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.     

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.     

Novel efficient methods for measuring mesophyll anatomical characteristics from fresh thick sections using stereology and confocal microscopy: application on acid rain-treated Norway spruce needles

Recent design-based stereological methods that can be applied to thick sections cut in an arbitrary direction are presented and their implementation for measuring mesophyll anatomical characteristics is introduced. These methods use software-randomized virtual 3D probes, such as disector and fakir test probes, in stacks of optical sections acquired using confocal microscopy. They enable unbiased estimations of the mean mesophyll cell volume, mesophyll cell number in a needle, and for the first time an internal surface area of needles or other narrow leaves directly from the fresh tissue cross-sections cut using a hand microtome. Therefore, reliable results can be obtained much faster than when using a standard microtechnical preparation. The proposed methods were tested on Norway spruce needles affected for 1 year by acid rain treatment. The effect of acid rain resulted in changes of mesophyll parameters: the ratio of intercellular spaces per mesophyll cell volume increased, while needle internal surface area, total number of mesophyll cells, and number of mesophyll cells per unit volume of a needle decreased in the treated needles.     

The structure and hardening status of Scots pine needles at different potassium availability levels

 Scots pine (Pinus sylvestris L.) seedlings were exposed to three levels of potassium (low, medium and high) and their needle morphology, the cellular structure of the mesophyll and transfusion parenchyma, and the hardening status of the mesophyll cells were examined by light and transmission electron microscopy. The higher the potassium level the greater was the growth of the needles. The area of the mesophyll tissue increased slightly and those of the phloem, xylem and resin ducts decreased in the needles of the seedlings grown at the high K level. Cellular studies revealed that swelling of the chloroplast thylakoids, accumulation of starch in the chloroplasts, translucency of the cytoplasm and plasmolysis in the mesophyll cells were related to a low K level. The hardening status of the mesophyll cells was enhanced after 5 weeks of hardening treatment at high K as seen in changes in chloroplast shape and position and the structure of the endoplasmic reticulum, but the pines showed no major differences in the hardening status of their mesophyll cells between K levels at the end of the experiment, after 9 weeks of hardening. Frost resistance, as shown by the electrolyte leakage test, was nevertheless highest at low K, being related to the increase in the concentration of polyamine putrescine at this potassium level. Received: 23 December 1997 / Accepted: 30 March 1998     

Shading-induced changes in the leaf mesophyll of plants of different functional types

Changes in the structural characteristics of mesophyll induced by shading were investigated in ten species of wild plants of diverse functional types. In all plant types, shading reduced leaf thickness and density by 30–50% and total surface of mesophyll, by 30–70%. The extent and mechanisms of mesophyll structural rearrangement depended on the plant functional type. In the ruderal plants, integral parameters of mesophyll, such as the surface of cells and chloroplasts and mesophyll resistance, changed threefold predominantly because of changes in the dimensions of the cells and chloroplasts. In these plants, shading reduced the volume of chloroplasts by 30%, and the chloroplast numbers per cell declined. The competitor plants showed a twofold increase in mesophyll resistance due to a decrease in the number of photosynthesizing cells per leaf area unit. Moreover, these plants maintained constant dimensions of mesophyll cells, ratios mesophyll surface/mesophyll volume and chloroplast surface/cell surface. In stress-tolerant plants, diffusion resistance of mesophyll remained the same irrespective of the growing conditions, and mesophyll rearrangement was associated with inversely proportional changes in the dimensions of the cells and cell volume per chloroplast. Noteworthy of these plants were relatively constant chloroplasts number per cell, per leaf area unit and total surface area of chloroplasts. The nature of relationship between the mesophyll diffusion resistance and structural parameters of leaf mesophyll differed in plants of diverse functional types.     

Fluorescence Microscopy of Fresh Tissue as a Rapid Technique for Assessing Early Injury to Mesophyll

A technique was developed for sectioning fresh red spruce foliage (Picea rubens Sarg.) for use in fluorescence microscopy. This allowed rapid examination of mesophyll in 3-5 mm needle sections. Healthy, ozone treated and cold stressed needles were examined to assess the utility of this technique for early detection of damage. Healthy mesophyll cells fluoresced bright red, while injured cells fluoresced yellow-green in ozone treated needles, and yellow-orange in frozen needles. Shifts in fluorescence wavelengths may be useful for early detection of injury to mesophyll before it is evident by standard light or electron microscopy.     

兴安落叶松针叶解剖结构变化及其光合能力对气候变化的适应性

叶片易受环境因子影响,其形态解剖结构特征不但与叶片的生理功能密切相关,而且反映树木对环境变化的响应和适应。叶片结构的改变势必会改变树木的生理功能。同一树种长期生长在异质环境条件下,经过自然选择和适应,会在形态和生理特性等方面产生变异,形成特定的地理种群。另外,母体所经受的环境胁迫也会影响到其子代的生长、发育和生理等特征。因此,了解植物叶片形态结构对环境变化的响应与适应是探索植物对环境变化的响应适应机制的基础。兴安落叶松(Larix gmelinii Rupr.)是我国北方森林的优势树种,主要分布在我国东北地区,但日益加剧的气候变化可能会改变其现有的分布区。为了区分叶片对气候变化的可塑性和适应性,本研究采用同质园法比较测定了6个不同气候条件下的兴安落叶松种源的32年生树木的针叶解剖结构和光合生理相关因子,利用石蜡切片方法分析了针叶的解剖结构特征、光合能力（Pmax-a）、水分利用效率（WUE）之间的关系及其对气候变化的适应性。结果表明：表皮细胞厚度、叶肉细胞厚度、传输组织厚度、维管束厚度、内皮层厚度以及叶片总厚度均存在显著的种源间差异（P < 0.05）。叶肉细胞厚度与Pmax-a、气孔导度和WUE之间均存在显著的正相关关系（P < 0.05）。叶肉细胞厚度、表皮细胞厚度、叶片总厚度以及叶肉细胞厚度和表皮细胞厚度在叶片总厚度中所占比例均与种源地的干燥度指数（即年蒸发量与年降水量之比）呈正线性关系。这些结果说明：不同种源兴安落叶松针叶解剖结构因对种源原地气候条件的长期适应而产生显著的差异,从而引起其针叶光合作用、水分利用等生理功能发生相应的变化,从而有利于该树种在气候变化的情景下得以生存和繁衍。     

Effect of Anti-Microtubular Agents on Respiration and Ultrastructural Organization of Wheat Leaf Cells

The effect of anti-microtubular agents oryzalin (15 M) and colchicine (1 mM) on respiration and fine cellular organization of leaves was studied in wheat (Triticum aestivum L.) seedlings. Unlike oryzalin, colchicine considerably suppressed total respiration and the activities of cytochrome and cyanide-resistant pathways of electron transport, probably due to its rotenone-like effect since the latter was negated by vicasol that shunts the first complex of respiratory chain. Oryzalin-induced changes in the ultrastructure of mitochondria and polysomes corresponding to a decrease in their functional activity were detected. The changes in the shape of mitochondria were observed in the companion cells, whereas the mesophyll organelles did not respond to oryzalin. Disintegration of polysomes induced by oryzalin was detected in the cells of both tissues. Association of microtubules with mitochondria, polysomes, and plasma membrane was detected in situ. These data suggest that the intact cytoskeleton may participate in the spatial organization of energy transformation and protein synthesis machinery of the cells; the fact that the observed changes were tissue-specific points at the functional role of microtubules and/or to the number of targets for the inhibitor.     

X-Ray Microanalytical Studies on Mineral Concentrations in Vacuoles of Needle Tissues from Larix decidua (L.) Mill.

Elemental concentrations in the vacuoles of mesophyll, endodermis and transfusion parenchyma cells were determined by means of electron probe microanalysis on bulk frozen hydrated needles of an outdoor-grown larch tree. The investigations were done routinely throughout a life cycle of the deciduous larch needle, starting with bud burst in March and ending with leaf abscission in November. It could be shown that the endodermal vacuoles preferentially accumulate S, CI and Ca while the transfusion parenchyma vacuoles transiently store Mg, K and S. Between May and September the inorganic ion level of mesophyll cells is constantly lower than in the endodermal and transfusion parenchyma vacuoles. In autumn, when the degradation of chlorophylls becomes detectable, the elements Mg, S, CI and Ca are found increasingly in the vacuoles of the mesophyll cells. It is concluded that, in mature and intact larch needles, the vacuoles of the endodermis in cooperation with the transfusion parenchyma vacuoles act as efficient ion storage buffers which maintain a constant low ion level in the vacuoles of mesophyll cells.     

Closure of Stomata in Water-Stressed Pine Needles Results from the Decreased Water Potential of the Mesophyll Apoplast in the Substomatal Cavity

Pine (Pinus sylvestris L.) seedlings grown under controlled conditions were subjected to water deficit (external water potentials ranging from–0.15 to–1.5 MPa) by adding polyethylene glycol 6000 (PEG) to the nutrient solution. Following this treatment, the dry weights of plant shoots and roots, as well as the ratio of variable to maximum chlorophyll fluorescence (F_v/F_m), nonphotochemical quenching (NPQ) of chlorophyll excitations, photosynthetic CO₂/H₂O exchange, dark respiration of needles, and water potential of mesophyll apoplast in the substomatal cavity of pine needles, were measured. The imposed water deficit was followed by the inhibition of seedling growth, suppression of photosynthesis and transpiration, and by the decreased content of photosynthetic pigments. It is shown for the first time that the closure of stomata in the needles of water-stressed pine seedlings falls into the physiological reaction norm and is caused by the reduction of water potential in the mesophyll apoplast of the substomatal cavity.     

Needle ontogeny of mature Scots pines under enhanced UV-B radiation

The aim of this work was to test our hypothesis that pine needles protect themselves against UV-B radiation via anatomical changes in the epidermal layer. This could lead to needle growth reductions if large quantities of assimilates are allocated for the epidermal protective mechanisms at the expense of photosynthetic area. Effects of enhanced UV-B radiation on the needle ontogeny of mature Scots pines (Pinus sylvestris L.) were studied during the second season of a field experiment. Depending on the season and the time of the year (1996-1997), the enhanced UV-B irradiance varied from 0.92 to 5.09 kJ m^-2 day^-1 UV-B_BE compared to 0.54-2.44 kJ m^-2 day^-1 UV-B_BE of ambient radiation. It was found that UV-B treatment accelerated the early development of needles. In 6-day-old enhanced UV-B-treated needles, mesophyll and hypodermic cells were fully differentiated, whereas in ambient-treated needles, no lobate mesophyll cells were seen and hypodermic cells had not yet developed. In fully grown needles, no accelerated differentiation was seen, except that the epidermal cross-sectional area was smaller. The continuation of the experiment will show if such a significant difference only occurs irregularly and incidentally or if it is of consistent significance for needles.     

Changes in Morphology,Anatomy, and Photosynthetic Capacity of Needles of Japanese Larch (Larix kaempferi) Seedlings Grown in High CO2 Concentrations

Photosynthetic traits of two-year-old Japanese larch seedlings (Larix kaempferi Carr.) grown at elevated CO₂ concentrations were studied in relation to structural changes in the needles. Seedlings were grown at two CO₂ concentrations, 360 (AC) and 720 (EC) mol mol^–1 at high and low nutrient supply rates, high N (HN) and low N (LN). The photosynthetic capacity fell significantly in EC+LN, but increased significantly in EC+HN. Since the mesophyll surface area exposed to intercellular space per unit leaf area (A^mes/A) is correlated with the photosynthetic rate, we measured A^mes/A for larch needles growing in EC. Changes of A^mes/A in both EC+HN and EC+LN were very similar to the changes in photosynthetic capacity. This suggests that the changes of A^mes/A in EC probably caused the changes in the photosynthetic capacity. The changes of A^mes/A in EC were attributed to changes in the mesophyll cell size and mesophyll cell number. The photosynthetic capacity in EC can be explained by taking morphological and structural adaptations into account as well as biochemical factors.     

Histochemical and biochemical approaches to the study of phenolic compounds and peroxidases in needles of Norway spruce (Picea abies)

The three youngest age-classes of needles of Norway spruce ( Picea abies ) were collected from four sites in the Krusne Hory Mountains (Czech Republic) characterized by different levels of damage caused by environmental pollution. Histochemical methods did not reveal any differences in localization of phenolics among the needles. Mesophyll cells close to the epidermis of needles and cells around resin ducts and substomatal cavities often accumulated higher amounts of phenolics than the rest of the mesophyll cells, but this was independent of age and damage. Needles of different age- and damage-class did not show any marked changes in general lignification pattern. However, a lower intensity of histochemical detection of lignin was observed in needles from the most damaged site. This finding was confirmed by chemical analysis using thioglycolic acid. Generally, the amount of lignin in mesophyll cells was lower in damaged trees than in healthy ones. Using the Folin–Ciocalteau method, no significant differences in the total content of phenolics were observed in the needles, although HPLC revealed marked alterations in the forms of seven phenolic acids. Concentrations of conjugated forms of phenolic acids (esters and glycosides) were higher in damaged needles (255.9 μg g⁻¹ f. wt) than in healthy needles (189.8 μg g⁻¹ f. wt). By contrast, content of esterified phenolic acids incorporated into cell walls was higher in needles from healthy trees (101.1 μg g⁻¹ f. wt) than in damaged needles (78.3 μg g⁻¹ f. wt). Marked differences were also observed in the activity of soluble peroxidases, although the activity of ionically bound forms was approximately the same in healthy and damaged needles. The total amounts of chlorophylls and carotenoids decreased as environmental damage increased.     

The Loss of Structural Integrity in Damaged Spruce Needles from Locations Exposed to Air Pollution I. Mesophyll and Central Cylinder

Abstract In connection with the new type of forest damage, the individual disease situation of two-year-old spruce ( Picea abies ) needles was analyzed histopathologically in forest areas exposed to different levels of O₃-, SO₂- and NO₃- pollution.
Early damage results from losses of chlorophyll in the mesophyll cells. The bleaching is more intensive towards the apex in severely damaged needles. The cytoplasm is aggregated at the cell wall and the chloroplasts show definite structural damage as well.
The mesophyll cells below the epidermis, or the cells adjacent to the vascular bundle sheath, appear to be particularly susceptible. Collapsed cells (bone cells), which increase in number with damage, can lead to tissue death in certain needle areas, (brown tips, transverse bands).
Necrotic spots are manifested as groups of dissociated cells in which hypertrophic and collapsed cells as well as abnormal proliferations can be observed.
Hypertrophy and cell collapse appear in the central cylinder in addition to severe phenol deposits.
Bone cells and chlorophyll losses can already be detected in the green needles of damaged trees, indicating latent damage, which becomes macroscopically visible only after more extensive damage.
Our results indicate that no biotic stress factors take part in the damage of the spruce needles investigated here. Anthropogenic air pollutants in addition to abiotic stress factors must be regarded as a main cause of damage.     

OZONE DAMAGE TO PONDEROSA PINE: A HISTOLOGICAL AND HISTOCHEMICAL APPRAISAL

Histological and histochemical changes occurred in current-year needles of sensitive clonal selections of Pinus ponderosa Laws under natural summer growing conditions in the San Bernardino National Forest near Los Angeles, California due to fumigations with 0.45 ppm ozone for 12 hr/day. Within five days after the start of fumigation, chloroplasts and carbohydrate stain accumulated in the peripheral portions of mesophyll cells. Concurrently, the homogenous distribution of proteins and nucleic acids was disrupted. Succinate dehydrogenase was localized mostly in guard cells, resin duct epithelial cells, albuminous cells, and differentiating vascular tissues within unfumigated and fumigated leaves. Acid phosphatase activity increased within mesophyll cells during ozone exposure, but there was no association of acid phosphatase or ozone injury with stomata. Wall destruction occurred in mesophyll cells after appreciable intracellular damage. These histological and histochemical changes occurred within 5–7 days, but visual symptoms were not evident until 2–3 weeks after fumigation. It is thus possible to assay ozone damage very soon after exposure if no other external agents cause similar results.     

Characterization of the short needle phenomenon in red spruce

Red spruce trees (Picea rubens Sarg.) occasionally produce short twigs bearing short needles. The frequency of short needle cohorts is positively correlated with both elevation and defoliation and they are found in greater numbers on trees that regularly experience winter injury. Short needles are smaller, have lower fresh and dry weights, and reduced volumes compared with normal needles. They have reduced cross-sectional areas due to smaller areas of stelar and mesophyll tissue systems. Individual mesophyll cells, however, have the same cross- and longitudinal sectional areas. On a weight or volume basis, both short and normal needles contain similar amounts of chlorophyll and carotenoid pigments. When pigment concentration was calculated on a unit needle or a needle area base, short needles contain more pigment than normal needles. Short needles appear to have a greater photosynthetic efficiency as determined by fluorescence measurements.     

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.     

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.     

Structural injury underlying mottling in ponderosa pine needles exposed to ambient ozone concentrations in the San Bernardino Mountains near Los Angeles, California

For several decades, southern California experienced the worst ozone pollution ever reported. Peak ozone concentrations have, however, declined steadily since 1980. In this study, the structural injuries underlying ozone symptoms in needles of ponderosa pine (Pinus ponderosa Dougl. ex Laws.) collected in summer 2006 from one of the most polluted sites in the San Bernardino Mountains were investigated using serial sections examined by light and electron microscopy. Ozone-specific light-green diffuse mottling was observed in the current-year needles, whereas older foliage showed brownish mottling similar to winter fleck injury. Especially, within the outer layers of mesophyll, many markers of oxidative stress, typical for ozone, were observed in both apoplast and symplast. Altogether within cells of mottles, these markers were indicative of hypersensitive-like response, whereas degenerative structural changes were diagnosed in the surrounding mesophyll. Evidence of drought stress and frost injury to older needles was also detected. Hence, mottling injury appeared to be primarily caused by ozone stress, however, other environmental stressors also determined the symptom morphology and distribution, especially within the older foliage.     

Manometrische Atmungsmessungen an Protoplasten vonPetunia

Respiration of protoplasts isolated from the leaf mesophyll ofPetunia hybrida was estimated by the Warburg method. The time course of respiration rate shows an initial decrease followed by a prominent increase which is caused by shaking of the protoplast suspension. The absolute respiration rates are low and show an unusual spread, but the temporal form of the curve is always similar to that one shown in Fig. 1. The RQ-values shift from below 1 during the initial phase to far above 1 during the increase of the respiration. For isolated protoplasts from petals the same is true. Feeding the protoplasts with DNA does not influence the respiration rate significantly. The physiological quality of protoplasts seems to suffer in a high degree from the isolation process.