related to the developing nutrient imbalance of needles.     

Influence of elevated ozone and limited nitrogen availability on conifer seedlings in an open-air fumigation system: effects on growth, nutrient content, mycorrhiza, needle ultrastructure, starch and secondary compounds

Scots pine (Pinus sylvestris L.) and Norway spruce (Picea abies Karst.) seedlings were exposed to realistically elevated O₃ levels in open‐air experiments over three growing seasons. The total O₃ exposure doses were 1.2 × (1991), 1.5 × (1992) and 1.7 × (1993) ambient levels. During the 1992 and 1993 growing seasons pine and spruce seedlings received two different levels of nitrogen supply. Effects on growth, mycorrhiza formation, needle ultrastructure, primary and secondary compounds were studied. Ozone exposure had only slight effects on biomass production, growth height and nutrient content of studied conifers. Higher nitrogen availability improved growth of the seedlings and resulted in higher concentration of nitrogen in needles. In Scots pine O₃ exposure did not have effects on quantity of total mycorrhizas and short roots, while higher nitrogen availability decreased quantity of mycorrhizas and short roots. In both tree species O₃ exposure induced O₃‐related ultrastructural symptoms, e.g. granulation and dark staining of the chloroplast stroma in the needle mesophyll cells, at both nitrogen availability levels. Ozone exposure and nitrogen availability did not have significant effects on starch concentrations in either tree species. Concentrations of some individual terpenes were higher in O₃‐exposed needles, while concentrations of individual and total resin acids, total phenolics and catechins were not affected by O₃ exposure. Nitrogen availability did not have substantial effects on concentrations of monoterpenes. By contrast, concentrations of some individual and total resin acids were lower in pine needles and higher in spruce needles with higher nitrogen availability, while phenolic concentration in spruce needles decreased at higher nitrogen availability. The results suggest that realistically elevated levels of O₃ in the field can have some negative effects on the mesophyll ultrastructure of conifer needles, but carbon allocation to root and shoot growth and secondary metabolites are not affected substantially.     

Two different modes of early development and nitrogen assimilation in gymnosperm seedlings†

Light-independent chloroplast development and expression of genes encoding chloroplast proteins occur in many but not all species of gymnosperms. Early development in maritime pine (Pinus pinaster) seedlings was strongly light-independent, whereas Ginkgo biloba seedlings exhibited a typical angiosperm-like morphogenesis with differentiated patterns in light and dark. In pine, chloroplast polypeptides were undetectable in the seed embryo and accumulated in cotyledons of both light- and dark-grown plants in good correlation with light-independent chlorophyll synthesis. In contrast, chlorophyll and chloroplast proteins were only detected in light-grown ginkgo. Pine cytosolic glutamine synthetase (GS) and ferredoxin glutamate synthase (Fd-GOGAT) were present at low levels in the seeds and accumulated at comparable amounts in light- and dark-grown seedlings. Fd-GOGAT was also barely detectable in the seeds of ginkgo and only accumulated in green plants with mature chloroplasts. In G. biloba seeds and etiolated plants only cytosolic GS was identified, while in light-grown seedlings this molecular form was present at low abundance and choroplastic GS was the predominant isoenzyme. The above results have been confirmed by immunolocalization of GS protein in pine and ginkgo plantlets. In pine, GS was present in the peripheral cytoplasm of mesophyll cells and also in the phloem region of the vascular bundle. Immunocytochemical analysis showed that the labelling of mesophyll and phloem cells was only cytoplasmic. In developing ginkgo, GS antigens were present in the chloroplasts of mesophyll parenchyma cells of leaflets and green cotyledons. In contrast, a weak labelling of GS was observed in the parenchyma and phloem cells of non-green cotyledons enclosed in the seed coat. Taking all this into account, our data indicate the existence of two different modes of GS and GOGAT regulation in gymnosperms in close correlation with the differential response of plants to light. Furthermore, the results suggest that glutamine and glutamate biosynthesis is confined to the chloroplast of mesophyll cells in species with light-dependent chloroplast, development whereas compartmentation would be required in species with light-independent plastid development.     

Structure and CO2 Exchange in the Needles of Pinus sylvestris and Abies sibirica

The morphological and functional organization of the needles of Scotch pine (Pinus sylvestris L.) and Siberian fir (Abies sibirica Ledeb.), which differ in their light requirement were studied. The characteristic properties of the high-light-requiring pine included high rates of apparent photosynthesis and dark respiration, high assimilation number, numerous folds in mesophyll cell walls, and increased partial volume of intercellular spaces and hyaloplasm in the mesophyll. In the needles of shade-enduring fir, the higher efficiency of photosynthesis at low light intensities depended on the higher number of membranes and higher pigment content in the chloroplasts. The low assimilation number in fir indicated a shortage of photosynthetic reaction centers. The relative volume of the vascular cylinder and the vascular bundles in the needles and the partial volume of chloroplasts in the hyaloplasm, are considered as indices of the rate of assimilate export from mesophyll cells and their possible damping at different levels of structural organization.     

茉莉酸甲酯对马尾松萜类合成酶活性及其细胞化学定位的影响

马尾松(Pinus massoniana)是我国南方生态建设与造林用材的主要树种,为了揭示马尾松抗虫机理尤其是诱导抗虫性的分子机制,该研究以马尾松幼苗为材料,通过外源喷施茉利酸甲酯(Me JA),分析了处理与对照间植株针叶显微结构、萜类合成酶活性及其细胞化学定位的变化。结果表明:在0.2 mmol·L~(-1)Me JA处理下马尾松植株松针中萜类物质,尤其是单萜、二萜的相对含量增加,马尾松毛虫拒食性明显,诱导抗性增强。显微观测中,针叶叶肉细胞内树脂道分泌物增加,叶绿体数目减少,但叶绿体体积增大,叶绿体片层结构增加。Me JA处理4周后,针叶中萜类合成酶活性增加,通过电镜酶细胞化学观察,膜系统尤其是叶绿体膜上萜类合成酶活性定位明显增强。这说明Me JA诱导的马尾松诱导抗性可能与改变的叶绿体结构及绿色质体萜类合成酶活性密切相关。     

Seasonal and Experimentally Induced Changes in the Ultrastructure of Chloroplasts of Pinus silvestris

The ultrastructure of chloroplasts in mesophyll cells of Pinus silvesris was examined under the electron microscope. Secondary needles were regularly sampled from a tree in a natural stand for one year. Primary needles from one-year-old seedlings exposed to frost hardening and dehardening conditions in a controlled environment chamber were also studied. These seedlings were exposed to 8 or 55 W m^-2. All needles were put in fixative at the different sampling dates and stored in a refrigerator until they were prepared for electron microscopy at the end of the experimental period. During the summer the choroplasts were symmetrically shaped and heavily loaded with starch. The membrane systems were well developed and consisted of both grana and stroma thylakoids. In autumn and during early artificial frost hardening the starch content was reduced, the chloroplasts appeared amoeboid and membrane-free stroma regions were seen. Later the chloroplasts became swollen and aggregated in one part of the cell. Starch was lost and the chloroplasts aggregated earlier at 8 W m^-2 than at 55 W m^-2. During winter the stroma thylakoids were first reduced in number and later even the grana thylakoids were damaged, resulting in mostly disorganized single membranes. Also the chloroplast envelope disappeared. In spring and early summer the chloroplasts migrated to the proximity of the cell walls. The membrane systems were reorganized and starch accumulated. During the first days of artificial dehardening the photosynthetic membranes were severely damaged, especially at 55 W m^-2, but soon new membranes were formed. Starch accumulated earlier at 55 than at 8 W m^-2. The reported ultrastructural variations are discussed in relation to functional and biochemical fluctuations caused by the season or by artificial variations in the climate as demonstrated earlier.     

Rapid Image Analysis Screening Procedure for Identifying Chloroplast Number Mutants in Mesophyll Cells of Arabidopsis thaliana (L.) Heynh

To analyze the genetic control of the process of chloroplast division, a direct image analysis screening procedure has been developed in which mutants of Arabidopsis thaliana (L.) Heynh. var Landsberg erecta are selected on the basis of abnormal chloroplast number. The selection procedure is based on image analysis thresholding after iodine staining, which facilitates the automatic counting of chloroplasts in isolated mesophyll cells. M2 seedlings are screened for significant deviation from the wild type relationship between mesophyll cell size and chloroplast number. Mutants with both abnormally high and abnormally low chloroplast numbers were identified. Of 3500 individual M2 seedlings screened, 18 mutant lines have been isolated and shown to be stably inherited in three subsequent generations. The most extreme phenotypes show an 80% reduction or a 50% increase in chloroplast number per mesophyll cell.     

Cellular Levels of Ribulose 1,5 Bisphosphate Carboxylase and Chloroplast Compartment Size in Wheat Mesophyll Cells

Pyke, K. A. and Leech, R. M. 1987. Cellular levels of ribulose1,5 bisphosphate carboxylase and chloroplast compartment sizein wheat mesophyll cells.—J. exp. Bot. 38: 1949–1956. The amount of the photosynthetic enzyme ribulose 1,5 bisphosphatecarboxylase (RUBISCO),as determined in mesophyll cells in primarywheat leaves was related to the size of the chloroplast compartmentwithin the cell for wheat species of three ploidy levels. Asimilar comparison was made for several genotypes of the hexaploidbreadwheat Triticum aestivum. Estimation of total chloroplastvolume per mesophyll cell was made assuming chloroplasts tobe oblate spheroid in shape. A significant correlation was found between the amount of RUBISCOper cell and the total chloroplast volume per cell for diploid,tetraploid and hexaploid wheat species. A significant correlationbetween cellular RUBISCO level and total chloroplast volumeper cell was also observed for a range of genotypes of the hexaploidT. aestivum but these genotypes of T. aestivutn accumulate agreater amount of RUBISCO per unit chloroplast volume than doany other wheat species. For these genotypes of T. aestivumthe stromal concentration of RUBISCO was estimated at 0·5mol m^–3 with a ribulose Msphosphate binding site concentrationof 4·0 mol m^–3. These results are discussed with respect to a gene dosage hypothesisto explain the accumulation of RUBISCO in leaf mesophyll cells. Key words: Ribulose, bisphosphate carboxylase, wheat chloroplasts, mesophyll cells     

Elevated atmospheric CO2 differentially affects needle chloroplast ultrastructure and phloem anatomy in Pinus palustris: interactions with soil resource availability

The response of forest species to increasing atmospheric CO₂, particularly under resource limitations, will require study in order to predict probable changes which may occur at the plant, community and ecosystem levels. Longleaf pine (Pinus palustris Mill.) seedlings were grown for 20 months at two levels of CO₂ (365 and 720 μol mol¹) in two levels of soil nitrogen (4 and 40 g m^?2), and with two levels of soil moisture (–0·5 and –1·5 MPa xylem pressure potential). Leaf tissue was collected in the spring (12 months exposure) and autumn (20 months exposure) and examined using transmission electron microscopy (TEM) and light microscopy. During early spring, elevated CO₂ magnified effects of N and water treatment on starch accumulation and in some cases contributed to altered organization of mesophyll chloroplasts. Disruption of chloroplast integrity was pronounced under elevated CO₂, low N and water stress. In autumn, needles contained little starch; however, chloroplasts grown under high CO₂ exhibited stress symptoms including increased plastoglobuli and shorter grana. A trend for reduced needle phloem cross-sectional area resulting from fewer sieve cells was also observed under elevated CO₂. These results suggest that, in nature, longleaf pine seedlings may not benefit from a doubling of CO₂, especially when soil resources are limiting.     

Shoot Growth and Changes in the Mesophyll Cell Structure in the Biennial Needles of Abies sibirica Ledeb.

Structural aspects of source–sink relations in current-year and second-year shoots of Siberian fir were investigated. The period of linear growth of current-year shoots was accompanied by a decrease in the size of starch granules in chloroplasts and the number of mitochondria, as well as by the disappearance of lipid inclusions in the mesophyll cells of second-year needles. Growth processes did not affect seasonal changes in the structure of chloroplast grana in these cells. After the termination of shoot growth, large polysaccharide globules appeared in the cytoplasm of mesophyll cells in second-year needles. It is possible that the formation of these inclusions prevents the inhibition of the photosynthesis by starch, which was accumulated in chloroplasts.     

Leaf age as a factor in anatomical and physiological acclimative responses of Taxus baccata L. needles to contrasting irradiance environments

To evaluate the acclimative ability of current-year and previous-year needles of a shade tolerant conifer Taxus baccata L. to contrasting irradiance conditions, seedlings were raised under 27% solar irradiance and at 3 years of age they were transferred to an experimental garden and grown for one season under full irradiance (HL), 18% irradiance (ML) or 5% irradiance (LL). Whereas previous year needles did not change anatomically, current year needles in HL were thicker and had a thicker palisade and spongy mesophyll, and greater leaf mass per area than ML or LL needles. LL needles had greater nitrogen concentration than HL needles irrespective of age but only previous year LL needles also had an increased N per area content, thanks to their lack of reduction in LMA. Adjustment of chlorophyll and carotenoid content occurred in both needle age classes with LL and ML needles having much higher concentrations but, in current year needles, only slightly higher per area content than HL needles. Chlorophyll a/b ratio was not affected by age or irradiance. These modifications had no significant effect on photosynthetic capacities, which did not significantly differ between the age classes in HL or LL treatment and between treatments. On the other hand, high growth irradiance resulted in a greater photochemical yield, photochemical quenching, apparent electron transport rate and inducible non-photochemical quenching in needles formed in the current season. In previous year needles, however, only inducible NPQ was enhanced by high irradiance with other parameters remaining identical among treatments. To test sensitivity to photoinhibition, at the end of the summer plants from the three irradiance levels were transferred to a HL situation and F _v/F _M was determined over the following 18 days. Sensitivity to photoinhibition was negatively related to growth irradiance and previous year needles were less photoinhibited than current year needles. Thus, differences in acclimation ability between needle age classes were most pronounced at the level of anatomy and light reactions of photosynthesis, both of which showed almost no plasticity in previous year needles but were considerably modified by irradiance in current year needles.     

Free amino acids and total nitrogen during shoot development in Scots pine seedlings

The concentration of free amino acids and total nitrogen was studied in needles, stems and roots of seedlings of Pinus sylvestris L. for five weeks during the second growth period ("summer"). In one group of seedlings the source/sink relation was disturbed through removal of the terminal buds. The seedlings were cultivated in artificial year-cycles in a climate chamber.
Total nitrogen increased in needles and sterns of intact seedlings in the beginning of the "summer" and decreased during shoot growth. In seedlings, from which the buds had been removed, nitrogen remained at high levels in the primary needles and accumulated in steins and roots. The results are consistent with utilization of nitrogen in older needles and in the stem during shoot elongation.
The pool of free amino acids increased in the beginning of the "summer" and decreased after bud break in primary needles, stems and roots. Arginine and glutamine, in the roots also asparagine, were the dominating amino acids (amides included). Together, these compounds (plus glutamate and aspartate) contributed about 90% of the nitrogen in the amino acid pool in all organs. In primary needles and in the stem, arginine predominated at the end of hardening (75–85% of the amino acid nitrogen). Free amino acids contributed at most ca 10% of the total nitrogen in primary needles, where the ratio of free amino acid nitrogen: total nitrogen was highest at the end of dormancy and in the early "summer". Free amino acids accumulated after bud removal in primary needles and especially in stems and roots. Glutamine became relatively more dominant than arginine in the different organs.
The observations are consistent with the role of arginine and glutamine for storage and transport of nitrogen in conifers. Because of the low concentrations of amino acid nitrogen in the primary needles, arginine is not considered a major nitrogen reserve in needles of Scots pine seedlings.     

Effects of carbon dioxide enrichment on response of mycorrhizal pitch pine (Pinus rigida) to aluminum: growth and mineral nutrition

 Carbon dioxide enrichment may increase the Al tolerance of trees by increasing root growth, root exudation and/or mycorrhizal colonization. The effect of elevated CO₂ on the response of mycorrhizal pitch pine (Pinus rigida Mill.) seedlings to Al was determined in two experiments with different levels of nutrients, 0.1- or 0.2-strength Clark solution. During each experiment, seedlings inoculated with the ectomycorrhizal fungus Pisolithus tinctorius (Pers.) Coker & Couch were grown 13 weeks in sand irrigated with nutrient solution (pH 3.8) containing 0, 6.25, 12.5, or 25 mg/l Al (0, 232, 463, or 927 μM Al) in growth chambers fumigated with 350 (ambient) or 700 (elevated) μl/l CO₂. At ambient CO₂, in the absence of Al, mean total dry weights (DW) of seedlings at the high nutrient level were 164% higher than those at the low level. Total DW at elevated CO₂, in the absence of Al, was significantly greater than that in ambient CO₂ at the low (+34%) and high (+16%) nutrient levels. Root and shoot DW at both nutrient levels decreased with increasing Al concentrations with Al reducing root growth more than shoot growth. Although visible symptoms of Al toxicity in roots and needles were reduced by CO₂ enrichment, there were no significant CO₂ × Al interactions for shoot or root DW. The percentage of seedling roots that became mycorrhizal was negatively related to nutrient level and was greater at elevated than at ambient CO₂ levels. Generally, elevated CO₂ had little effect on concentration of mineral nutrients in roots and needles. Aluminum reduced concentrations of most nutrients by inhibiting uptake. Received: 18 June 1997 / Accepted: 8 December 1997     

Fluctuation in free and conjugated polyamines in Scots pine seedlings after changes in temperature and daylength

Free, soluble and insoluble conjugated polyamines from the needles, roots and stem of five month old Scots pine (Pinus sylvestris L.) seedlings inoculated with Suillus variegatus (Fr.) O. Kuntze and seedlings without inoculation were analysed during decrease in daylength and temperature. Temporary changes in free, soluble and insoluble conjugated polyamine pools caused by a decrease in daylength or temperature were observed. Inoculation of pine seedlings affected significantly the polyamine levels of five month old pine seedlings. The roots of inoculated seedlings contained significantly higher levels of free and soluble conjugated purtrescine and free, soluble conjugated and insoluble conjugated spermidine than the roots of noninoculated seedlings. The needles of inoculated seedlings contained significatly higher concentrations of free putrescine and soluble conjugated spermidine but lower amount of free spermine than the needles of noninoculated seedlings. The stems of inoculated seedlings contained higher concentrations of free putrescine but lower amounts of insoluble conjugated spermine. Changes in polyamine levels in noninoculated seedlings were observed after shortening of the daylength, whereas in inoculated ones changes were induced mainly by the decrease in temperature. The possible role of polyamines in the initial stage of cold hardening process is discussed.     

Microscopic structure of Scots pine (<Emphasis Type="Italic">Pinus sylvestris</Emphasis> (L.)) needles during ageing and autumnal senescence

Needle ageing and senescence were studied in Scots pine (Pinus sylvestris) trees growing in natural conditions with minimal anthropogenic influence. The four existing needle generations were analyzed by light and transmission electron microscopy before and during autumnal yellowing of the oldest needle generation. The change from green to yellow occurred within less than 4 days in central Finland. The structure of the oldest needles remained largely intact as long as they were green. Increase in mitochondrion size and hypertrophy of the phloem parenchyma were the only changes, probably related to the approaching senescence. In the yellow needles, the structure of the mesophyll tissue varied from nearly intact with reduced chloroplasts and higher numbers of plastoglobuli, to totally disintegrated cells. In the disintegrated cells, peroxisomes were absent, and chloroplasts were smaller with a patchy appearance and degraded, eventually empty-looking stroma. Mitochondria were enlarged, but retained integrity until the last stages of deterioration, and lipids increased. At the light microscopic level, vacuolar volume in mesophyll cells and cavity formation in transfusion tissue increased. Ageing was characterized by increases in the vacuolar volume and cytoplasmic lipids, altered appearance of vacuolar tannin from homogenous ‘sandy’, to large spherical drops and finally to a large mass in the mesophyll, and by hypertrophy and tannin accumulation in the phloem parenchyma. Changes related to needle ageing, senescence and cell location in the mesophyll tissue were discussed relative to findings with stress by strong light, weather conditions and ozone.