DESICCATION OR FREEZING? MECHANISMS OF WINTER INJURY TO RED SPRUCE FOLIAGE

Selected branches of red spruce {Picea rubens Sarg.) saplings growing at 900 m in elevation on Camels Hump Mountain, Vermont, were treated with an acrylic polymer antitranspirant (Adkar Cloud Cover) in October 1989. Winter injury was subsequently apparent on first-year foliage on 1 February 1990. Addition of antitranspirant significantly increased chloroform extractable wax (plus antitranspirant) weight and cuticle (plus antitranspirant) thickness. The treatment reduced cuticular transpiration under laboratory conditions, but did not lessen the severity of visually apparent winter injury. Xylem water potential of damaged and undamaged branches did not differ. Fresh to dry weight ratios revealed a tendency for damaged needles to desiccate rapidly following winter injury. Foliar pigment concentrations were negatively correlated with severity of winter damage. These results suggest that winter desiccation plays a minor role in inducing winter injury to red spruce foliage.     

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.     

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.     

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.     

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.     

Electron Microscopic Studies of Spruce Needles in Connection with the Occurrence of Novel Forest Decline

Needles of four spruce trees showing different degrees of novel kinds of forest decline were investigated by electron microscopy. Green needles appearing at least superficially still intact were selected for the present investigation. Most of the mesophyll appeared to be undamaged. However, groups of atypical mesophyll cells were found close to the endodermis or the hypodermis. The chloroplasts of the apparently damaged cells were particularly affected. Changes in the matrix of the chloroplasts, i.e,. increased affinity to osmium, occurrence of extensive nests of plastoglobuli, as well as damage to the membranes, i.e. lesions in the envelope and abnormal thylakoid membranes, were observed. Signs of decomposition of other cellular structures including mitochondria were also detectable. There appeared to be a close correlation between the degree of damage at the whole tree level and the degree of damage occurring at the cellular level. It is concluded that particularly the lipids and the proteinsof, the membranes are affected by anthropogenic air pollutants and natural stressors. The altered membrane structure may for instance cause abnormal osmotic conditions for the cellular compartments and may impair transport processes and thus lead to lossof function not only of the cells but also of the whole needle.     

Diverse responses of root cell structure to aluminium stress

Plant cells respond to a certain stress factor in different ways depending on their developmental stage and type of tissue. Structural damage may be severe or even lethal in individual cells within a tissue that exhibits moderate or no effects of stress. In the case of aluminium toxicity, detailed observations of root tips of 3 day old Zea mays L., cv. TO360 seedlings revealed differences in the response of some cells. Two different structural changes appeared within root epidermis just behind the root cap. Cells with dark and shrunken cytoplasm occurred next to swollen cells with preserved cellular compartments. Within the root cortex, individual cells or a few cells of a file have severely damaged cytoplasm, in contrast to almost undisturbed cytoplasm of adjacent cells. Such extremely sensitive cells appear irregularly within the root apex. Their structural similarity with cells that are observed after a hypersensitive response in infected plant tissues suggests a role to accumulate aluminium, in order to allow the surrounding tissue to survive the stress.     

Accumulation of Phenolic Compounds in the Necrotic Areas of Austrian and Red Pine Needles Due to Salt Spray

Austrian pine (Pinus nigra) and red pine (P resinosa) were testedfor needle damage caused by salt spray. Morphology and histologyof newly grown needles were compared in 3- and 6-year-old plantsgrowing in the greenhouse under controlled conditions. Pottedplants covered with plastic bags were sprayed daily either withdistilled H₂O or saturated salt solution. Needle tips, mid-partsand bases were fixed after 1, 3 and 24 h, and 3, 7 and 30 din glutaraldehyde+0·1 % caffeine for precipitation ofphenolics. Histochemical reactions and electron microscope observationsshowed that the morphological damage is linked to increasedphenolic compound deposition in mesophyll cells, beginning directlyunder the stomata. Disorganization of membranes leads to deathof the mesophyll cells, even before morphological damage isrecognizable as brown spots on the needle surface. Thus thehistochemical reactions and observations of cell structure revealeddamage earlier than macroscopic observations. Salt injury, phenolic compounds, histological damage, ultrastructural damage, salt spray, Pinus nigra, Austrian pine, Pinus resinosa, red pine     

Hrp Mutant of Pseudomonas syringae pv phaseolicola Induces Cell Wall Alterations but Not Membrane Damage Leading to the Hypersensitive Reaction in Lettuce

Both wild-type (S21-WT) and hrpD- (S21-533) strains of Pseudomonas syringae pv phaseolicola induced the formation of large paramural papillae in lettuce (Lactuca sativa) mesophyll cells adjacent to bacterial colonies. Localized alterations to the plant cell wall included deposition of hydroxyproline-rich glycoproteins, phe-nolics, and callose, and were associated with proliferation of the endoplasmic reticulum and multivesicular bodies. Tissue collapse during the hypersensitive reaction caused by S21-WT was associated with electrolyte leakage and rapid accumulation of the phy-toalexin lettucenin A, both of which followed membrane damage indicated by the failure of mesophyll cells to plasmolyze. A few cells lost the ability to plasmolyze after inoculation with S21-533, and low levels of lettucenin A were recorded, but neither leakage of electrolytes nor tissue collapse were detected. Dysfunction of the plasma membrane in cells adjacent to colonies of S21-WT led to extensive vacuolation of the cytoplasm, organelle disruption, and cytoplasmic collapse[mdash]changes unlike those occurring in cells undergoing apoptosis. Strain S21-533 remained viable within symptomless tissue, whereas cells of S21-WT were killed as a consequence of the hypersensitive reaction. Our observations emphasize the subtle coordination of the plant's response occurring at the subcellular level.     

苜蓿假盘菌侵染苜蓿叶片的细胞学研究

采用微分干涉相差显微镜、扫描和透射电镜技术系统研究了苜蓿假盘菌Pseudopeziza medicaginis在苜蓿叶片的侵染过程及超微结构特征。结果表明,接种4h后,子囊孢子萌发产生芽管;12h后,芽管以直接侵入的方式进入表皮细胞形成侵染菌丝;24h后,表皮细胞中侵染菌丝向相邻表皮细胞扩展,同时侵入到叶肉细胞以胞内生长方式扩展;接种72h后,侵染菌丝在表皮细胞下的叶肉组织中形成初始菌落;第5d后,菌丝扩展至整个叶片组织,大量菌丝聚集形成子座组织,并进一步形成子囊盘与子囊。病菌菌丝在侵入寄主细胞初期,并不     

苜蓿假盘菌侵染苜蓿叶片的细胞学研究

采用微分干涉相差显微镜、扫描和透射电镜技术系统研究了苜蓿假盘菌Pseudopeziza medicaginis在苜蓿叶片的侵染过程及超微结构特征。结果表明,接种4h后,子囊孢子萌发产生芽管;12h后,芽管以直接侵入的方式进入表皮细胞形成侵染菌丝;24h后,表皮细胞中侵染菌丝向相邻表皮细胞扩展,同时侵入到叶肉细胞以胞内生长方式扩展;接种72h后,侵染菌丝在表皮细胞下的叶肉组织中形成初始菌落;第5d后,菌丝扩展至整个叶片组织,大量菌丝聚集形成子座组织,并进一步形成子囊盘与子囊。病菌菌丝在侵入寄主细胞初期,并不穿透寄主质膜与原生质,而是被其所包围。但随着菌丝进一步扩展,叶片组织发生了一系列的病理变化,其中包括叶肉细胞肿胀、细胞质消解、叶绿体等细胞器解体以及寄主细胞坏死塌陷,并最终在叶表面产生典型的褐斑病症状。     

Light and Electron Microscopy of the Compatible Interaction Between Arabidopsis and the Downy Mildew Pathogen Peronospora parasitica

In this study, we focused on compatible interactions between Peronospora parasitica isolate Emoy‐2 and wild‐type (Oy‐0) and mutant (Ws‐eds1) Arabidopsis thaliana accessions by using light and transmission electron microscopy (TEM). Light microscopy of compatible interactions revealed that conidia germinated and penetrated through the anticlinal cell walls of two epidermal cells. Rapid spreading of the hyphal growth with formation of numerous haustoria within the mesophyll cells was subsequently followed by profuse sporulation in the absence of host cell necrosis on both wild‐type and mutant accessions. TEM observations revealed that coenocytic intercellular hyphae ramified and spread intercellularly throughout the host tissue forming several haustoria in host mesophyll cells. Intracellular haustoria were lobed with the diameter of 6–7 μm. Each haustorium was connected to intercellular hyphae in the absence of apparent haustorial neck. The cytoplasm of the haustorium included the organelles characteristic of the pathogen. Callose‐like deposits were frequently observed at sites of penetration around the proximal region of the haustorial neck. Apart from a few callose ensheatments, no obvious response was observed in host cells following formation of haustoria. Most of mesophyll cells contained normal haustoria and the host cytoplasm displayed a high degree of structural integrity. Absence of host cell wall alteration and cell death in penetrated host cell of both accessions suggest that the pathogen exerts considerable control over basic cellular processes and in this respect, response to this biotroph oomycete differs considerably from responses to other pathogens such as necrotrophs.     

Sudden Collapse of Vacuoles in Saintpaulia sp. Palisade Cells Induced by a Rapid Temperature Decrease

It is well known that saintpaulia leaf is damaged by the rapid temperature decrease when cold water is irrigated onto the leaf surface. We investigated this temperature sensitivity and the mechanisms of leaf damage in saintpaulia (Saintpaulia sp. cv. ‘Iceberg’) and other Gesneriaceae plants. Saintpaulia leaves were damaged and discolored when subjected to a rapid decrease in temperature, but not when the temperature was decreased gradually. Sensitivity to rapid temperature decrease increased within 10 to 20 min during pre-incubation at higher temperature. Injury was restricted to the palisade mesophyll cells, where there was an obvious change in the color of the chloroplasts. During a rapid temperature decrease, chlorophyll fluorescence monitored by a pulse amplitude modulated fluorometer diminished and did not recover even after rewarming to the initial temperature. Isolated chloroplasts were not directly affected by the rapid temperature decrease. Intracellular pH was monitored with a pH-dependent fluorescent dye. In palisade mesophyll cells damaged by rapid temperature decrease, the cytosolic pH decreased and the vacuolar membrane collapsed soon after a temperature decrease. In isolated chloroplasts, chlorophyll fluorescence declined when the pH of the medium was lowered. These results suggest that a rapid temperature decrease directly or indirectly affects the vacuolar membrane, resulting in a pH change in the cytosol that subsequently affects the chloroplasts in palisade mesophyll cells. We further confirmed that the same physiological damage occurs in other Gesneriaceae plants. These results strongly suggested that the vacuoles of palisade mesophyll cells collapsed during the initial phase of leaf injury.     

香樟叶肉含晶细胞季节变化研究

为探讨香樟(Cinnamomum camphora)叶肉含晶细胞超微结构的季节变化,阐明香樟叶肉中草酸钙晶体在春夏秋冬的变化规律。该研究以多年生香樟(C. camphora)叶片为材料,分别于春夏秋冬四个季节露地取样,制作超薄切片,用透射电子显微镜(TEM)观察叶肉含晶细胞超微结构的变化。结果表明:春季时香樟叶肉中只有少数细胞有草酸钙晶体,数量较少,晶体结构多为柱状晶、方晶; 夏季时香樟叶肉细胞中随机分布于液泡的草酸钙晶体明显比春季的数量多、体积大、形态丰富,晶体多为柱状晶、方晶、针晶、簇晶; 秋季时香樟叶肉细胞草酸钙晶体和夏季的类似,数量较多,形态多样,以方晶和柱状晶针晶为主,伴有晶簇; 冬季时香樟叶肉含晶细胞晶体形态为柱状晶、方晶、针晶,数量比夏季和秋季的数量略有减少。该研究结果表明在一年四季中香樟叶肉细胞液泡中均有草酸钙晶体结构存在。     

Allograft rejection, autograft fusion and inflammatory responses to injury in Callyspongia diffusa (Porifera; Demospongia)

Sponges exhibit a variety of swift, cellular defence responses to protect self integrity. The sponge Callyspongia diffusa has been used to characterize the cytological changes that occur during allograft rejection, autograft fusion, and inflammation. Allogeneic contact results in fusion of the two exopinacoderms followed by an infiltration of mesohyl cells into the graft zone. As mesohyl cells accumulate, they form tissue bridges that span the graft interface. After a few days, the tissue bridges and the nearby cellular infiltrate become necrotic and slough off, which separates the allogeneic tissues. Autograft fusion begins similarly but cellular infiltration does not follow exopinacoderm fusion. Contacted exopinacocytes are redistributed, the endopinacoderms and choanosomes come into contact, and the grafted sponge tissues merge. Tissue damage exposes internal regions of the sponge to the external environment. In many areas of injury, exposed choanosome is sealed by infiltrating mesohyl cells. In other areas, exposed endopinacoderm appears to serve as new exopinacoderm. Cellular debris is removed by phagocytic archaeocytes and new exopinacoderm is regenerated over the damaged choanosome. No scars remain once the inflammatory infiltrate has dispersed. In general, mesohyl cells are involved in defence responses without an observed enrichment of any specific cell type. However, archaeocytes from rejecting sponges appear to line both sides of the allogeneic interface.     

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.     

Pine needle growth and fine structure after prolonged acid rain treatment in the subarctic

The growth and morphology of Scots pine needles were studied in a long-term acid rain experiment in the far north of Finnish Lapland. Pine trees 5 m tall of age 50–70 years were exposed, by spraying the foliage and soil from a height of 2 m, to either clean water (IC) or acidified water over the period 1985–1992, the acidification site being divided into sub-areas in which the precipitation contained two levels of either sulphuric (Sm, Sh) or nitric (Nm, Nh) acid, or both (SNm, SNh). The treatments with medium and high sulphate-S over eight consecutive years yielded a total sulphur deposition of 3·4 and 17·1 gm⁻², respectively, and those with medium and high nitrate-N a total nitrogen deposition of 1·1 and 5·9 g m⁻². Needles were collected for light and electron microscopy, growth measurements and morphometry. Growth in branch height had decreased by about 40% after 6 years of SNm or SNh treatment, and needle growth by 15% in the SNh trees as compared with the irrigated control trees (IC), although decreases were statistically significant only with respect to the non-irrigated control trees (DC). Growth of branches and needles was slightly better in the Nh treatment than in the IC group. The areas of the whole needle, the mesophyll and the phloem decreased in response to SNh treatment as compared with IC or DC, and a statistically significant decrease of about 30–40% was seen in the area of the xylem in comparison with DC. Cellular damage was observed following the acid treatments, especially with a high acid load. The damage was manifested in collapse of the cellular compartments, increases in lipid accumulations and swelling or disorganization of the protoplast. Increased vacuolization of the cytoplasm, plasmalemma irregularities and chilling-type damage to the mitochondria were also observed.     

感染大麦黄花叶病毒(BaYMV)的大麦细胞质内含体及细胞器病变的研究

超薄切片电镜观察表明,在感染大麦黄花叶病毒(BaYMV)的大麦(品种“早熟3号”)叶肉细胞中,液泡周围偶而可看到病毒颗粒束,在发病后期黄化或坏死的叶肉细胞中,可见到散布的病毒颗粒。在所有表现症状的病叶叶肉细胞,表皮细胞和木质部薄壁细胞中均可观察到风轮体、束状体、板状集结体以及膜状体等细胞质内含体,未见 卷简体和细胞核内含体。感病初期细胞中,细胞质丰富,核糖体数量增加,内质网肥大,随着病毒症状发喂,叶绿体、线粒体等细胞器逐渐肿大,外膜破裂直至解体。     

Ultrastructural characterisation of the host–pathogen interface in white blister-infected Arabidopsis leaves

In this study transmission electron microscopy (TEM) was used to examine details of the host–pathogen interface in Arabidopsis thaliana cotyledons infected by Albugo candida, causal agent of white blister. After successful entry through stomatal pores, the pathogen developed a substomatal vesicle and subsequently produced intercellular hyphae. TEM observations revealed that coenocytic intercellular hyphae ramified and spread intercellularly throughout the host tissue forming several haustoria in host mesophyll cells. Intracellular haustoria were spherical and 4.5 μm in diameter. Each haustorium was connected to intercellular hyphae by a narrow, slender haustorium neck. The cytoplasm of the haustorium included the organelles characteristic of the pathogen. No obvious response was observed in host cells following formation of haustoria. Most of the mesophyll cells contained normal haustoria and the host cytoplasm displayed a high degree of structural integrity. Absence of host cell wall alteration and cell death in penetrated host cells suggest that the pathogen exerts considerable control over basic cellular processes and in this respect, response to this biotrophic Oomycete differs considerably from responses to other pathogens such as necrotrophs. Modification of the host plasma membrane (PM) along the cell wall and around the haustoria, was detected by applying the periodic acid-chromic acid-phosphotungstic acid (PACP) staining technique. After staining with PACP, the host PM was found to be intensely electron dense where it was adjacent to the host cell wall and the distal region of the haustorial neck. By contrast, the extrahaustorial membrane, where the host PM surrounded the haustorium, was consistently very lightly stained.     

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.