The uncommon cavitated secretory trichomes in Bauhinia s.s. (Fabaceae): the same roles in different organs

Cavitated secretory trichomes are characterized by a short or absent stalk that is connected to a secretory hollow head. They are rare structures in angiosperms; in Fabaceae, they have been recorded in only seven genera, including Bauhinia s.s. Because B. curvula and B. rufa exhibit glands that are responsible for attracting pollinators to flowers, this study aimed to test whether the cavitated secretory trichomes present in the flowers of these species have an attraction function. As leaf trichomes are commonly related to plant defence, comparative analyses of the morphology, ontogeny, ultrastructure and chemical profile of the secretory trichomes present in flowers and leaves were conducted. It was found that cavitated secretory trichomes are similar in their external morphology and development, regardless of the organ or species analysed. However, interspecific differences were found in the secretion process and chemical profile of the exudate. The differences found in the cavitated secretory trichomes between species indicate that they secrete distinct compounds, whereas the similarities found in these structures between vegetative and reproductive organs indicate that the cavitated trichomes have equivalent ecological functions within a species, probably in plant defence during organ development. © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2016, 180 , 104–122.     

Floral ontogeny in Dipterygeae (Fabaceae) reveals new insights into one of the earliest branching tribes in papilionoid legumes

Flowers of Dipterygeae (Fabaceae, Papilionoideae) exhibit an unusual petaloid calyx. The two adaxial sepals are large and petaloid, and the three abaxial sepals form a three‐toothed lobe. The goal of this study was to elucidate the ontogenetic pathways of this peculiar calyx in light of the floral development of the three genera that comprise the tribe. Floral buds of Dipteryx alata, Pterodon pubescens and Taralea oppositifolia were analysed using scanning electron microscopy and light microscopy. The order of bracteole and sepal initiation varies among the species. The androecium is asymmetric. The carpel cleft is positioned to the right or to the left, and is opposite the adaxial antepetalous stamen. The peculiarity of the calyx becomes noticeable in the intermediate stages of floral development. It results from the differential growth of the sepal primordia, in which the abaxial and lateral primordia remain diminutive during floral development, compared with the adaxial ones that enlarge and elongate. Bracteoles, abaxial sepals, petals and anthers are appendiculate, except in T. oppositifolia, in which the appendices were not found in bracteoles or anthers. These appendices comprise secretory canals or cavities. Considering that the ontogenetic pathway for the formation of the petaloid calyx is similar and exclusive for Dipterygeae, it might be a potential synapomorphy for the group, with the presence of secretory canals in the appendices of abaxial and lateral sepals and petals. © 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 174 , 529–550.     

Identification of anti-inflammatory constituents in Hypericum perforatum and Hypericum gentianoides extracts using RAW 264.7 mouse macrophages

Hypericum perforatum (St. John’s wort) is an herb widely used as supplement for mild to moderate depression. Our prior studies established synergistic anti-inflammatory activity associated with 4 bioactive compounds in a fraction of a H. perforatum ethanol extract. Whether these 4 compounds also contributed to the ethanol extract activity was addressed in the research reported here. Despite the popularity of H. perforatum, other Hypericum species with different phytochemical profiles could have their anti-inflammatory potentials attributed to these or other compounds. In the current study, ethanol extracts of different Hypericum species were compared for their inhibitory effect on LPS-induced prostaglandin E2 (PGE2) and nitric oxide (NO) production in RAW 264.7 mouse macrophages. Among these extracts, those made from H. perforatum and H. gentianoides demonstrated stronger overall efficacy. LC–MS analysis established the 4 compounds were present in the H. perforatum extract and pseudohypericin in all active fractions. The 4 compounds accounted for a significant part of the extract’s inhibitory activity on PGE2, NO, tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β) in RAW 264.7 as well as peritoneal macrophages. Pseudohypericin was the most important contributor of the anti-inflammatory potential among the 4 compounds. The lipophilic fractions of H. gentianoides extract, which did not contain the previously identified active constituents, decreased PGE2 and NO potently. These fractions were rich in acylphloroglucinols, including uliginosin A that accounted for a proportion of the anti-inflammatory activity observed with the active fractions. Overall, the current study established that a different group of major anti-inflammatory constituents were present in H. gentianoides, while showing that the previously identified 4 compound combination was important for H. perforatum’s anti-inflammatory potential.     

Metabolite profiling and fingerprinting of Hypericum species: a comparison of MS and NMR metabolomics

Hypericum perforatum, commonly known as St. John’s wort, is a popular herbal supplement used for the treatment of mild to moderate depression. The major secondary metabolites of St. John’s wort extracts include phenylpropanoids, flavonoids, xanthones, phloroglucinols, and naphthodianthrones. There are over 400 species in the genus Hypericum world-wide, most of which are little or not characterized in terms of phytochemical or pharmacological properties. Metabolomics techniques were used to investigate the natural product diversity within the genus Hypericum (Hypericaceae) and its correlation to bioactivity, exemplified by cytotoxic properties. Utilizing nuclear magnetic resonance (NMR) fingerprinting and mass spectrometry (MS) metabolic profiling techniques, MS and NMR spectra of extracts from H. perforatum, H. polyphyllum, H. tetrapterum, H. androsaemum, H. inodorum, H. undulatum and H. kouytchense were evaluated and submitted to statistical multivariate analyses. Although comparable score plots in principal component analysis were derived from both MS and NMR datasets, loading plots reveal, that different set of metabolites contribute for species segregation in each dataset. Major peaks in ¹H NMR and MS spectra contributing to species discrimination were assigned as those of hyperforins, lipids, chlorogenic and shikimic acid. Shikimic acid and its downstream phenylpropanoids were more enriched in H. perforatum, H. androsaemum, H. kouytchense and H. inodorum extracts; whereas a novel hyperforin was found exclusively in H. polyphyllum. Next to H. perforatum, H. polyphyllum and H. tetrapterum show the highest levels of hypericins, and H. perforatum and H. polyphyllum are highest in phloroglucinols, suggesting that the latter species might be used as an alternative to St. John’s wort. However, the major hyperforin-type compound in H. polyphyllum possesses a novel constitution of yet unknown bioactivity. Anti-cancer in vitro assays to evaluate the ability of extracts from Hypericum species in inhibiting prostate and colon cancer growth suggest that such bioactivity might be predicted by gross metabolic profiling.     

Chemical Composition and Antimicrobial Activity of the Essential Oils from Several Hypericum Taxa (Guttiferae) Growing in Central Italy (Appennino Umbro‐Marchigiano)

The chemical composition of the essential oils of nine taxa from seven sections of Hypericum L. (Guttiferae; H. perforatum subsp. perforatum, H. perforatum subsp. veronense, H. calycinum, H. montanum, H. richeri subsp. richeri, H. hyssopifolium, H. hirsutum, H. hircinum subsp. majus, and H. tetrapterum) occurring in central Italy (Appennino Umbro‐Marchigiano) was analyzed by GC/FID and GC/MS. A total of 186 compounds were identified in the different species and subspecies, accounting for 86.9–92.8% of the total oils. The major fraction of the oil was always represented by sesquiterpene hydrocarbons (30.3–77.2%), while quantitative differences occurred between the other classes of volatiles depending on the taxa considered. Chemical composition of the nine Hypericum entities with respect to the taxonomical classification was discussed. Essential oils obtained from six taxa, i.e., H. perforatum subsp. perforatum, H. perforatum subsp. veronense, H. calycinum, H. richeri subsp. richeri, H. hirsutum and H. tetrapterum, were also tested for their antimicrobial properties against five different microbial strains by the broth‐microdilution method, and they were found to have significant activity (expressed as MIC) on B. subtilis, moderate activity on C. albicans and S. aureus, and weak activity on E. coli and E. faecalis, the most active being those from H. hirsutum, H. richeri subsp. richeri, and H. tetrapterum.     

Idioblasts and other unusual internal foliar secretory structures in Scrophulariaceae

 Leaf samples of mostly herbarium specimens (237 species of 172 genera) were cleared. Internal secretory structures of large size or unusual shape were detected and observed. Selected samples were processed into resin and sectioned for light microscopy or prepared for scanning electron microscopy. Adding results from two earlier publications, our survey includes 365 species from 174 genera. Five types of internal secretory structure, mostly unknown previously in the traditional Scrophulariaceae, occur in only nine genera: 1) single-celled subepidermal idioblasts, empty at maturity, in Scrophularia and Verbascum (Lersten and Curtis 1997), 2) 2–16 (or more)-celled nodules, with fibrous contents, in mid-mesophyll strata of Graderia and Radamaea, 3) epithelium-lined oil cavities in Leucophyllum (Lersten and Beaman 1998) and Capraria, 4) Kranz-type enlarged bundle sheath in Anticharis, and 5) paraveinal mesophyll (PVM) in Picria and Bonnaya. Received April 24, 2000 Accepted October 27, 2000     

Cytogenetic characterization of three Hypericum species by in situ hybridization

The chromosomal positions of the 5S/25S rRNA genes of Hypericum perforatum (2n=32), H. maculatum (2n=16) and H. attenuatum (2n=32) were comparatively determined by FISH, and six, three and seven chromosome pairs of the respective karyotypes were subsequently distinguished. The rDNA loci between H. perforatum and H. maculatum seem to be identical (with respect to the ploidy difference), indicating that H. perforatum probably arose by autotetraploidization from an ancestor closely related to H. maculatum. The positional differences between the 5S rRNA gene loci of H. perforatum and H. maculatum on the one hand and H. attenuatum on the other argue against a previous hypothesis according to which H. perforatum originated from a remote interspecific hybridization between H. maculatum and H. attenuatum. Received: 7 October 1999 / Accepted: 23 November 1999     

Effects of Oenothera biennis L. and Hypericum perforatum L. extracts on some central nervous system myelin proteins,brain histopathology and oxidative stress in mice with experimental autoimmune encephalomyelitis

We investigated the effects of Oenothera biennis L. and Hypericum perforatum L. extracts on brain tissue histopathology, myelin oligodendrocyte glycoprotein (MOG), myelin basic protein (MBP), total antioxidant status (TAS), total oxidant status (TOS) and oxidative stress index (OSI) in mice with experimental autoimmune encephalomyelitis (EAE). Forty-seven C57BL/6J mice were divided into the following groups: multiple sclerosis (MS), control (healthy mice), MS + H. perforatum treated (MS + HP), MS + O. biennis treated (MS + OB). All groups except the control group were immunized by EAE methods. Two weeks after the immunization, the mice in the MS + HP group were fed normal food containing 18 ? 21 g/kg H. perforatum extract, the mice in MS + OB group were fed normal food containing 18 ? 21 g/kg O. biennis extract, and the mice in control and MS groups were fed normal food for six weeks. Brain tissue samples were collected from all mice for histopathological and biochemical analysis. Clinical signs of the disease were scored using functional systems scores (FSS) daily. The H. perforatum and O. biennis extracts ameliorated the increased brain tissue MOG and MBP values for animals with MS. H. perforatum and O. biennis extract decreased the TOS and OSI values for brain tissue and increased TAS levels in brain tissue of animals with MS. In addition, H. perforatum and O. biennis extracts decreased the clinical signs at the end of the experiment compared to the beginning of extract administration. We found that myelin was lost in MS group vs. control group. H. perforatum and O. biennis extract treatments decreased the amount of myelin loss in the MS + HP and MS + OB groups. We also observed amyloid deposition on vascular walls, in the cytoplasm of the neurons and in the intercellular space in the MS group. O. biennis and H. perforatum treated groups exhibited neither abnormal amyloid deposition nor obvious cell infiltration. The beneficial effects of O. biennis and H. perforatum for attenuating myelin loss and amyloid deposition suggest their therapeutic utility for treatment of MS.     

Essential oil composition of Hypericum L. species from Southeastern Serbia and their chemotaxonomy

The essential oils of the aerial parts of nine species of Hypericum (Hypericum barbatum, Hypericum hirsutum, Hypericum linarioides, Hypericum maculatum, Hypericum olympicum, Hypericum perforatum, Hypericum richeri, Hypericum rumeliacum and Hypericum tetrapterum), collected from different locations in Southeast Serbia, were obtained by steam distillation and analyzed by GC and GC–MS. The essential oils investigated were characterized by a high content of non-terpene compounds and a low content of monoterpenes. The contents of non-terpenes, monoterpenes and sesquiterpenes in oils of the species H. barbatum, H. richeri and H. rumeliacum (section Drosocaprium) were similar and these oils were characterized by high contents of fatty acids. The oils of H. hirsutum and H. linarioides (section Taeniocarpium) contained a high percentage of n-nonane. There were similarities in contents of non-terpenes and sesquiterpenes in oils of species that belong to the section Hypericum (H. maculatum, H. perforatum and H. tetrapterum). The oil of H. olympicum differed from others by higher terpene content. A comparison was also carried out of the chemical composition of the essential oils from flower, leaf and stem of H. perforatum and it revealed that the highest concentration of non-terpene compounds was found in the flower and stem oil, while a high concentration of sesquiterpenes was characteristic for leaf oil. There were significant differences in the concentrations of the same compounds in the essential oils of H. maculatum, H. olympicum and H. perforatum, collected in different years from the same location which could be explained by seasonal differences. All data were statistically processed with principal component analysis and cluster analysis. The main conclusion from the above data is that genetic and environmental factors both play a role in determining the composition of essential oils of the Hypericum species studied.     

RAPD analysis: a method to investigate aspects of the reproductive biology of Hypericum perforatum L.

Recent interest in breeding strategies for Hypericum perforatum L. requires a better understanding of the floral biology of this medicinal plant. The aim of the present study was to check, whether RAPD fingerprinting may be a useful tool for research on the mode of reproduction of this species. Progenies from three defined single plants of two accessions, as well as progenies from a random sample of seeds of a wild population, of H. perforatum were characterized by RAPD analyses using six primers. The results obtained by DNA fingerprints indicate the predominance of an identical mode of reproduction for this species, obviously due to apomixis. Nevertheless, non-identical reproduction was evident as a minor effect in H. perforatum, as could be demonstrated by significant deviations in the RAPD fingerprints of progenies from one single plant. It is concluded that RAPD fingerprint analysis is a suitable technique to discover identity or non-identity in H. perforatum populations. Therefore, RAPDs may be used in addition to cytological studies to confirm the mode of reproduction by apomixis versus self-pollination, haploid parthenogenesis or cross-fertilization. Received: 12. August 1999 / Accepted: 27 August 1999     

Comparative anatomy of secretory structures of leaves in Hypericum L.

The structure, type, morphology and location of secretory structures in leaves of 43 species, 1 subspecies and 1 variety of 9 sections in Hypericum L. were comparatively studied using tissue clearing, paraffin sectioning and thin sectioning. The results have shown that the presence of secretory structures is a common feature of leaves in this genus. According to their anatomical characteristics, the secretory structures can be divided into nodules, secretory cavities (canals) and tiny secretory tubes. In their distribution in leaves the nodules fall into two types: the leaf edge type and the scattered type. According to the location of cavities in the cross sections of leaves, the cavities can be divided into 4 types: the median type which is situated between the palisade tissue and spongy tissue, the palisade tissue type, the spongy tissue type and the across-mesophyll type. Based on the location of cavities and nodules in leaves, the species in Hypericum can be divided into 3 groups: group Ⅰ , in which only cavities are present; group Ⅱ, in which only nodules are present; group Ⅲ, in which both cavities and nodules are present. The type, location, distribution density and morphology of secretory structures are of some taxonomic value at the level of species and of section in Hypericum L. From these observations, the evolutionary trends concerning the morphology and anatomy of secretory structures and the affinity among sections in the genus Hypericum ate dis-cussed.     

The Intrasectional Chemotaxonomic Placement of Hypericum elegans Stephan ex Willd. Inferred from the Essential‐Oil Chemical Composition

Here we report, for the first time, the results of detailed GC and GC/MS analyses of the essential oil of a rare taxon in Serbia, Hypericum elegans Stephan ex Willd . One hundred and sixty two constituents identified accounted for 98.6% of the oil. The major components of the oil were undecane (31.9%), α‐pinene (16.7%), nonane (6.1%), bicyclogermacrene (5.8%), 2‐methyloctane (3.7%), and germacrene D (3.6%). Non‐terpenoids as chemotaxonomic markers constituted the main fraction of H. elegans oil, whereby n‐alkanes were the most abundant contributors of this fraction. Based on these results and previously published ones, we performed an intrasectional multivariate statistical comparison of corresponding essential‐oil chemical compositions. Principal component analysis (PCA) and agglomerative hierarchical clustering (AHC) of the data on the volatile profiles of section Hypericum taxa revealed that H. elegans either represents an oil chemotype of its own (AHC) or could be considered related to H. perforatum (PCA).     

Effects of cytokinins and elicitors on the production of hypericins and hyperforin metabolites in Hypericum sampsonii and Hypericum perforatum

Hypericum perforatum L. (St. John’s wort) and Hypericum sampsonii Hance are medicinal plants used in China in the treatment of viruses and other disorders. In the current study, we investigated the effects of cytokinins 6-benzylaminopurin (BA), zeatin (ZT) and thidiazuron (TDZ) on plant growth and production of hypericins (pseudohypericin and hypericin) and hyperforin. Our data suggested that culture of H. perforatum in modified MS (Murashige and Skoog) medium, with a 50% reduction in ammonium nitrate and potassium nitrate, and supplemented with BA (0.44 μM) and indolebutyric acid (IBA, 0.049 μM), resulted in increased production of hypericins. Similar results were noted with H. sampsonii with minor changes to the medium (0.46 μM ZT and 0.049 μM IBA). There were approximately 2.95-, 2.62-fold increases in H. perforatum pseudohypericin and hypericin production by TDZ (0.45 μM) induction compared to the controls. No enhancement of hypericins and hyperforin production was elicited by TDZ in H. sampsonii. The elicitor methyl jasmonate (MJA, 50 μM) and its analog, 2,3-dihydroxypropyl jasmonate (DHPJA, 50 μM), were also used in H. perforatum and H. sampsonii shoot culture to increase secondary metabolite production, eliciting an increase in the production of hypericins and hyperforin. While leaf senescence and biomass inhibition were observed in cultures induced by MJA, no such effects were observed with DHPJA.     

Rutin as Deoxyribonuclease I Inhibitor

DNase I inhibitory potential of water extract of nine Hypericum species (H. umbellatum, H. barbatum, H. rumeliacum, H. rochelii, H. perforatum, H. tetrapterum, H. olympicum, H. hirsutum, H. linarioides) and the most important Hypericum secondary metabolites (hypericin, hyperforin, quercetin, and rutin) was investigated. All examined Hypericum extracts inhibited DNase I with IC₅₀ below 800 μg/ml, whereby H. perforatum was the most potent (IC₅₀=391.26±68.40 μg/ml). Among the investigated Hypericum secondary metabolites, rutin inhibited bovine pancreatic DNase I in a non‐competitive manner with IC₅₀ value of 108.90±9.73 μm . DNase I inhibitory ability of rutin was further confirmed on DNase I in rat liver homogenate (IC₅₀=137.17±16.65 μm ). Due to the involvement of DNase I in apoptotic processes the results of this study indicate the importance of frequent rutin and H. perforatum consumption in daily human nutrition. Rutin is a dietary component that can contribute to male infertility prevention by showing dual mechanism of sperm DNA protection, DNase I inhibition and antioxidant activity.     

Leaf blade structure of Verbesina macrophylla (Cass.) F. S. Blake (Asteraceae): ontogeny,duct secretion mechanism and essential oil composition

• Secretory structures are common in Asteraceae, where they exhibit a high degree of morphological diversity. The species Verbesina macrophylla, popularly known as assa‐peixe, is native to Brazil where it is widely used for medicinal purposes. Despite its potential medical importance, there have been no studies of the anatomy of this species, especially its secretory structures and secreted compounds. This study examined leaves of V. macrophylla with emphasis on secretory structures and secreted secondary metabolites.
• Development of secretory ducts and the mechanism of secretion production are described for V. macrophylla using ultrastructure, yield and chemical composition of its essential oils.
• Verbesina macrophylla has a hypostomatic leaf blade with dorsiventral mesophyll and secretory ducts associated with vascular bundles of schizogenous origin. Histochemistry identified the presence of lipids, terpenes, alkaloids and mucopolysaccharides. Ultrastructure suggests that the secretion released into the duct lumen is produced in plastids of transfer cells, parenchymal sheath cells and stored in vacuoles in these cells and duct epithelial cells. The essential oil content was 0.8%, and its major components were germacrene D, germacrene D‐4‐ol, β‐caryophyllene, bicyclogermacrene and α‐cadinol.
• Secretory ducts of V. macrophylla are squizogenous. Substances identified in tissues suggest that both secretions stored in the ducts and in adjacent parenchyma cells are involved in chemical defence. The essential oil is rich in sesquiterpenes, with germacrene D and its derivatives being notable components.

     

Toxic Effects of Pb on Anatomy and Hypericin Content in Hypericum perforatum L.

Lead (Pb) is one of the most common heavy metal contaminants in the environment. The present study was therefore undertaken to determine the effects of Pb on structural characteristics and hypericin production in Hypericum perforatum. Mature plants were treated with contaminated soil in seven treatments (75, 150, 300, 600, 800, 1000, and 1500 mg/kg Pb in soil) with three repeats per treatment every 14 days. Maximum observed Pb content in shoot parts was observed in the treatments with 600 and 1500 mg/kg Pb. The Pb concentration in roots was higher than in shoot parts, enhanced with increasing Pb concentration in the soil. In this study, Pb treatment significantly influenced the morphology, anatomy, and hypericin content in the plant. Anatomical characteristics of leaf, stem, and root affected by Pb contamination, as well as scanning electron microscopy (SEM) studies, revealed structural changes in stomata and epicuticular waxes. Under Pb toxicity, anatomical symptoms occurred in leaves, including increase in sizes of epidermal cells, mesophyll tissue, and diameter of stems and roots, as well as amplified vascular bundles and pith area. This, therefore, indicated that metal contamination can change the chemical composition of this plant. Maximum hypericin content was observed in the treatment containing 600 mg/kg Pb in soil, which then decreased.     

RFLP analysis of cpDNA in the genus <Emphasis Type="Italic">Hypericum</Emphasis>

The chloroplast DNA of 43 species including 16 sections from the genus Hypericum was studied by PCR-RFLP analysis. The PCR-amplified products of four cpDNA regions, trnC-trnD, psbC-trnS, trnL-trnF and rbcL were digested with four restriction endonucleases. A high level of interspecific variation was detected while intraspecific diversity was not observed. The resulting parsimony analysis indicated the monophyletic assemblage of the sections Androsaemum, Olympia, Drosocarpium and Trigynobrathys. Monophyly of Hypericum is weakly supported, but close relationships of H. perforatum and H. maculatum are indicated. The members of Ascyreia are weakly resolved, but clustering of H. kouytchense and H. oblongifolium is well supported, however, H. reptans is nested with Olympia. CpDNA profiles and the positions on the parsimony tree indicate that the chloroplast donor among the putative parents of the hybrid species H. ×inodorum is H. androsaemum.     

Anatomy of the vegetative organs and secretory structures of Rhaponticum carthamoides (Asteraceae)

Roots, stems, rhizomes and leaves of Rhaponticum carthamoides (Willd.) Iljin (a Siberian adaptogenic plant, originating from the Altai and Saian Mountains) of different ages were investigated by means of light and electron microscopy. Schizogenous secretory reservoirs occurred in every organ, and were located within the secondary xylem (adventitious roots and rhizome of young plants), at the interface of endodermis/cortical parenchyma (roots and hypocotyl), along phloem and primary xylem (older rhizome), around the vascular bundles (inflorescence stem, petiole and leaf midrib veins) and along phloem (cotyledonary and leaf veins). At the interface of endodermis/inner parenchyma, secretion accumulated in the intercellular spaces prior to the formation of proper epithelial cells. The secretion as observed by transmission electron microscopy comprised three components: soluble (i.e. absent from sections; probably phenolic), insoluble and strongly osmiophilic (probably phenolic) and insoluble, moderately osmiophilic (probably lipidic). Numerous osmiophilic oil droplets, similar to the lipidic secretion inside the reservoirs, local proliferation of rough endoplasmic reticulum and numerous multivesicular bodies characterized epithelial cells in all organs. Leucoplasts (in subterranean organs) with osmiophilic inclusions and peroxisomes with crystalloid inclusions were specific for parenchyma cells. Peltate glandular hairs were formed on leaf blades.  © 2004 The Linnean Society of London, Botanical Journal of the Linnean Society , 2004, 144 , 207–233.     

Subcellular concentrations of sugar alcohols and sugars in relation to phloem translocation in <Emphasis Type="Italic">Plantago major,Plantago maritima</Emphasis>, <Emphasis Type="Italic">Prunus persica</Emphasis>, and <Emphasis Type="Italic">Apium graveolens</Emphasis>

Sugar and sugar alcohol concentrations were analyzed in subcellular compartments of mesophyll cells, in the apoplast, and in the phloem sap of leaves of Plantago major (common plantain), Plantago maritima (sea plantain), Prunus persica (peach) and Apium graveolens (celery). In addition to sucrose, common plantain, sea plantain, and peach also translocated substantial amounts of sorbitol, whereas celery translocated mannitol as well. Sucrose was always present in vacuole and cytosol of mesophyll cells, whereas sorbitol and mannitol were found in vacuole, stroma, and cytosol in all cases except for sea plantain. The concentration of sorbitol, mannitol and sucrose in phloem sap was 2- to 40-fold higher than that in the cytosol of mesophyll cells. Apoplastic carbohydrate concentrations in all species tested were in the low millimolar range versus high millimolar concentrations in symplastic compartments. Therefore, the concentration ratios between the apoplast and the phloem were very strong, ranging between 20- to 100-fold for sorbitol and mannitol, and between 200- and 2000-fold for sucrose. The woody species, peach, showed the smallest concentration ratios between the cytosol of mesophyll cells and the phloem as well as between the apoplast and the phloem, suggesting a mixture of apoplastic and symplastic phloem loading, in contrast to the herbal plant species (common plantain, sea plantain, celery) which likely exhibit an active loading mode for sorbitol and mannitol as well as sucrose from the apoplast into the phloem.