Nutrient supplementation-induced metabolic profile changes and early appearance of free N-glycans in nutrient deficient tomato plants revealed by mass spectrometry.

Inorganic fertilizers are routinely used in large scale crop production for the supplementation of nitrogen, phosphorus, and potassium in nutrient poor soil. To explore metabolic changes in tomato plants grown on humic sand under different nutritional conditions, matrix-assisted laser desorption ionization (MALDI) mass spectrometry was utilized for the analysis of xylem sap. Variations in the abundances of metabolites and oligosaccharides, including free N-glycans (FNGs), were determined. Statistical analysis of the sample-related peaks revealed significant differences in the abundance ratios of multiple metabolites, including oligosaccharides, between the control plants, grown with no fertilizers, and plants raised under "ideal" and "nitrogen deficient" nutritional conditions, i.e., under the three treatment types. Among the 36 spectral features tentatively identified as oligosaccharides, the potential molecular structures for 18 species were predicted based on their accurate masses and isotope distribution patterns. To find the spectral features that account for most of the differences between the spectra corresponding to the three different treatments, multivariate statistical analysis was carried out by orthogonal partial least squares-discriminant analysis (OPLS-DA). They included both FNGs and non-FNG compounds that can be considered as early indicators of nutrient deficiency. Our results reveal that the potential nutrient deficiency indicators can be expanded to other metabolites beyond FNGs. The m/z values for 20 spectral features with the highest variable influence on projection (VIP) scores were ranked in the order of their influence on the statistical model.

Profiling of widely targeted metabolomics for the identification of chemical composition in epidermis, xylem and pith of Gleditsiae Spina.

Gleditsiae Spina, the thorn of Gleditsia sinensis Lam., has a long history of being used as a traditional medicine in East Asian countries. However, only a few biologically active substances have been identified from it. In this study, the epidermis, xylem and pith of Gleditsiae Spina, respectively Gs-E, Gs-X and Gs-P, were studied. We used a widely targeted metabolomics method to investigate the chemical composition of Gs-E, Gs-X and Gs-P. A total of 728 putative metabolites were identified from Gleditsiae Spina, including 211 primary metabolites and 517 secondary metabolites. These primary and secondary metabolites could be categorized into more than 10 different classes. Flavonoids, phenolic acids, lipids, amino acids and derivatives, and organic acids constituted the main metabolite groups. Multivariate statistical analysis showed that the Gs-E, Gs-X and Gs-P samples could be clearly separated. Differential accumulated metabolite (DAM) analysis revealed that more than half of the DAMs exhibited the highest relative concentrations in Gs-E, and most of the DAMs showed the lowest relative concentrations in Gs-X. Moreover, 11 common differential primary metabolites and 79 common differential secondary metabolites were detected in all comparison groups. These results further our understanding of chemical composition and metabolite accumulation of Gleditsiae Spina.

Transfer of Metal(loid)s from Soil to Leaves and Trunk Xylem Sap of Medicinal Plants and Possible Health Risk Assessment.

The objective of the present study was to investigate metal(loid)s in soils, in the trunk xylem sap and in the leaves of the Dipteryx alata plant located near the highway with high vehicle traffic in agricultural regions and near landfills, and to assess the transfer of metal(loid)s from soil to plant and possible health risk assessment. Trunk xylem sap, leaves and soil samples were collected at three sites near the highway. The analysis of trace elements was carried out using inductively coupled plasma optical emission spectroscopy (ICP OES). In the three soil sampling sites far from the highway edge, 15 elements were quantified. The concentrations of elements in the soil presented in greater proportions in the distance of 5 m in relation to 20 and 35 m. The metal(loid)s content in the study soil was higher than in other countries. The concentrations of Al, Cu, Fe, Mg, Mn, P, Se and Zn in the xylem sap were much higher than the leaves. The values of transfer factor of P, Mg and Mn from soil to the xylem sap and transfer factor of P from soil to leaf were greater than 1, indicating that the specie have a significant phytoremediation and phytoextraction potential. This plant has a tendency to accumulate As, Cd and Cr in its leaf tissues. The chronic hazard index (HI) values recorded in this study were above 1 for adults and adolescents. It is concluded that the soil, the trunk xylem sap and leaves of this plant are contaminated by heavy metals. Ingestion of the trunk xylem sap of this plant can cause toxicity in humans if ingested in large quantities and in the long term; therefore, its consumption should be avoided.

Positron-emitting tracer imaging of fluoride transport and distribution in tea plant.

BACKGROUND: Tea (Camellia sinensis (L.) O. Kuntze) is a hyper-accumulator of fluoride (F). To understand F uptake and distribution in living plants, we visually evaluated the real-time transport of F absorbed by roots and leaves using a positron-emitting (18 F) fluoride tracer and a positron-emitting tracer imaging system. RESULTS: F arrived at an aerial plant part about 1.5 h after absorption by roots, suggesting that tea roots had a retention effect on F, and then was transported upward mainly via the xylem and little via the phloem along the tea stem, but no F was observed in the leaves within the initial 8 h. F absorbed via a cut petiole (leaf 4) was mainly transported downward along the stem within the initial 2 h. Although F was first detected in the top and ipsilateral leaves, it was not detected in tea roots by the end of the monitoring. During the monitoring time, F principally accumulated in the node. CONCLUSION: F uptake by the petiole of excised leaf and root system was realized in different ways. The nodes indicated that they may play pivotal roles in the transport of F in tea plants. © 2020 Society of Chemical Industry.

Comparative Investigation for Rotten Xylem (kuqin) and Strip Types (tiaoqin) of Scutellaria baicalensis Georgi Based on Fingerprinting and Chemical Pattern Recognition.

Scutellaria baicalensis Georgi (SBG) is not just as a traditional herbal medicine but also a popular functional food in China and other Asian countries. A sensitive simple strategy was developed for the first time to analyze SBG from eight different geographical sources using high-performance liquid chromatography (HPLC) coupled with multivariate chemometric methods. Two unsupervised pattern recognition models, hierarchical cluster analysis (HCA) and principal components analysis (PCA), and a supervised pattern recognition model, partial least squares discriminant analysis (PLS-DA), were used to analyze the chemical compositions and physical traits of SBG. The important chemical markers baicalin, baicalein, and wogonoside were analyzed quantitatively and with PLS-DA. These methods distinguished rotten xylem (kuqin) and strip types (tiaoqin) of SBG and found that the thickness of the slice had a significant impact on the classification of SBG. Two classes of strip types were identified: one as the uncut pharmaceutical, which was sectioned with a thickness >3 mm; the other as a thin-sectioned strip type, with a thickness of <2 mm. This fingerprinting technique coupled to a chemometric analysis was used for the simultaneous quantitation of three components (chemical markers) of SBG, and greatly simplified the complicated identification of the multiple components of this plant relative to traditional methods. The strategy can clearly distinguish between kuqin and tiaoqin of SBG, and suggests that the thickness of the slice can be used as the basis for evaluation of SBG. These data provide a theoretical basis and scientific evidence for the development and utilization of SBG.

Characterization of Chemical Component Variations in Different Growth Years and Tissues of Morindae Officinalis Radix by Integrating Metabolomics and Glycomics.

Morindae Officinalis Radix (MOR), the dried root of Morinda officinalis F.C. How (Rubiaceae), is a popular food supplement in southeastern China for bone protection, andrological, and gynecological healthcare. In clinical use, 3-4 year old MOR is commonly used and the xylem is sometimes removed. However, there is no scientific rationale for these practices so far. In this study, metabolomics and glycomics were integrated using multiple chromatographic and mass spectrometric techniques coupled with multivariate statistical analysis to investigate the qualitative and quantitative variations of secondary metabolome and glycome in different growth years (1-7 years) and tissues (xylem and cortex) of MOR. The results showed that various types of bioactive components reached a maximum between 3 and 4 years of growth and that the xylem contained more potentially toxic constituents but less bioactive components than the cortex. This study provides the chemical basis for the common practice of using 3-4 year old MOR with the xylem removed.

Tissue-Specific Metabolite Profiling on the Different Parts of Bolting and Unbolting Peucedanum praeruptorum Dunn (Qianhu) by Laser Microdissection Combined with UPLC-Q/TOF⁻MS and HPLC⁻DAD.

BACKGROUND: Qianhu is a traditional Chinese medicine. It is thought that Qianhu roots will harden after bolting and not be suitable for medicinal purposes. Bolting Qianhu and unbolting Qianhu are referred to as "Xiong Qianhu" and "Ci Qianhu," respectively. In this study, the properties, microscopic and chemical characteristics of Ci Qianhu and Xiong Qianhu roots were compared using fluorescence microscopy, laser microdissection coupled with ultra-high-performance liquid chromatography-quadrupole/time-of-flight mass spectrometry, and high-performance liquid chromatography with diode-array detection. RESULTS: Microscopy results showed that the area of secondary xylem in the root increased after bolting, with the cork and secretory canals showing strong fluorescence intensity. A total of 34 peaks, mostly pyranocoumarins, were identified in the tissues of Ci Qianhu and Xiong Qianhu. The secretory canals contained the highest variability of coumarins, whereas the secondary xylem contained the least coumarins. Moreover, seven coumarins, especially the pyran- coumarin, decreased after bolting. Generally, both before and after bolting, coumarin level was the highest in the bark, followed by the middle part, and the lowest in the inner part. CONCLUSION: Thus, it was indicated that the area of secondary xylem increased after bolting, however the coumarin variant and content decreased in the secondary xylem of Qianhu. The result shows that the quality of Qianhu decreases after bolting, which supports the viewpoint that Xiong Qianhu is not suitable for medicinal use.

Elevated CO2 does not affect stem CO2 efflux nor stem respiration in a dry Eucalyptus woodland, but it shifts the vertical gradient in xylem [CO2 ].

To quantify stem respiration (RS ) under elevated CO2 (eCO2 ), stem CO2 efflux (EA ) and CO2 flux through the xylem (FT ) should be accounted for, because part of respired CO2 is transported upwards with the sap solution. However, previous studies have used EA as a proxy of RS , which could lead to equivocal conclusions. Here, to test the effect of eCO2 on RS , both EA and FT were measured in a free-air CO2 enrichment experiment located in a mature Eucalyptus native forest. Drought stress substantially reduced EA and RS , which were unaffected by eCO2 , likely as a consequence of its neutral effect on stem growth in this phosphorus-limited site. However, xylem CO2 concentration measured near the stem base was higher under eCO2 , and decreased along the stem resulting in a negative contribution of FT to RS , whereas the contribution of FT to RS under ambient CO2 was positive. Negative FT indicates net efflux of CO2 respired below the monitored stem segment, likely coming from the roots. Our results highlight the role of nutrient availability on the dependency of RS on eCO2 and suggest stimulated root respiration under eCO2 that may shift vertical gradients in xylem [CO2 ] confounding the interpretation of EA measurements.

A Method to Study the Distribution Patterns for Metabolites in Xylem and Phloem of Spatholobi Caulis.

Spatholobi Caulis (SC), the vine stem of Spatholobus suberectus Dunn, is a widely used traditional Chinese medicine (TCM) for the treatment of blood stasis syndrome and related diseases. Xylem and phloem are the main structures of SC and the color of xylem in SC is red brown or brown while the phloem with resin secretions is reddish brown to dark brown. They are alternately arranged in a plurality of concentric or eccentric rings. In order to investigate the distribution patterns of metabolites in xylem and phloem of SC, an analytical method based on UFLC-QTRAP-MS/MS was established for simultaneous determination of 22 constituents including four flavanols, nine isoflavones, two flavonols, two dihydroflavones, one flavanonol, one chalcone, one pterocarpan, one anthocyanidin and one phenolic acid in the samples (xylem and phloem) from Laos. Furthermore, according to the contents of 22 constituents, heat map, principal components analysis (PCA), orthogonal partial least squares discriminant analysis (OPLS-DA) and t-test were used to evaluate the samples and discover the differences between xylem and phloem of SC. The results indicated that the measured ingredients in xylem and phloem were significantly different. To be specific, the contents of flavonoids in xylem were higher than that in phloem, while the content of protocatechuic acid showed a contrary tendency. This study will not only reveal the distribution patterns of metabolites in xylem and phloem of SC but also facilitate further study on their quality formation.

Illumination on "Reserving Phloem and Discarding Xylem" and Quality Evaluation of Radix polygalae by Determining Oligosaccharide Esters, Saponins, and Xanthones.

The root of Polygala tenuifolia Willd. or Polygala sibirica L. exhibits protective effects on the central nervous system and is frequently used to treat insomnia, amnesia, and other cognitive dysfunction. In our study, we studied nine bioactive compounds spanning oligosaccharide esters, saponins, and xanthones by using a sensitive, efficient, and validated method established on ultra-performance liquid chromatography coupled with triple quadrupole mass spectrometry. The quantified result of interesting compounds proved that accumulation of those compounds were found in phloem rather than in xylem. By taking the standardized result of nine compound contents into account, the "Spider-web" analytical result of xylem and phloem from Radix polygalae (RP) unveiled the rationality of RP's classical use in clinic including discarding the xylem and reserving the phloem. Moreover, the remarkable variation was also revealed from the quantitative result of 45 samples with different diameters from the different origins, which did not significantly correlate with the variation of RP's diameter. Our study could shed the light on the quality assessment of RP for further research and illustrate the scientific connotation of the processing method of "discarding the xylem and reserving the phloem".

Growth rings in roots of medicinal perennial dicotyledonous herbs from temperate and subtropical zones in China.

Growth years are closely related to the quality of Chinese medicinal materials, and they can provide a reliable basis for quality evaluation on crude drugs in accordance with the suitable growth years. Herbchronology, the numerical and pattern analysis of annual growth rings in herbaceous perennials, has emerged to be a recognized science that is applied in age determination of perennial forbs. Fifty medicinal species growing in temperate and subtropical zones of China were examined by microstructure observation to determine the presence of growth rings in the secondary root xylem of medicinal perennial dicotyledonous herbs. Nearly half of the surveyed species showed clearly or relatively clearly demarcated growth rings in their roots, and additional anatomical patterns have been observed in different species. Clear variations in growth rings of different genus or species were characterized in this study, and these findings proved the feasibility of applying herbaceous plant growth rings in age identification of traditional Chinese medicine.

Enzymatic activities for lignin monomer intermediates highlight the biosynthetic pathway of syringyl monomers in Robinia pseudoacacia.

Most of the known 4-coumarate:coenzyme A ligase (4CL) isoforms lack CoA-ligation activity for sinapic acid. Therefore, there is some doubt as to whether sinapic acid contributes to sinapyl alcohol biosynthesis. In this study, we characterized the enzyme activity of a protein mixture extracted from the developing xylem of Robinia pseudoacacia. The crude protein mixture contained at least two 4CLs with sinapic acid 4-CoA ligation activity. The crude enzyme preparation displayed negligible sinapaldehyde dehydrogenase activity, but showed ferulic acid 5-hydroxylation activity and 5-hydroxyferulic acid O-methyltransferase activity; these activities were retained in the presence of competitive substrates (coniferaldehyde and 5-hydroxyconiferaldehyde, respectively). 5-Hydroxyferulic acid and sinapic acid accumulated in the developing xylem of R. pseudoacacia, suggesting, in part at least, sinapic acid is a sinapyl alcohol precursor in this species.

Binding of transition metals to monosilicic acid in aqueous and xylem (Cucumis sativus L.) solutions: a low-T electron paramagnetic resonance study.

The supplementation of monosilicic acid [Si(OH)4] to the root growing medium is known to protect plants from toxic levels of iron (Fe), copper (Cu) and manganese (Mn), but also to mitigate deficiency of Fe and Mn. However, the physicochemical bases of these alleviating mechanisms are not fully understood. Here we applied low-T electron paramagnetic resonance (EPR) spectroscopy to examine the formation of complexes of Si(OH)4 with Mn(2+), Fe(3+), and Cu(2+) in water and in xylem sap of cucumber (Cucumis sativus L.) grown without or with supply of Si(OH)4. EPR, which is also useful in establishing the redox state of these metals, was combined with measurements of total concentrations of metals in xylem sap by inductive coupled plasma. Our results show that Si(OH)4 forms coordination bonds with all three metals. The strongest interactions of Si(OH)4 appear to be with Cu(2+) (1/1 stoichiometry) which might lead to Cu precipitation. In line with this in vitro findings, Si(OH)4 supply to cucumber resulted in dramatically lower concentration of this metal in the xylem sap. Further, it was demonstrated that Si(OH)4 supplementation causes pro-reductive changes that contribute to the maintenance of Fe and, in particular, Mn in the xylem sap in bioavailable 2+ form. Our results shed more light on the intertwined reactions between Si(OH)4 and transition metals in plant fluids (e.g. xylem sap).

Online induction heating for determination of isotope composition of woody stem water with laser spectrometry: a methods assessment.

Application of stable isotopes of water to studies of plant-soil interactions often requires a substantial preparatory step of extracting water from samples without fractionating isotopes. Online heating is an emerging approach for this need, but is relatively untested and major questions of how to best deliver standards and assess interference by organics have not been evaluated. We examined these issues in our application of measuring woody stem xylem of sagebrush using a Picarro laser spectrometer with online induction heating. We determined (1) effects of cryogenic compared to induction-heating extraction, (2) effects of delivery of standards on filter media compared to on woody stem sections, and (3) spectral interference from organic compounds for these approaches (and developed a technique to do so). Our results suggest that matching sample and standard media improves accuracy, but that isotopic values differ with the extraction method in ways that are not due to spectral interference from organics.

The transcriptome of sesquiterpenoid biosynthesis in heartwood xylem of Western Australian sandalwood (Santalum spicatum).

The fragrant heartwood oil of West Australian sandalwood (Santalum spicatum) contains a mixture of sesquiterpene olefins and alcohols, including variable levels of the valuable sesquiterpene alcohols, α- and ß-santalol, and often high levels of E,E-farnesol. Transcriptome analysis revealed sequences for a nearly complete set of genes of the sesquiterpenoid biosynthetic pathway in this commercially valuable sandalwood species. Transcriptome sequences were produced from heartwood xylem tissue of a farnesol-rich individual tree. From the assembly of 12,537 contigs, seven different terpene synthases (TPSs), several cytochromes P450, and allylic phosphatases were identified, as well as transcripts of the mevalonic acid and methylerythritol phosphate pathways. Five of the S. spicatum TPS sequences were previously unknown. The full-length cDNA of SspiTPS4 was cloned and the enzyme functionally characterized as a multi-product sesquisabinene B synthase, which complements previous characterization of santalene and bisabolol synthases in S. spicatum. While SspiTPS4 and previously cloned sandalwood TPSs do not explain the prevalence of E,E-farnesol in S. spicatum, the genes identified in this and previous work can form a basis for future studies on natural variation of sandalwood terpenoid oil profiles.

[Study on origin and authentication of medicinal materials of Dalbergiae Lignum in crude drug's market].

The study is aimed to distinguish morphological characteristics of Dalbergiae Lignum collected from crude drug's markets and establish a identification methods and the quality standard for Dalbergiae Lignum. The macroscopic and microscopic features of Dalbergiae Lignum from crude drug's market were observed, analyzed and compared according to Hongmu specification issued by the People's Republic of China in 2000, and by the characteristics recorded in domestic monograph of Mucai Shibie (wood identification). The redwood of Dalbergiae Lignum cut into small pieces as medicinal material are dry heart wood of mahogany (trees from Dalbergia sp.), which characteristics of the small pieces as crude drug are different. There are differences in macroscopic and microscopic features about texture of wood and color, odor, taste, transverse section, radial section, tangential section. The results can provide basis for identification, application and improment of the quality standard of Dalbergiae Lignum as medicinal material.

[Dynamic changes of polysaccharides content in Salvia miltiorrhiza root from different provenances].

OBJECTIVE: To detect the content of polysaccharides in the root of Salvia miltiorrhiza collected from different provenances, and to discover the dynamic change rules of polysaccharides at various growing stages. METHODS: Polysaccharides were extracted by hot water extraction method, and the content of polysaccharides was measured by phenol-sulfuric acid. RESULTS: The content of polysaccharides changed at different growing stages. The variation trend of different provenances were different, and the polysaccharides content of different provenances were different at the same growing stage. At different growing periods, the distribution of polysaccharides in the xylem and cortex of roots were different. The content of polysacchrides in the root was minimum at maturity. At harvest time, the content of polysaccharides in the root was the highest in Beijing, the second was in Jiangsu, and the lowest was in Henan. CONCLUSION: The experiment provides theoretical basis for the selection of Salvia miltiorrhiza with the highest content of polysaccharides and the development and utilization of the polysaccharides.

Arabinogalactan protein-rich cell walls, paramural deposits and ergastic globules define the hyaline bodies of rhinanthoid Orobanchaceae haustoria.

BACKGROUND AND AIMS: Parasitic plants obtain nutrients from their hosts through organs called haustoria. The hyaline body is a specialized parenchymatous tissue occupying the central parts of haustoria in many Orobanchaceae species. The structure and functions of hyaline bodies are poorly understood despite their apparent necessity for the proper functioning of haustoria. Reported here is a cell wall-focused immunohistochemical study of the hyaline bodies of three species from the ecologically important clade of rhinanthoid Orobanchaceae. METHODS: Haustoria collected from laboratory-grown and field-collected plants of Rhinanthus minor, Odontites vernus and Melampyrum pratense attached to various hosts were immunolabelled for cell wall matrix glycans and glycoproteins using specific monoclonal antibodies (mAbs). KEY RESULTS: Hyaline body cell wall architecture differed from that of the surrounding parenchyma in all species investigated. Enrichment in arabinogalactan protein (AGP) epitopes labelled with mAbs LM2, JIM8, JIM13, JIM14 and CCRC-M7 was prominent and coincided with reduced labelling of de-esterified homogalacturonan with mAbs JIM5, LM18 and LM19. Furthermore, paramural bodies, intercellular deposits and globular ergastic bodies composed of pectins, xyloglucans, extensins and AGPs were common. In Rhinanthus they were particularly abundant in pairings with legume hosts. Hyaline body cells were not in direct contact with haustorial xylem, which was surrounded by a single layer of paratracheal parenchyma with thickened cell walls abutting the xylem. CONCLUSIONS: The distinctive anatomy and cell wall architecture indicate hyaline body specialization. Altered proportions of AGPs and pectins may affect the mechanical properties of hyaline body cell walls. This and the association with a transfer-like type of paratracheal parenchyma suggest a role in nutrient translocation. Organelle-rich protoplasts and the presence of exceptionally profuse intra- and intercellular wall materials when attached to a nitrogen-fixing host suggest subsequent processing and transient storage of nutrients. AGPs might therefore be implicated in nutrient transfer and metabolism in haustoria.

Antifungal and cytotoxicity activities of the fresh xylem sap of Hymenaea courbaril L. and its major constituent fisetin.

BACKGROUND: The great potential of plants as Hymenaea courbaril L (jatoba) has not yet been throughly explored scientifically and therefore it is very important to investigate their pharmacological and toxicological activities to establish their real efficacy and safety. This study investigated the cytotoxicity of xylem sap of Hymenaea courbaril L and its bioactivity against the fungi Cryptococcus neoformans species complex and dermatophytes. METHODS: The fresh xylem sap of H. courbaril was filtered resulting in an insoluble brown color precipitate and was identified as fisetin. In the filtrate was identified the mixture of fisetinediol, fustin, 3-O-methyl-2,3-trans-fustin and taxifolin, which were evaluated by broth microdilution antifungal susceptibility testing against C. neoformans species complex and dermatophytes. The fresh xylem sap and fisetin were screened for cytotoxicity against the 3T3-A31 cells of Balb/c using neutral red uptake (NRU) assay. RESULTS: The fresh xylem sap and the fisetin showed higher in vitro activity than the filtrate. The xylem sap of H. courbaril inhibited the growth of dermatophytes and of C. neoformans with minimal inhibition concentration (MIC) < 256 µg/mL, while the fisetin showed MIC < 128 µg/mL for these fungi. Fisetin showed lower toxicity (IC50 = 158 µg/mL) than the fresh xylem sap (IC50 = 109 µg/mL). CONCLUSION: Naturally occurring fisetin can provide excellent starting points for clinical application and can certainly represent a therapeutic potential against fungal infections, because it showed in vitro antifungal activity and low toxicity on animal cells.

A new approach for the study of the chemical composition of bordered pit membranes: 4Pi and confocal laser scanning microscopy.

PREMISE OF THE STUDY: Coniferous bordered pits are some of the most unique and fascinating microstructures of the lignified cell wall. The pit membrane consists of a margo and a torus region, hence facilitating both xylary water transport and also limiting air intrusion by pit aspiration. Additionally, bordered pits have been reported to play a decisive role in the control of rapid liquid flow via the shrinkage and swelling of pectin. The study of the nanostructural chemical composition of pit membranes has been difficult with common imaging/chemical techniques, which involve drying and/or coating of the samples. • METHODS: Using fluorescent tagging and antibodies specific to pectin, and a His-tagged cellulose-binding module that reacts with crystalline cellulose, in combination with confocal laser scanning microscopy (CLSM) and 4Pi microscopy, we generated three-dimensional images of intact pit membranes. • KEY RESULTS: With enhanced resolution in the z-direction of the 4Pi microscope, it was possible to distinguish cellulose in the torus and the margo strands of Pinus strobus. The torus was surrounded by pectin, and a pectin ring was found at the margin of the torus. We also found differences in the structure of the pit membrane between aspirated and unaspirated pits, with a displacement of pectin to form a ring-like structure, the collapse of a void in the interior of the torus, and an apparent change in the chemical structure of cellulosic components, during the aspiration process. • CONCLUSIONS: The 4Pi microscope is well suited to scanning pit membranes to discover previously undescribed anatomical features in bordered pits and can provide information on chemical composition when used in combination with appropriate probes.