Sample preparation for mass spectrometry imaging of leaf tissues: a case study on analyte delocalization.

Appropriate sample preparation is pivotally important to obtain high-quality mass spectrometry imaging (MSI) data. Unlike mammalian tissues, preparation of cryosections from plant tissues for MSI measurement is quite challenging due to its intrinsic complex texture and cellular structure. This is especially true for leaf samples which are generally thin, water-rich, and fragile. In this work, a systematic study was performed, aiming to evaluate three embedding materials and five mounting approaches for matrix-assisted laser desorption ionization (MALDI) MSI of secondary metabolites in cross sections of the ginkgo leaf. Delocalization of endogenous metabolites was chosen as a major indicator for evaluation of three embedding materials including ice, carboxymethyl cellulose (CMC), and gelatin and different mounting approaches. Image distortion and analyte delocalization were observed when ice was used as an embedding medium. CMC embedding provided better results compared to the ice by using modified mounting approach. Among three embedding materials, no delocalization was observed in specimens embedded with gelatin, and gelatin embedding is the least affected by different mounting approaches. An alternative approach to mitigate analyte delocalization is the removal of embedding media embraced the tissue sections before mounting, which is particularly suitable for ice-embedded samples. Additionally, the extent of analyte delocalization was closely related to their lipophilicity/hydrophilicity properties, and less analyte diffusion was observed for hydrophobic analytes than for the water-soluble compounds.

Transcriptome profile analysis reveals the ontogenesis of rooted chichi in Ginkgo biloba L.

The Ginkgo biloba L. chichi is a unique organ. To explore the molecular mechanisms underlying the ontogenesis of G. biloba chichi, we used RNA-seq to analyse the transcriptome profile of rooted chichi at two developmental stages (ch1 and ch2) and nearby tissues (ck), and each sample had three biological replicates. A total of 57.74 Gb of clean bases were generated in nine cDNA libraries. These bases were de novo assembled into 68,277 unigenes with average length of 844 bp, and 51.47% of the unigenes had a match in at least one public database. The differentially expressed genes (DEGs) in ch1 vs. ck and ch2 vs. ck were 2748 and 8594, respectively. The DEGs involved in the auxin signal pathway, auxin polar transport, storage-related proteins, and the cell cycle pathway might play roles in the ontogenesis of chichi. The quantitative real-time PCR results were closely correlated with transcriptome data. The transcriptome resources generated in the current study provide gene expression profiles and differential expression profiles of G. biloba chichi and offer an essential resource to probe the molecular mechanisms underlying the ontogenesis of G. biloba chichi.

Phloem networks in leaves.

The survival of all vascular plants depends on phloem and xylem, which comprise a hydraulically coupled tissue system that transports photosynthates, water, and a variety of other molecules and ions. Although xylem hydraulics has been extensively studied, until recently, comparatively little is known quantitatively about the phloem hydraulic network and how it is functionally coupled to the xylem network, particularly in photosynthetic leaves. Here, we summarize recent advances in quantifying phloem hydraulics in fully expanded mature leaves with different vascular architectures and show that (1) the size of phloem conducting cells across phylogenetically different taxa scales isometrically with respect to xylem conducting cell size, (2) cell transport areas and lengths increase along phloem transport pathways in a manner that can be used to model Münch's pressure-flow hypothesis, and (3) report observations that invalidate da Vinci's and Murray's hydraulic models as plausible constructs for understanding photosynthate transport in the leaf lamina.

The presumed ginkgophyte Umaltolepis has seed-bearing structures resembling those of Peltaspermales and Umkomasiales.

The origins of the five groups of living seed plants, including the single relictual species Ginkgo biloba, are poorly understood, in large part because of very imperfect knowledge of extinct seed plant diversity. Here we describe well-preserved material from the Early Cretaceous of Mongolia of the previously enigmatic Mesozoic seed plant reproductive structure Umaltolepis, which has been presumed to be a ginkgophyte. Abundant new material shows that Umaltolepis is a seed-bearing cupule that was borne on a stalk at the tip of a short shoot. Each cupule is umbrella-like with a central column that bears a thick, resinous, four-lobed outer covering, which opens from below. Four, pendulous, winged seeds are attached to the upper part of the column and are enclosed by the cupule. Evidence from morphology, anatomy, and field association suggests that the short shoots bore simple, elongate Pseudotorellia leaves that have similar venation and resin ducts to leaves of living GinkgoUmaltolepis seed-bearing structures are very different from those of Ginkgo but very similar to fossils described previously as Vladimaria. Umaltolepis and Vladimaria do not closely resemble the seed-bearing structures of any living or extinct plant, but are comparable in some respects to those of certain Peltaspermales and Umkomasiales (corystosperms). Vegetative similarities of the Umaltolepis plant to Ginkgo, and reproductive similarities to extinct peltasperms and corystosperms, support previous ideas that Ginkgo may be the last survivor of a once highly diverse group of extinct plants, several of which exhibited various degrees of ovule enclosure.

The hydraulic architecture of Ginkgo leaves.

PREMISE OF THE STUDY: The hydraulics of xylem has been widely studied in numerous species and organ types. However, comparatively little is known about how phloem and xylem are hydraulically coupled or about many of the basic structural properties of phloem (such as conducting cell numbers and conductive areas), which nevertheless have direct bearing on understanding phloem loading and unloading. METHODS: Using a combination of light, epifluorescence, confocal, and transmission electron microscopy, we quantified the hydraulic architecture of Ginkgo biloba leaf laminae and examined the scaling relationships between phloem and xylem in five fully mature leaves. KEY RESULTS: The conductive areas and lengths of sieve cells and tracheids increase basipetally toward the petiole in a manner that is consistent with Münch's pressure flow hypothesis for phloem transport. This trend holds true for individual veins, the sum of conductive areas across all veins at any distance from the petiole, and for individual sieve cells and tracheids. Further, the conductive areas of phloem and xylem are isometrically correlated across the entire vasculature of the leaf lamina. The data for conducting cell areas do not conform with the predictions of the hydraulic models of da Vinci and Murray. CONCLUSIONS: The scaling of Ginkgo lamina hydraulics complies with that observed in leaves of other gymnosperms and most angiosperms and is inconsistent with theoretical models that assume that the volume of transported incompressible fluids is conserved.

A Jurassic wood providing insights into the earliest step in Ginkgo wood evolution.

The fossil record of Ginkgo leaf and reproductive organs has been well dated to the Mid-Jurassic (170 Myr). However, the fossil wood record that can safely be assigned to Ginkgoales has not yet been reported from strata predating the late Early Cretaceous (ca. 100 Myr). Here, we report a new fossil wood from the Mid-Late Jurassic transition deposit (153-165 Myr) of northeastern China. The new fossil wood specimen displays several Ginkgo features, including inflated axial parenchyma and intrusive tracheid tips. Because it is only slightly younger than the oldest recorded Ginkgo reproductive organs (the Yima Formation, 170 Myr), this fossil wood very probably represents the oldest bona fide fossil Ginkgo wood and the missing ancestral form of Ginkgo wood evolution.

Root development and structure in seedlings of Ginkgo biloba.

PREMISE OF THE STUDY: The popular, highly recognizable, well-known gymnosperm, Ginkgo biloba, was studied to document selected developmental features, which are little known in its primary root system from root tips to cotyledonary node following seed germination. METHODS: Using seedlings grown in soil, vermiculite, or a mixture, we examined sections at various distances from the root cap to capture a developmental sequence of anatomical structures by using standard brightfield, epifluorescence, and confocal microscopic techniques. KEY RESULTS: The vascular cylinder is usually a diarch stele, although modified diarchy and triarchy are found. Between exarch protoxylem poles, metaxylem usually develops into a complete disc, except near the transition region, which has irregularly arranged tracheary cells. The disc of primary xylem undergoes secondary growth on its metaxylem flanks with many tracheids added radially within a few weeks. Production of fibers in secondary phloem also accompanies secondary growth. In the cortex, endodermis produces Casparian bands early in development and continues into the upper transition region. Phi cells with phi-thickenings (bands of lignified walls) of a layer of inner cortex are often evident before endodermis, and then adjoining, additional layers of cortex develop phi cells; phi cells do not occur in the upper transition region or stem. An exodermis is produced early in root development and is continuous into the transition region and cotyledonary node. CONCLUSIONS: Seedling root axes of Ginkgo biloba are more complex than the literature suggests, and our findings contribute to our knowledge of root structure of this ancient gymnosperm.

Cellulose structure and lignin distribution in normal and compression wood of the Maidenhair tree (Ginkgo biloba L.).

We studied in detail the mean microfibril angle and the width of cellulose crystals from the pith to the bark of a 15-year-old Maidenhair tree (Ginkgo biloba L.). The orientation of cellulose microfibrils with respect to the cell axis and the width and length of cellulose crystallites were determined using X-ray diffraction. Raman microscopy was used to compare the lignin distribution in the cell wall of normal/opposite and compression wood, which was found near the pith. Ginkgo biloba showed a relatively large mean microfibril angle, varying between 19° and 39° in the S2 layer, and the average width of cellulose crystallites was 3.1-3.2 nm. Mild compression wood without any intercellular spaces or helical cavities was observed near the pith. Slit-like bordered pit openings and a heavily lignified S2L layer confirmed the presence of compression wood. Ginkgo biloba showed typical features present in the juvenile wood of conifers. The microfibril angle remained large over the 14 annual rings. The entire stem disc, with a diameter of 18 cm, was considered to consist of juvenile wood. The properties of juvenile and compression wood as well as the cellulose orientation and crystalline width indicate that the wood formation of G. biloba is similar to that of modern conifers.

Direct observation of bulk and surface chemical morphologies of Ginkgo biloba leaves by Fourier transform mid- and near-infrared microspectroscopic imaging.

Fourier transform infrared microspectroscopy is a powerful tool to obtain knowledge about the spatial and/or temporal distributions of the chemical compositions of plants for better understanding of their biological properties. However, the chemical morphologies of plant leaves in the plane of the blade are barely studied, because sections in this plane for mid-infrared transmission measurements are difficult to obtain. Besides, native compositions may be changed by chemical reagents used when plant samples are microtomed. To improve methods for direct infrared microspectroscopic imaging of plant leaves in the plane of the blade, the bulk and surface chemical morphologies of nonmicrotomed Ginkgo biloba leaves were characterized by near-infrared transmission and mid-infrared attenuated total reflection microspectroscopic imaging. A new self-modeling curve resolution procedure was proposed to extract the spectral and concentration information of pure compounds. Primary and secondary metabolites of secretory cavities, veins, and mesophylls of Ginkgo biloba leaf blades were analyzed, and the distributions of cuticle, protein, calcium oxalate, cellulose, and ginkgolic acids on the adaxial surface were determined. By the integration of multiple infrared microspectroscopic imaging and chemometrics methods, it is possible to analyze nonmicrotomed leaves and other plant samples directly to understand their native chemical morphologies in detail.

Branch architecture in Ginkgo biloba: wood anatomy and long shoot-short shoot interactions.

PREMISE: Ginkgo, centrally placed in seed plant phylogeny, is considered important in many phylogenetic and evolutionary studies. Shoot dimorphism of Ginkgo has been long noted, but no work has yet been done to evaluate the relationships between overall branch architecture and wood ring characters, shoot growth, and environmental conditions. • METHODS: Branches, sampled from similar canopy heights, were mapped with the age of each long shoot segment determined by counting annual leaf-scar series on its short shoots. Transverse sections were made for each long shoot segment and an adjacent short shoot; wood ring thickness, number of rings, and number of tracheids/ring were determined. Using branch maps, we identified wood rings for each long shoot segment to year and developmental context of each year (distal short shoot growth only vs. at least one distal long shoot). Climate data were also analyzed in conjunction with developmental context. • KEY RESULTS: Significantly thicker wood rings occur in years with distal long shoot development. The likelihood that a branch produced long shoots in a given year was lower with higher maximum annual temperature. Annual maximum temperature was negatively correlated with ring thickness in microsporangiate trees only. Annual minimum temperatures were correlated differently with ring thickness of megasporangiate and microsporangiate trees, depending on the developmental context. There were no significant effects associated with precipitation. • CONCLUSIONS: Overall, developmental context alone predicts wood ring thickness about as well as models that include temperature. This suggests that although climatic factors may be strongly correlated with wood ring data among many gymnosperm taxa, at least for Ginkgo, correlations with climate data are primarily due to changes in proportions of shoot developmental types (LS vs. SS) across branches.

Fossil ginkgophyte seedlings from the Triassic of France resemble modern Ginkgo biloba.

BACKGROUND: Fossil evidence of ginkgophyte ontogeny is exceedingly rare. Early development in the extant Ginkgo biloba is characterized by a series of distinct ontogenetic stages. Fossils providing insights into the early ontogeny of ancient ginkgophytes may be significant in assessing the degree of relatedness between fossil ginkgophytes and G. biloba. RESULTS: An assemblage of seedlings from the early Middle Triassic of France is assigned to the ginkgophytes based on leaf morphology. The specimens represent an ontogenetic sequence consisting of four stages: (I) formation of the cotyledons in the seed and germination; (II) development of primary leaves and taproot; (III) thickening of the taproot and appearance of secondary roots; and (IV) development of the first differentiated leaves and absence of the seed remnants. CONCLUSIONS: The fossil seedlings provide a rare opportunity to examine the early ontogeny of a Triassic ginkgophyte. Germination and seedling development in the fossil are nearly identical to that of the extant gymnosperm G. biloba. We hypothesize that the fossil may be closely related biologically to G. biloba, and that certain developmental processes in seedling development were in place by the Middle Triassic.

Jurassic mimicry between a hangingfly and a ginkgo from China.

A near-perfect mimetic association between a mecopteran insect species and a ginkgoalean plant species from the late Middle Jurassic of northeastern China recently has been discovered. The association stems from a case of mixed identity between a particular plant and an insect in the laboratory and the field. This confusion is explained as a case of leaf mimesis, wherein the appearance of the multilobed leaf of Yimaia capituliformis (the ginkgoalean model) was accurately replicated by the wings and abdomen of the cimbrophlebiid Juracimbrophlebia ginkgofolia (the hangingfly mimic). Our results suggest that hangingflies developed leaf mimesis either as an antipredator avoidance device or possibly as a predatory strategy to provide an antiherbivore function for its plant hosts, thus gaining mutual benefit for both the hangingfly and the ginkgo species. This documentation of mimesis is a rare occasion whereby exquisitely preserved, co-occurring fossils occupy a narrow spatiotemporal window that reveal likely reciprocal mechanisms which plants and insects provide mutual defensive support during their preangiospermous evolutionary histories.

Structure and function of the tentpole in the reproductive process of Ginkgo biloba L.

The tentpole is a unique structure of the female gametophyte in Ginkgo biloba; however, its exact functions in the reproductive process are unclear. In the present study, we used semi-thin sectioning and electron microscopy to study the structure and function of the tentpole during fertilization in G. biloba. The tentpole was always initiated between two or more deeply immersed archegonia. Before fertilization, the tentpole had developed into a column-like structure, protruding toward the archegonial chamber; cells at the periphery of tentpole were loosely ranged, and abundant lipid droplets and starch grains were accumulated in the tentpole cells. After fertilization, the tentpole degenerated, and some membranous debris was overlaid on its surface. In addition, there were significant decreases in the lipids and starch grains. These results suggested that the tentpole led to the degeneration of the megaspore membrane and then supported the pliable apex of the nucellar tissues. Importantly, the tentpole also contributed to supplying nutrition for fertilization and embryo development.

Patterns of diversity in leaves from canopies of Ginkgo biloba are revealed using Specific Leaf Area as a morphological character.

PREMISE OF THE STUDY: The difference reported in the literature for the Specific Leaf Area (SLA, cm(2)/g) of leaves on short- and long-shoots of Acer rubrum could mean that SLA can serve as a quantitative morphological trait. Our survey of SLA in canopies of Ginkgo biloba sampled a different clade of seed plants to investigate this morphological phenomenon. Such a survey in this dioecious taxon, and one in which a single canopy may have juvenile and reproductive portions, as well as one where canopies bear leaves of several shapes, examine these additional morphological factors as well as any long-shoot short-shoot differences. METHODS: We measured SLA for a set of 642 dried leaves, a sampling across all morphological levels in canopies of large landscape specimens. The tabulated values were analyzed as distributions. KEY RESULTS: Populations of leaves of G. biloba, sorted by morphological features of canopy structure, differ between long- and short-shoots (175%), on the two genders of tree (131%), in the juvenile and reproductive portions of a canopy (183%), and with the presence or absence of seed on short-shoots in the reproductive portion of megasporangiate canopies (114%). Basipetal leaves of long-shoots and leaves of short-shoots have similar values of SLA. CONCLUSIONS: With the exception of the acropetal decrease in SLA along long-shoots, the differences among the several classes of leaf seem to reflect local sink strength, even though the sink itself develops after leaves mature. The large overall range in the values of SLA in Ginkgo underscores the relevance of the details of canopy structure to parsing ecological phenomena.

Structural and hydraulic correlates of heterophylly in Ginkgo biloba.

This study investigates the functional significance of heterophylly in Ginkgo biloba, where leaves borne on short shoots are ontogenetically distinct from those on long shoots. Short shoots are compact, with minimal internodal elongation; their leaves are supplied with water through mature branches. Long shoots extend the canopy and have significant internodal elongation; their expanding leaves receive water from a shoot that is itself maturing. Morphology, stomatal traits, hydraulic architecture, Huber values, water transport efficiency, in situ gas exchange and laboratory-based steady-state hydraulic conductance were examined for each leaf type. Both structure and physiology differed markedly between the two leaf types. Short-shoot leaves were thinner and had higher vein density, lower stomatal pore index, smaller bundle sheath extensions and lower hydraulic conductance than long-shoot leaves. Long shoots had lower xylem area:leaf area ratios than short shoots during leaf expansion, but this ratio was reversed at shoot maturity. Long-shoot leaves had higher rates of photosynthesis, stomatal conductance and transpiration than short-shoot leaves. We propose that structural differences between the two G. biloba leaf types reflect greater hydraulic limitation of long-shoot leaves during expansion. In turn, differences in physiological performance of short- and long-shoot leaves correspond to their distinct ontogeny and architecture.

Seeing the forest with the leaves - clues to canopy placement from leaf fossil size and venation characteristics.

Although a variety of leaf characteristics appear to be induced by light environment during development, analysis of ontogenetic changes in living broad leaved trees has suggested that a number of other traits also lumped into the classic 'sun' versus 'shade' morphological distinctions, including leaf size, shape, and vein density, are instead controlled largely by local hydraulic environment within the tree canopy. The regularity in how these traits vary with canopy placement suggests a method for addressing a classic paleobotanical quandary: the stature of the source plant - from herb or shrub to canopy tree - is typically unknown for leaf fossils. The study of Ginkgo here complements previous work on Quercus that indicated that leaves throughout the crown are identical in size and venation at the time of bud break and that morphological adaptation to the local microenvironment takes place largely during the expansion phase after the determination of the vascular architecture is complete. Hence, variation in vein density does not reflect differential vein production so much as the distortion of similar vein networks over different final surface areas driven by variation in local hydraulic supply during expansion. Unlike the diffusely growing leaves of the angiosperm, Quercus, the marginally growing leaves of Ginkgo do show some potential for differential vein production, but expansion effects still dominate. The approach suggested here may prove useful for assessing the likelihood that two distinct fossil morphospecies actually represent leaves of the same plant and to gather information concerning canopy structure from disarticulated leaves.

Biology and chemistry of Ginkgo biloba.

Ginkgo biloba has been existing on earth since 200 million years and is considered as a "living fossil". It is among the most sold medicinal plants in the world. A number of secondary metabolites representing terpenoids, polyphenols, allyl phenols, organic acids, carbohydrates, fatty acids and lipids, inorganic salts and amino acids have been isolated from the plant. However, the main bioactive constituents are terpene trilactones and flavonoid glycosides which are considered responsible for the pharmacological activities of its standardized leaf extract. Scattered information is available on the extraction and analysis of these pharmacologically important constituents which have been compiled in the present review.

[Study on pharmacognosy of Ginkgo leaf].

The primary study of Ginkgo leaf such as crude drug macroscopic and powder characteristics were carried out, and the flavonoids content in the leaf of Ginkgo in different areas of Gansu province was determined by HPLC, in order to provide scientific references for the exploitation of Ginkgo in Gansu province.

Hydraulic design of leaves: insights from rehydration kinetics.

We examined the leaf hydraulic design in 10 species based on their rehydration kinetics. In all cases, a biphasic response described the temporal pattern of water uptake, with time constants of approximately 30 to 800 s and approximately 800 to 8000 s. The time constants of the fast phase were significantly shorter in the six angiosperms (30 to 110 s) compared with the two single-veined conifer species (>400 s) examined, while the two multi-veined gymnosperm species, Gnetum gnemon and Ginkgo biloba, had time constants for the fast phase of approximately 150 s. Among angiosperm species, the fast phase constituted 50-90% of the total water absorbed, whereas in gymnosperms 70-90% of the water uptake could be assigned to the slow phase. In the four gymnosperms, the relative water uptake corresponding to the fast phase matched to a good degree the relative volume of the venation and bundle sheath extension; whereas in the angiosperm species, the relatively larger water influx during the fast phase was similar in relative volume to the combined venation, bundle sheath extension, epidermis and (in four species) the spongy mesophyll. This suggests a general trend from a design in which the epidermis is weakly connected to the veins (all four gymnosperms), to a design with good hydraulic connection between epidermis and veins that largely bypasses the mesophyll (four of six angiosperms), to a design in which almost the entire leaf appears to function as a single pool.