Functional Magnetic Resonance Imaging in Intact Plants—Quantitative Observation of Flow in Plant Vessels

Quantitative magnetic resonance (MR) images of flow velocities in intact corn plants were acquired using magnetization-prepared MR microscopy. A phase contrast flow imaging technique was used to quantitate water flow velocities and total volume flow rates in small xylem vessels. The simultaneous measurement of the transpiration of the whole plant was achieved by using a closed climate chamber within the MR magnet. The total volume flow rate and the transpiration values were in close correlation. Functional magnetic resonance imaging in intact plants was performed by light stimulation of the transpiration inside of the magnet. The change in the flow velocities in the xylem vessels of single vascular bundles was in correlation with the changes in the transpiration. Significant differences were observed between the xylem vessels in different vascular bundles. Furthermore, flow velocity measurements were performed on excised plant stems and visualized by the uptake of the MR contrast agent, gadolinium-diethylenetriamine pentaacetic acid (Gd-DTPA). A comparison between the phase contrast flow imaging and the contrast media uptake showed to be in good agreement with each other.     

Chromium localization in plant tissues of Lycopersicum esculentum Mill using ICP-MS and ion microscopy (SIMS)

High-resolution imaging secondary ion mass spectrometry (HRI-SIMS) in combination with inductively coupled plasma mass spectrometry (ICP-MS) were utilised to determine specific sites of chromium concentration in tomato plant tissues (roots, stems and leaves). The tissues were obtained from plants grown for 2 months in hydroponic conditions with Cr added in a form chromium salt (CrCl₃·6H₂O) to concentrations of 25 and 50 mg/L. The chemical fixation procedure used permit to localize only insoluble or strongly bound Cr components in tomato plant tissue. In this work no quantitative SIMS analysis was made. HRI-SIMS analysis revealed that the transport of chromium is restricted to the vascular system of roots, stems and leaves. No Cr was detected in epidermis, palisade parenchyma and spongy parenchyma cells of the leaves. The SIMS-300 spectra obtained from the tissues confirm the HRI-SIMS observations. The roots, and especially walls of xylem vessels, were determined as the principal site of chromium accumulation in tomato plants.     

Role of intracellular contents to facilitate supercooling capability in beech (Fagus crenata) xylem parenchyma cells

In order to find the possible role of intracellular contents in facilitating the supercooling capability of xylem parenchyma cells, changes in the temperature of supercooling levels were compared before and after the release of intracellular substances from beech xylem parenchyma cells by DTA. Various methods were employed to release intracellular substances from xylem parenchyma cells and all resulted in a reduction of supercooling ability. It was concluded that the reduction of supercooling ability primarily resulted from changes of intracellular conditions, including the release of intracellular contents or their mixing with extracellular solutions, rather than due to changes of cell wall structures. It is therefore suggested that any unidentified intracellular contents may function to facilitate supercooling capability in xylem parenchyma cells.     

Perfusion imaging of the human lung using flow-sensitive alternating inversion recovery with an extra radiofrequency pulse (FAIRER)

Pulmonary perfusion is an important parameter in the evaluation of lung diseases such as pulmonary embolism. A noninvasive MR perfusion imaging technique of the lung is presented in which magnetically labeled blood water is used as an endogenous, freely diffusible tracer. The perfusion imaging technique is an arterial spin tagging method called Flow sensitive Alternating Inversion Recovery with an Extra Radiofrequency pulse (FAIRER). Seven healthy human volunteers were studied. High-resolution perfusion-weighted images with negligible artifacts were acquired within a single breathhold. Different patterns of signal enhancement were observed between the pulmonary vessels and parenchyma, which persists up to TI = 1400 ms. The T1s of blood and lung parenchyma were determined to be 1.46s and 1.35 s, respectively.     

Biotrophic interaction of Sporisorium scitamineum on a new host—Saccharum spontaneum

Sporisorium scitamineum is a biotrophic smut fungus harbored inside the smut gall on the top internodal region of Saccharum spontaneum, a wild relative of sugarcane (Saccharum officinarum). The interactions of spined conidia of S. scitamineum with S. spontaneum were examined during the different stages of plant growth starting from the bud stage to the decaying stage. The spores in the soil from the polyetic inocula grew into confined epidermal cells of the buds and finally sporulated in the topmost internodal region. Hyphae invasion of the plant tissues were restricted to the point of infection. Culms of infected plants in late October sporulated, notably; hyphal sporulation produced shorter hyphal stolons. Remarkably, the nodal regions of infected plants had no spores and fragmented hyphae. On the basis of microscopic analyses, hyphae and spores were absent in all internodes above the ground till the topmost smut gall region. This result indicated that, S. scitamineum undergoes tissue-confined invasion of S. spontaneum. By associating culture medium method with polymerase chain reaction (PCR) on plant portions void of smut gall, S. scitamineum was not detected, indicating that colonization was not systemic. It was observed that the biotrophic interaction resulted in structural reorganization in the restricted region of infection forming erect cylindrical structure, in which the fungus was sandwiched between the central stalk and sheath, and possibly played a key role in preventing inflorescence. Comparatively, a significant difference in the rate of teliospores germination between reference Ustilago esculenta (26.6%, P < 0.05) and S. scitamineum (62.9%, P < 0.05) at 20° C was observed. This study also provides insights on the effect of different temperature regimes on the germination of S. scitamineum teliospores in vitro.     

Ultrasound processing to enhance drying of cashew apple bagasse puree: Influence on antioxidant properties and in vitro bioaccessibility of bioactive compounds

The present study has evaluated the effects of power ultrasound pre-treatment on air-drying and bioactive compounds of cashew apple bagasse. The sonication induced the disruption of cashew bagasse parenchyma, which resulted in lower resistance to water diffusion, less hysteresis, and increased rehydration rate. The processing did not affect the lignocellulose fibers or the sclerenchyma cells. For sonicated samples, water activity reached values below 0.4, after 2 h of drying, which is appropriate to prevent bacterial and fungi growth. The sorption isotherms of cashew apple bagasse presented sigmoid-shape for all samples and followed the type II according to BET classification. Sonicated cashew apple bagasse showed higher antioxidant activity, higher total phenolic compounds (TPC) and higher vitamin C content when compared to the non-sonicated sample. The increase in TPC and vitamin C contributed to the product antioxidant activity. A slight reduction on Vitamin C bioaccessibility was observed, but the TPC bioaccessibility has increased. Sonication reduced the quality loss of conventional drying treatments improving the quality of the dried product.     

Compositional contrast of uncoated fungal spores and stained section-face by low-loss backscattered electron imaging

Comparative surface imaging was performed on uncoated fungal spores and stained section-face by field emission scanning electron microscopy with an in-column energy-selective backscattered electron detector. Epoxy resin thin sections (ca. 200 and 500 nm thick) of the osmicated and uranyl acetate/lead citrate-stained fungus were examined with the microscope. Topographical contrast was evident in secondary electron imaging by either a below-lens or an in-lens detector. Meanwhile, low-loss backscattered electron images showed mainly compositional contrast at low accelerating voltages (mostly below 1 kV). With attenuated topographical contrast, several different electron densities could be detected, exhibiting several levels of electron density even on a flat plane of spines. Minute differences in topography on epoxy resin sections as seen by secondary electron imaging represented the periphery of the fungal spores and hyphae. On the other hand, the compositional contrast could be retrieved from stained section-face in low-loss BSE imaging, revealing subcellular entities after contrast inversion. The resolution of low-loss BSE imaging was sufficient to resolve plasma membrane, and various types of vacuoles and vesicles. These results suggest that low-loss backscattered electron imaging could potentially provide compositional information to resolve surface chemical features of uncoated microbial cells and stained section-face with heterogeneous surface compositions.     

Nonhost-associated proliferation of intrahyphal hyphae of citrus scab fungus Elsinoe fawcettii: refining the perception of cell-within-a-cell organization

Ultrastructural aspects of intrahyphal hyphae formation were investigated in Elsinoe fawcettii by transmission electron microscopy. Desiccated and hydrated cultures of E. fawcettii hyphae in liquid and solid media were prepared to determine the effects of water/nutrient availability and media fluidity on the formation of intrahyphal hyphae of the fungus. In all the culture conditions, intrahyphal hyphae were observed in enclosing hyphae. Electron-transparent hyphal cell walls clearly delimited intrahyphal hyphae from the cytoplasm of enclosing hyphae. Intrahyphal hyphae occupied most of the lumen of the enclosing hyphae, and showed intact hyphal cytoplasm with distinct organelles and inclusions. Intrahyphal hyphae were found to grow out of the degenerated hyphae that were almost devoid of cellular contents (simple intrahyphal hyphae). Some intrahyphal hyphae appeared to push aside a septum and passed into the adjacent hyphal cell. Besides a single intrahyphal hypha, instances were noted where enclosing hyphae contained several individual intrahyphal hyphae (multiple intrahyphal hyphae). Other enclosing hyphae contained intrahyphal hyphae, which also had intrahyphal hyphae (compound intrahyphal hyphae). The cell wall of intrahyphal hyphae showed the continuity with the cell wall of the enclosing hyphae. Concentric bodies typical of ascomycetes occurring in dry habitats were not found in all the types of hyphae. These results suggest that intrahyphal hyphae formation of E. fawcettii does not require plant defense responses. The fungus is thought to form intrahyphal hyphae during the saprophytic phase in ex planta ecological niches as well as the parasitic phase in host parts.     

Ultrastructure of spined conidia and hyphae of the rice false smut fungus Ustilaginoidea virens

Spined conidia and hyphae of Ustilaginoidea virens were examined by light and electron microscopy. Bright-field light microscopy showed that conidia were round to elliptical and warty on the surface with diameters approximately ranging from 3 to 5 microm. Scanning electron microscopy revealed the globose to irregularly rounded and ornamented conidia with prominent spines. The spines were pointed at the apex or irregularly curved, and approximately 200-500 nm long. Ultrastructure of spined conidia and hyphae revealed by transmission electron microscopy showed lipid globules and vacuoles in the cytoplasm enclosed by an electron-transparent cell wall. Conspicuous electron-dense spines were evident on the surface of conidia, and had obclavate or irregularly protruding shapes with varying heights along the conidial cell wall. Microfibrillar structures with stretching or branching patterns were evident in the spine matrix. Some conidia were interconnected by spines from the neighboring conidia by their extended outgrowth. Hyphae had concentric bodies that showed an electron-transparent core surrounded by an electron-dense layer. One or more intrahyphal hyphae were found in hyphal cytoplasm. The fungus is thought to form concentric bodies and intrahyphal hyphae as survival mechanisms against the water- and nutrient-deficient environments that may occur in the necrotic regions of host plants.     

Ultrastructural analysis of Vitis vinifera leaf tissues showing atypical symptoms of Plasmopara viticola

In an abandoned farm in Tuscany a year by year regression of downy mildew disease on grapevines was observed and a decrease in virulence as well as vigor and fertility of the causal fungus, Plasmopara viticola. Anomalous spots of the fungus (i.e. atypical coloration of leaves or mosaic) on leaf tissues of a sensitive Vitis vinifera grapevine were observed. The anomalous symptoms were often associated with the typical 'oil spots' and were present under environmental conditions favourable for a normal development of the disease. An ultrastructural study was carried out on leaf tissues of grapevine plants aimed at clarifying the cause of this phenomenon and detecting whether there were alterations in P. viticola mycelium and endophytes present. ELISA was also performed to check the presence of grapevine viruses in the plants. TEM results demonstrated that characteristic P. viticola was present in leaf samples showing oil spots, while, both the fungus and the host tissues showed cytological alterations in leaves with mosaic symptoms. Finally, hyphae were absent in leaf tissues without downy mildew spots, but showing severe ultrastructural modifications. Several plant virus infections were found in these grapevines. Literature reports that the development and sporulation of some phytopathogenic fungi inside their hosts can be limited by virus infections. Further experimental approaches are required to determine if resistance to P. viticola can be induced by viral infections in grapevines.     

Ultrastructural insights into tomato infections caused by three different pathotypes of Pepino mosaic virus and immunolocalization of viral coat proteins

This paper presents studies on an ultrastructural analysis of plant tissue infected with different pathotypes of Pepino mosaic virus (PepMV) and the immunolocalization of viral coat proteins. Because the PepMV virus replicates with a high mutation rate and exhibits significant genetic diversity, therefore, isolates of PepMV display a wide range of symptoms on infected plants. In this work, tomato plants of the Beta Lux cultivar were inoculated mechanically with three pathotypes representing the Chilean 2 (CH2) genotype: mild (PepMV-P22), necrotic (PepMV-P19) and yellowing (PepMV-P5-IY). The presence of viral particles in all infected plants in the different compartments of various cell types (i.e. spongy and palisade mesophyll, sieve elements and xylem vessels) was revealed via ultrastructural analyses. For the first time, it was possible to demonstrate the presence of crystalline inclusions, composed of virus-like particles. In the later stage of PepMV infection (14 dpi) various pathotype-dependent changes in the structure of the individual organelles (i.e. mitochondria, chloroplasts) were found. The strongest immunogold labeling of the viral coat proteins was also observed in plants infected by necrotic isolates. A large number of viral coat proteins were marked in the plant conductive elements, both xylem and phloem.     

Fast NMR flow measurements in plants using FLASH imaging

A fast method for quantitative NMR imaging of flow velocities in intact plants is described. The purpose of this method is to observe dynamic changes of flow velocity in the xylem of plants after fast changes of environmental conditions. The spatial image resolution is 47 x 188 micrometer(2) in-plane. The method applies a fast gradient echo sequence (FLASH). Compared to other flow NMR imaging sequences, the imaging time was reduced by a factor of 6 with comparable signal-to-noise ratio. A complete flow measurement consists of a set of 8 different flow weighted images with a total acquisition time of 3.5 min.     

Use of light and scanning electron microscopy to examine colonisation of barley rhizosphere by the nematophagous fungus Verticillium chlamydosporium

Barley roots were readily colonised by the nematophagous fungus Verticillium chlamydosporium. Light microscopy (LM) but also low temperature scanning electron microscopy (LTSEM) revealed details of the colonisation process. Hyphae were found on the rhizoplane often with dictyochlamydospores. Hyphae of V. chlamydosporium penetrated epidermal cells, often by means of appressoria. A hyphal network was formed in epidermal and cortical cells. Likewise, hyphal coils were found within root cells next to transverse cell walls. Cortical cells were the limits of fungal colonisation, since no hyphae were seen in the vascular cylinder. Modifications of root cell contents (phenolic droplets and callose appositions) were common three weeks after inoculation with V. chlamydosporium. These features may indicate induction of plant defence reactions in late stages of root colonisation by the fungus. Both LTSEM and LM have proved extremely useful to describe root colonisation by the fungus. The results found may have implications in the mode action of nematophagous fungi against plant parasitic nematodes.     

BOLD signal compartmentalization based on the apparent diffusion coefficient

Functional MRI (fMRI) can detect blood oxygenation level dependent (BOLD) hemodynamic responses secondary to neuronal activity. The most commonly used method for detecting fMRI signals is the gradient-echo echo-planar imaging (EPI) technique because of its sensitivity and speed. However, it is generally believed that a significant portion of these signals arises from large veins, with additional contribution from the capillaries and parenchyma. Early experiments using diffusion-weighted gradient-echo EPI have suggested that intra-voxel incoherent motion (IVIM) weighting inherent in the sequence can selectively attenuate contributions from different vessels based on the differences in the mobility of the blood within them. In the present study, we used similar approach to characterize the apparent diffusion coefficient (ADC) distribution within the activated areas of BOLD contrast. It is shown that the voxel values of the ADCs obtained from this technique can infer various vascular contributions to the BOLD signal.     

Effects of gamma irradiation on morphological changes and biological responses in plants

This review discusses the morphological changes and biological responses of plants irradiated with gamma rays. Seedlings exposed to relatively low doses of gamma rays (1-5 Gy) developed normally, while the growth of plants irradiated with a high dose gamma ray (50 Gy) was significantly inhibited. Based on TEM observations, chloroplasts were extremely sensitive to gamma irradiation compared to other cell organelles, particularly thylakoids being heavily swollen. In addition, some portions of the mitochondria and endoplasmic reticulum were structurally altered, for example, distortion and swelling. The cerium perhydroxide deposition, as a maker for H(2)O(2) deposition, was typically manifest on the plasma membranes and cell walls of the tissues from both the control and irradiated plants. However, the intensities of cerium perhydroxide deposits (CPDs) were remarkably increased in the plasma membranes and cell walls of pumpkin tissues such as petiole, cotyledon, hypocotyl and especially leaf after gamma irradiation. These observations are in good agreement with the results of H(2)O(2) content in all tissues. The immuno-localization analysis for peroxidase (POD) on the tissues from pumpkin plant showed the same pattern between the control and irradiated plants, but the density of gold particles as indication of POD localization was significantly increased on the cell corner middle lamellae of parenchyma cells, especially in the petiole after gamma irradiation. However, accumulation and localization of H(2)O(2) and POD in vessels were not significantly different between both plants. The accumulation and localization of both H(2)O(2) and POD were differentially affected by gamma irradiation depending on the different tissue types. The deposition of both H(2)O(2) and POD in parenchyma cells appeared much higher than in vessels, suggesting that the former is more sensitive than the latter against gamma rays.     

可见光显微分光光度法测定毛竹发育过程中木素微区分布

运用光学显微镜和可见光显微分光光度计以及组织化学染色对毛竹发育过程中的木质素微区分布进行了研究。木质素在各组织中均有分布,其含量因竹龄,组织及细胞壁微区不同而有差异。细胞壁各微区中存在愈创木基(G)和紫丁香基(S)两种木质素组成单元。纤维次生壁,细胞角隅区和复合胞间层的木质素含量12个月内逐渐增加,而后变化较小,但是各微区的木质化速率因竹龄变化而变化。同一竹龄竹壁径向和纤维帽不同位置的木质素含量未有明显的规律性变化。薄壁组织次生壁,胞间层以及导管次生壁在12个月内木质化程度不断加强,而后变化较小。     

The role of voxel aspect ratio in determining apparent vascular phase behavior in susceptibility weighted imaging

Susceptibility weighted imaging (SWI) uses apparent phase contrast to enhance the contrast-to-noise ratio (CNR) in the magnitude image. In theory, the apparent phase will depend on the aspect ratio when both venous blood and parenchyma occupy the same voxel. To demonstrate the maximal expected effect of the external field from a vein, we model the vein as an infinitely long cylinder perpendicular to the main magnetic field. The results show that the apparent phase of a voxel in the image is a function of resolution, vessel size and, to a lesser degree, vessel center within the voxel. The simulations explain why a negative-phase mask has worked in SWI processing of high-resolution images collected in the transverse direction, despite the expected positive-phase behavior for vessels perpendicular to the main field. The predicted phase behavior from the simulations is in good agreement with that observed from human brain datasets.     

基于共聚焦显微拉曼的真菌菌丝中几丁质的原位检测研究

几丁质是真菌细胞壁中一种重要的结构多糖,本文首次采用共聚焦显微拉曼技术对山茶刺盘孢菌的气生菌丝进行原位检测研究,首先确定了采集菌丝拉曼光谱的最优实验参数,并获得了菌丝,几丁质标准品和背景三种物质的典型拉曼光谱,对其中的特征峰进行归属分析,发现菌丝光谱中有明显的几丁质特征峰。然后对置于载玻片上菌丝的感兴趣区域进行拉曼光谱面扫描,通过主成分分析法发现面扫描区域中,菌丝和背景两种信号可以明显区分开来,结合主成分的载荷因子图得到了菌丝的两个主要的特征差异峰1 622和1 368 cm-1,1 622 cm-1属于菌丝中几丁质的特征峰,而1 368 cm-1是来源于菌丝中的果胶多糖。最后通过对几丁质在1 622 cm-1特征峰波段附近范围积分,绘制了几丁质在菌丝中二维和三维的化学成像图,直观且无损的再现了几丁质在菌丝中的空间分布。     

Morphological analysis of mouse lungs after treatment with magnetite-based magnetic fluid stabilized with DMSA

Mouse lungs injected with magnetic fluids based on magnetite nanoparticles stabilized by 2,3-dimercaptosuccinic acid were studied. We observed clusters of magnetic nanoparticles inside blood vessels, within the organ parenchyma and cells, as well as increased numbers of leukocytes in the organ. Both the particle concentration and organ inflammation diminished in a time-dependent manner.     

First evidence of an intimate symbiotic association between fungi and larvae in basal attine ants

The most conspicuous ants in all of tropical America are those that belong to the tribe Attini which cultivate fungus. The objective of the present study is to verify the alterations that occur in the cuticle of the worker larvae from Myrmicocrypta, Mycetarotes and Trachymyrmex with the purpose of trying to establish the degree of this association. Attine ants from the Atta genus were used as a control group. The analysis of histological sections showed results about the distribution of the cells and tissues on the epidermis of A. sexdens rubropilosa ant larvae and Mycetarotes parallelus, Trachymyrmex fuscus and Myrmicocrypta sp. A cuticle covering the ants was observed in the larvae of all the species, and this is formed by a simple cubic epithelium, whose cells possibly change its shape to prismatic, depending on their secretory activity. Just under this epithelium large adipose cells with reserve granules in their cytoplasm were found. The presence of a space filled by granulose and acellular material was also observed, indicating that the larvae were in a molt period. The presence of fungal hyphae is observed both on the external side of the basal attine larvae as well as emitting projections to the interior of the cuticle reaching the epithelium and the adipocyte cells. Data obtained in the present study demonstrated that the fungus deposited on the surface of immature ants from attine basal species maintain a close relationship with them, once the fungus hyphae have the ability to disorganize the cuticle lamellas, penetrating the interior of the insect cells through the emission of prolongations transporting the cuticle and epithelium barriers and making substances exchanges between larvae and fungus.