两种类型栓皮栎软木细胞微观排列与形态特征

采用扫描电镜对秦岭北坡楼观台地区的厚皮、薄皮两种类型栓皮栎软木进行细胞微观构造观察分析,并与欧洲栓皮槠进行比较,以阐明厚皮、薄皮栓皮栎软木的相关特性,为中国栓皮栎软木的合理利用提供依据。结果表明:(1)两种类型栓皮栎软木细胞的排列结构较一致,均由内部中空的封闭型薄壁细胞紧密排列组成;在弦切面上呈蜂窝状排列,径切面和横切面上呈砖墙状排列;在径切面上,软木细胞侧高整齐地排列成行,且与树干轴向垂直;在横切面上,软木细胞侧高整齐地处于以树干轴为中心散发出来的射线上。(2)栓皮栎软木细胞大小、细胞壁和侧壁褶皱等受生长季节的影响;从软木细胞形态特征上看,厚皮类型软木细胞壁薄、细胞体积大,其软木质量优于薄皮类型。(3)与欧洲栓皮槠比较,发现厚皮类型栓皮栎早软木细胞棱柱高较小(20.6μm vs.(对比)30~40μm),软木细胞壁略厚(1.7μm vs.1~1.5μm),细胞实体积(细胞壁体积占细胞总体积比例)略大(18.75%vs.10%),厚皮类型栓皮栎软木比欧洲栓皮槠的软木质量差一些。(4)受树皮生长应力的影响,两种类型栓皮栎软木细胞侧高壁上多发生褶皱,早软木细胞褶皱严重,晚软木细胞没有褶皱,但在早晚软木交界或含有杂质处褶皱特别严重,表明厚皮类型软木细胞的侧壁褶皱程度高于薄皮类型。(5)对细胞形态特征及软木特性等的分析表明,薄皮类型栓皮栎软木质量比厚皮类型差,未来对软木资源的开发利用应更注重厚皮类型。     

THE WOOL-LIKE COVERING OF THE ROOTS OF LANNEA ALATA I. THE ORIGIN AND MORPHOLOGY OF THE WOOLY COAT

Lannea alata (Engl.) Engl., Anacardiaceae, a tree or shrub of East Africa, has roots covered with dense wool-like hair. Cork cambium of the root produces a closely appressed cork from which the hairs (modified cork cells) arise. Cork cells and wooly hair are rich in sterol and carotin-oid-like compounds and have thick walls. It is suggested that the root wool plays a role in the soil-air, soil-water relations of Lannea alata and other plants.     

Viability of free and encapsulated Escherichia coli overexpressing cyclopentanone monooxygenase monitored during model Baeyer–Villiger biooxidation by confocal laser scanning microscopy

Baeyer–Villiger biooxidation of 4-methylcyclohexanone–5-methyloxepane-2-one catalysed by recombinant Escherichia coli overexpressing cyclopentanone monooxygenase encapsulated in polyelectrolyte complex capsules was used to investigate effect of substrate conversion on the viability of cells. Confocal laser scanning microscopy (CLSM) was used to assess cell viability using propidium iodide fluorescence marker for necrosis, and flavin autofluorescence to identify living bacteria. Viability of encapsulated cells decreased with increasing substrate concentration from 99 ± 1 to 83 ± 4%, while substrate conversions from decreased 100 to 6 ± 1%. Storage stabilization of encapsulated cells was observed by increased substrate conversion form 68 ± 2 to 96 ± 3%. Measurements by CLSM with standard deviations up to 5% may be regarded as powerful tool for recombinant cell viability determination during Baeyer–Villiger biooxidations.     

Development of Fern Sporangia: a Fluorescence Microscopy Study

The utility of fluorescence microscopy for studying development of fern spores is investigated. Changes in the fluorescence characteristics during the developmental stages of fern sporangia can be attributed to the changes in the chemical composition of the cell wall. Bright blue autofluorescence of the spores indicated the presence of sporopollenin. The sporan-gial walls and the spores autofluoresced yellow under blue light excitation. Fluorescence microscopy is a useful addition to light, scanning, and transmission electron microscopy because living specimens can be studied owing to their fluorescence properties.     

Combined histochemistry and autofluorescence for identifying lignin distribution in cell walls

Histological staining methods commonly used for detecting cellulose and lignin in cell walls were combined with epifluorescence microscopy to visualize differences in lignification between and within cellular elements. We tested our approach on sections of one-year-old branches of Fraxinus ornus L., Myrtus communis L., Olea europaea L., Pistacia lentiscus L. and Rhamnus alaternus L., containing both normal and tension wood. Sections were subjected to various staining techniques, viz. safranin O, safranin O/fast green FCF, and alcoholic solutions of safranin O/astra blue, according to the commonly accepted protocols. Stained and unstained sections were compared using both light and epifluorescence microscopy. Safranin O with or without counterstaining hid the strong fluorescence of vessel walls, cell corners and middle lamellae allowing the secondary wall fibers to fluoresce more clearly. Epifluorescence microscopy applied to stained sections showed more cell wall details than autofluorescence of unstained sections or white light microscopy of counterstained sections. This simple approach proved reliable and valuable for detecting differences in lignification in thick sections without the need for costly equipment.     

Definition and Evaluation of the Spatio‐Temporal Variations in Chlorophyll Fluorescence during the Phases of CAM and during Endogenous Rhythms in Continuous Light,in Thick Leaves of Kalanchoë daigremontiana5

Abstract: We used chlorophyll fluorescence imaging to examine the homogeneity of photosynthetic metabolism during CAM in the thick leaves of Kalanchoë daigremontiana Hamet et Perrier de la Bǎthie. Intense, persistent fluorescence from a DCMU treated thin leaf of Clematis sp placed beneath a K. daigremontiana leaf was readily detected through the thick leaf. Evidently reabsorption of fluorescence was qualitatively unimportant in the system used. Chlorophyll fluorescence images from 7 mm tissue discs excised from Kalanchoë leaves were collected at 60 s intervals during 20 min transients elicited by red excitation light. Information about patchiness and subsurface processes was gained by statistical factor analysis and Fourier transform. Although small, highly resolved rings of bright chlorophyll fluorescence surrounding discs of low fluorescence were observed from cells near the surface, no independent regional temporal variation in fluorescence was evident in the surface‐biased images. Temporally independent chlorophyll fluorescence was present in images biased towards sub‐epidermal sources, in most phases of CAM, and during endogenous rhythm. These asynchronous changes were several millimetres apart. This patchy fluorescence was confirmed when attached leaves were excited with blue light in a leaf chamber while CO₂ and H₂O exchange was monitored. Large spatio‐temporal variations in the efficiency of photosystem II were always observed during phases II and IV of CAM, when both CO₂ fixation cycles are active, and during the maximum rate of CO₂ fixation during the endogenous rhythm in continuous light. These data are discussed in terms of metabolic isolation in the thick but uniform tissues in which gas diffusion may be largely confined to wet cell walls, thereby rendering the tissue functionally heterobaric. Prolonged, but in some instances, reversible alterations in PSII efficiency could be produced by injection of metabolic inhibitors, confirming that patchy fluorescence may reflect the differing energy costs of photosynthesis in different CAM phases.     

An autofluorescence-based survey of late follicular atresia in ovaries of a teleost fish (Thunnus thynnus)

In this study, the authors examined late atretic follicles in the ovaries of Atlantic bluefin tuna, Thunnus thynnus (Linnæus 1758), at different times of the year using transmitted light and epifluorescence microscopy. Atresia (degeneration and resorption of developing ovarian follicles) is a natural process involved in fecundity downregulation in teleosts and is substantially enhanced in stressful conditions. Early (α and β) atretic stages of yolked oocytes have a relatively short duration in seasonally reproducing species, whereas later (γ and δ) atretic follicles (LAF) persist for longer time in the ovary, serving as a sign of previous vitellogenic activity. LAF can thus be used as reliable markers of maturity during non-reproductive periods. Lipofuscin granules accumulate in the cytoplasm of LAF cells as a result of lysosomal digestion of oocyte components. Taking advantage of the well-known autofluorescent properties of lipofuscins, LAF may be identified in unstained histological sections under fluorescence microscopy using appropriate excitation and emission wavelengths. The authors explore in this study the applicability of fluorescence microscopy to provide a fast and effective method to assess late atresia in fishes. This method may be particularly useful to determine sexual maturity in individuals sampled long after the spawning season, where LAF are difficult to detect in standard histological sections. Furthermore, LAF autofluorescence provides a rapid way to quantify late atresia in fishes using image analysis.     

How do trees grow? Response from the graphical and quantitative analyses of computed tomography scanning data collected on stem sections

Tree growth, as measured via the width of annual rings, is used for environmental impact assessment and climate back-forecasting. This fascinating natural process has been studied at various scales in the stem (from cell and fiber within a growth ring, to ring and entire stem) in one, two, and three dimensions. A new approach is presented to study tree growth in 3D from stem sections, at a scale sufficiently small to allow the delineation of reliable limits for annual rings and large enough to capture directional variation in growth rates. The technology applied is computed tomography scanning, which provides – for one stem section – millions of data (indirect measures of wood density) that can be mapped, together with a companion measure of dispersion and growth ring limits in filigree. Graphical and quantitative analyses are reported for white spruce trees with circular vs non-circular growth. Implications for dendroclimatological research are discussed.     

Localisation of fungal hyphae in wood using immunofluorescence labelling and confocal laser scanning microscopy

This study explored the feasibility of using immunofluorescence labelling in conjunction with confocal laser scanning microscopy (CLSM) for detection of common fungal colonisers of unseasoned radiata pine in New Zealand. Wood sections infected with Ophiostoma piceae were treated with monoclonal antibody IF3 (1), and then Oregon green 514 goat anti-mouse IgG, a fluorescent secondary antibody. Additional wood sections infected with other Ophiostoma spp., Sphaeropsis sapinea, Leptographium procerum, Trichoderma sp. and Phlebiopsis gigantea were treated similarly to determine whether the antibody was specific to O. piceae or was recognising other fungal species. Sections were examined using phase contrast and fluorescence light microscopy prior to CLSM. Immunolabelled fungal hyphae showed relatively weak fluorescence compared to the strong autofluorescence of wood cell walls and extractives. Labelled hyphae of O. piceae were detected in wood using CLSM but not with ordinary fluorescence microscopy. This is because CLSM has stronger illumination power and superior imaging ability compared with ordinary fluorescence microscopy. The monoclonal antibody did not cross-react with the other Ophiostoma species. However, non-specific antibody binding was observed with L. procerum and Trichoderma species. Furthermore, cell walls of L. procerum showed strong autofluorescence with optical properties similar to wood extractives when examined prior to incubation with the monoclonal and secondary antibody, therefore cross-reactivity tests were inconclusive for Leptographium and Trichoderma species. The current investigation demonstrated that CLSM provides possibilities for future investigations on in situ interactions of common radiata pine fungal colonisers, with one another and with wood.     

Structure of the endosperm in the mature seed of Melilotus alba]

The results of the exam at the light, the fluorescence and the scanning electron microscope of the endosperm of Melilotus alba mature impermeable seeds are reported. Cryostat sections, semithin sections and squashes are observed. Melilotus alba endosperm is variable in thickness and envelopes cotyledons and radicle. Its "aleurone" layer is one-cell thick, while the number of layers of its internal cells varies in relation to the location in the seed. In the aleurone cells, the cytoplasm and the outer portion of the wall are autofluorescent; tannic acid-ferric chloride stains the outer portion of the wall and allows to see clearly the inner thickenings, DAPI and haematoxylin demonstrate the presence of the nucleus. The cytoplasm of these cells is coloured by Sudan black b, and its fluorescence is enhanced by auramine and calcofluor white. Calcofluor white enhances the fluorescence of the outer portion of these walls, too, but is without effect on the non-autofluorescent thickening, indicating presence of cellulose only in the first case. Callose is absent. Also the thin autofluorescent walls of the endosperm inner cells react positively to calcofluor. These cells are very large, almost completely filled with "gelatinous" substances--the galactomannans--and very rarely contain a nucleus.     

Response of plasmodesmata formation in leaves of C4 grasses to growth irradiance

Rapid metabolite diffusion across the mesophyll (M) and bundle sheath (BS) cell interface in C₄ leaves is a key requirement for C₄ photosynthesis and occurs via plasmodesmata (PD). Here, we investigated how growth irradiance affects PD density between M and BS cells and between M cells in two C₄ species using our PD quantification method, which combines three‐dimensional laser confocal fluorescence microscopy and scanning electron microscopy. The response of leaf anatomy and physiology of NADP‐ME species, Setaria viridis and Zea mays to growth under different irradiances, low light (100 μmol m^?2 s^?1), and high light (1,000 μmol m^?2 s^?1), was observed both at seedling and established growth stages. We found that the effect of growth irradiance on C₄ leaf PD density depended on plant age and species. The high light treatment resulted in two to four‐fold greater PD density per unit leaf area than at low light, due to greater area of PD clusters and greater PD size in high light plants. These results along with our finding that the effect of light on M‐BS PD density was not tightly linked to photosynthetic capacity suggest a complex mechanism underlying the dynamic response of C₄ leaf PD formation to growth irradiance.     

The Effect of Cork Pieces on Pseudohypericin Production in Cells ofHypericum perforatumShoots

The stimulating effect of cork pieces on hypericin and pseudohypericin biosyntheses was studied in cells of shoots regenerated from the callus cultures of St. John's wort (Hypericum perforatumL.). The addition of the cork matrix slightly stimulated shoot growth and enhanced pseudohypericin biosynthesis about threefold (to 0.4 mg/g dry wt). Pseudohypericin production increased proportionally with the amount of cork material added (from 1 to 4 mg/ml of growth medium). Further increase in the amount of cork pieces inhibited both pseudohypericin production and shoot growth. Organic and aqueous extracts of cork pieces did not affect the production of these substances.     

Morphological and fine structural features of pit connections inCryptonemia sp., a highly differentiated marine red alga from Australia

Summary The thick and anatomically complex stalks of a Western Australian species ofCryptonemia (Cryptonemiales, Rhodophyta) are characterized by growth rings in cross-section. Cells of the medulla may die as the diameter of the stalks increases to maximum widths of over 2 centimeters, the remaining cell walls appearing to function as purely supportive tissue (a phenomenon hitherto unreported in the red algae). All cells of the stalk are enclosed by thick, compact cell walls and are interconnected by pit connections which become progressively more convoluted and fluted with increasing distance of the cells from the stalk surface. This is the first report of such a pit-connection morphology. It is suggested that the modification may serve to aid transport of solutes towards the more deeply-buried layers of living cells.     

Climate response of cork growth in the Mediterranean oak (Quercus suber L.) woodlands of southwestern Portugal

In the Mediterranean climate regions, drought events are expected to affect the growth of forests ecosystems by changing trees growth rates and eventually inducing shifts in their growth patterns. Cork oak (Quercus suber L.) is a strictly western Mediterranean tree species periodically harvested for its bark, the cork. So far, cork oak has received limited attention for dendroclimatological studies due to its typical faint and erratic tree wood rings. Moreover, its distinct cork rings chronologies have been completely neglected. In this study we introduce an approach using cork ring chronologies dated back 9–10 years for climate response. Despite enhancing interannual variability and increasing statistical response to short-term climatic variability, still poorly understood, this study will possibly allow infer long-term climate response. We analyzed the cork ring chronologies of 55 cork samples collected in mature (under exploitation) trees in three distinct locations in southwestern Portugal. Cork growth recorded a high climate signal, with highly significant and coherent responses to the yearly climate-related sources of variation. We successfully assessed trends of cork growth via correlation analysis including selected climate variables among mean monthly temperature, monthly precipitation and, on an annual basis, eight precipitation indices. The high mean sensitivities and inter-series correlations found for cork ring chronologies combined with the significant variance explained by climate variables suggest that climate is likely one dominant signal that affects cork growth, but local environmental stresses can decisively affect this (climate) signal. Assuming cork growth as a proxy for cork oak growth, it seems conceivable that despite the trees being highly resistant to drought stress, cork oak woodlands in southwestern Portugal would have to face lesser growth in a global warming scenario.     

Subcellular localization of flavonol aglycone in hepatocytes visualized by confocal laser scanning fluorescence microscope

Flavonoids are widely distributed in the plant kingdom and show various biological activities. The bioavailability of flavonoids in biological samples has conventionally been quantified by high-performance liquid chromatography and mass spectrometry, but with these analytical techniques it is difficult to estimate the subcellular localization of flavonoids in intact cells. In this study, we attempted to examine the localization of flavonoids in cultured cells using a confocal laser scanning fluorescence microscope and mouse hepatoma Hepa-1c1c7 cells. Five flavonol aglycones showed autofluorescence in the cells under the conditions (Ex. 488 nm to Em. 515–535 nm), whereas three flavonol glycosides and eight compounds belonging to other flavonoid subclasses, i.e., flavones, flavanones, and catechins, did not. The autofluorescence of galangin and kaempferol appeared stronger in the nucleus than cytoplasm, suggesting that they are incorporated into the cells and accumulated in the nucleus. The proposed method provided evidence that flavonol aglycones are incorporated into, and accumulated in the nucleus of, hepatocytes.     

THE CONNECTIVE BASE OF CIRSIUM HORRIDULUM (ASTERACEAE): DESCRIPTION AND COMPARISON WITH THE VISCOELASTIC FILAMENT

Structural, autofluorescent, and mechanical characteristics show that the so-called “collar” of the stamen is an elongated basal portion of the connective in Cirsium horridulum. The connective base/filament boundary is precisely demarcated by epidermal cells with thick, autofluorescent walls. In the region of the connective base near the anther lobes, the autofluorescence is less intense and the cells are longer, but wall thickness does not decrease. The autofluorescence can be assayed in fresh, frozen, or rehydrated materials, and is resistant to boiling in water and to acetone. Additional features delineate the connective base from the filament. The abaxial and lateral portions of the connective base surface are relatively flat due to the underlying thick secondary walls. Ultrathin sections taken at various points along the length of the connective base and the filament show that cuticle thickness gradually increases from approximately 30 nm in the connective base to 450 nm in the filament. The connective base/filament junction appears to be a weak mechanical link within the stamen. These data establish several new connective base characteristics in Cirsium and define the connective base/filament boundary.     

The core-microtome: A new tool for surface preparation on cores and time series analysis of varying cell parameters

A microtome designed for the surface preparation of entire increment cores allows cutting plane surfaces on cores up to a length of 40 cm. Compared to the common sanding procedure, the wood cells of the annual rings remain open, not filled with swarf, and the cell walls are smooth and hence clearly visible. This article aims at describing the functionality of the microtome and the procedures needed for an accurate surface preparation to achieve a good contrast for subsequent image analysis. Possible applications for a more detailed analysis of variations in the tracheid structure of conifers and vessel sizes of oak are presented, which can be included in time series analyses.     

Method for detecting tree ring boundary in conifers and broadleaf trees using Mask R-CNN and linear interpolation

As tree rings can reveal various information regarding climate and environmental factors, increasing research is being conducted on them. Although tree ring analysis software such as Windendro has been applied, research on the development of analysis software using image preprocessing algorithms and deep learning is recently being attempted as computer vision technology. In this study, Mask R-CNN and linear interpolation were applied from images collected using a smartphone (SM-G973, Samsung, Suwon) and a scanner (CanoScan 9000F, Canon, Otaku) to propose an effective method for detecting tree ring boundaries. Pitch pine (Pinus rigida), Korean pine (Pinus koraiensis), white birch (Betula platyphylla), and cork oak (Quercus variabilis) were selected as tree species. Of the 300 images, 240 were classified as training data and 60 as validation data. As a result of learning, smartphones detected 86.0 % (381 ring boundaries) of the rings in pitch pine, 82.1 % (367 ring boundaries) in Korean pine, 84.7 % (309 ring boundaries) in white birch, and 78.7 % (318 ring boundaries) in cork oak. The scanner detected 93.2 % (413 ring boundaries) of the rings in pitch pine, 90.8 % (405 ring boundaries) in Korean pine, 88.2 % (322 ring boundaries) in white birch, and 89.4 % (361 ring boundaries) in cork oak. In particular, the smartphone showed satisfactory results of 84.7 % and 78.7 % for detecting tree ring boundaries of white birch and cork oak, where the boundaries of the rings were unclear. In the annual growth analysis results, both smartphones and scanners were statistically insignificant, and there was no difference compared with those of Windendro. Therefore, Mask R-CNN might be an effective approach for tree ring boundary detection as it showed satisfactory results, even with smartphones. In addition, although there was distortion in cases where images were acquired with a circular lens, there was no statistically significant difference from Windendro results. Thus, Mask R-CNN and linear interpolation can be used for tree ring boundary detection and growth measurement.     

Nonannual tree rings in a climate‐sensitive Prioria copaifera chronology in the Atrato River,Colombia

In temperate climates, tree growth dormancy usually ensures the annual nature of tree rings, but in tropical environments, determination of annual periodicity can be more complex. The purposes of the work are as follows: (1) to generate a reliable tree‐ring width chronology for Prioria copaifera Griseb. (Leguminoceae), a tropical tree species dwelling in the Atrato River floodplains, Colombia; (2) to assess the climate signal recorded by the tree‐ring records; and (3) to validate the annual periodicity of the tree rings using independent methods. We used standard dendrochronological procedures to generate the P. copaifera tree‐ring chronology. We used Pearson correlations to evaluate the relationship of the chronology with the meteorological records, climate regional indices, and gridded precipitation/sea surface temperature products. We also evaluated 24 high‐precision ¹⁴C measurements spread over a range of preselected tree rings, with assigned calendar years by dendrochronological techniques, before and after the bomb spike in order to validate the annual nature of the tree rings. The tree‐ring width chronology was statistically reliable, and it correlated significantly with local records of annual and October–December (OND) streamflow and precipitation across the upper river watershed (positive), and OND temperature (negative). It was also significantly related to the Oceanic Niño Index, Pacific Decadal Oscillation, and the Southern Oscillation Index, as well as sea surface temperatures over the Caribbean and the Pacific region. However, ¹⁴C high‐precision measurements over the tree rings demonstrated offsets of up to 40 years that indicate that P. copaifera can produce more than one ring in certain years. Results derived from the strongest climate–growth relationship during the most recent years of the record suggest that the climatic signal reported may be due to the presence of annual rings in some of those trees in recent years. Our study alerts about the risk of applying dendrochronology in species with challenging anatomical features defining tree rings, commonly found in the tropics, without an independent validation of annual periodicity of tree rings. High‐precision ¹⁴C measurements in multiple trees are a useful method to validate the identification of annual tree rings.