What can reactive oxygen species (ROS) tell us about the action mechanism of herbicides and other phytotoxins?

Reactive oxygen species (ROS) are formed in plant cells continuously. When ROS production exceeds the antioxidant capacity of the cells, oxidative stress develops which causes damage of cell components and may even lead to the induction of programmed cell death (PCD). The levels of ROS production increase upon abiotic stress, but also during pathogen attack in response to elicitors, and upon application of toxic compounds such as synthetic herbicides or natural phytotoxins. The commercial value of many synthetic herbicides is based on weed death as result of oxidative stress, and for a number of them, the site and the mechanism of ROS production have been characterized. This review summarizes the current knowledge on ROS production in plants subjected to different groups of synthetic herbicides and natural phytotoxins. We suggest that the use of ROS-specific fluorescent probes and of ROS-specific marker genes can provide important information on the mechanism of action of these toxins. Furthermore, we propose that, apart from oxidative damage, elicitation of ROS-induced PCD is emerging as one of the important processes underlying the action of herbicides and phytotoxins.

Effects of Phytotoxic Nonenolides, Stagonolide A and Herbarumin I, on Physiological and Biochemical Processes in Leaves and Roots of Sensitive Plants.

Phytotoxic macrolides attract attention as prototypes of new herbicides. However, their mechanisms of action (MOA) on plants have not yet been elucidated. This study addresses the effects of two ten-membered lactones, stagonolide A (STA) and herbarumin I (HBI) produced by the fungus Stagonospora cirsii, on Cirsium arvense, Arabidopsis thaliana and Allium cepa. Bioassay of STA and HBI on punctured leaf discs of C. arvense and A. thaliana was conducted at a concentration of 2 mg/mL to evaluate phenotypic responses, the content of pigments, electrolyte leakage from leaf discs, the level of reactive oxygen species, Hill reaction rate, and the relative rise in chlorophyll a fluorescence. The toxin treatments resulted in necrotic and bleached leaf lesions in the dark and in the light, respectively. In the light, HBI treatment caused the drop of carotenoids content in leaves on both plants. The electrolyte leakage caused by HBI was light-dependent, in contrast with that caused by STA. Both compounds induced light-independent peroxide generation in leaf cells but did not affect photosynthesis 6 h after treatment. STA (10 µg/mL) caused strong disorders in root cells of A. thaliana leading to the complete dissipation of the mitochondrial membrane potential one hour post treatment, as well as DNA fragmentation and disappearance of acidic vesicles in the division zone after 8 h; the effects of HBI (50 µg/mL) were much milder. Furthermore, STA was found to inhibit mitosis but did not affect the cytoskeleton in cells of root tips of A. cepa and C. arvense, respectively. Finally, STA was supposed to inhibit the intracellular vesicular traffic from the endoplasmic reticulum to the Golgi apparatus, thus interfering with mitosis. HBI is likely to have another main MOA, probably inhibiting the biosynthesis of carotenoids.

Role of Autophagy in Haematococcus lacustris Cell Growth under Salinity.

The microalga Haematococcus lacustris (formerly H. pluvialis) is able to accumulate high amounts of the carotenoid astaxanthin in the course of adaptation to stresses like salinity. Technologies aimed at production of natural astaxanthin for commercial purposes often involve salinity stress; however, after a switch to stressful conditions, H. lacustris experiences massive cell death which negatively influences astaxanthin yield. This study addressed the possibility to improve cell survival in H. lacustris subjected to salinity via manipulation of the levels of autophagy using AZD8055, a known inhibitor of TOR kinase previously shown to accelerate autophagy in several microalgae. Addition of NaCl in concentrations of 0.2% or 0.8% to the growth medium induced formation of autophagosomes in H. lacustris, while simultaneous addition of AZD8055 up to a final concentration of 0.2 µM further stimulated this process. AZD8055 significantly improved the yield of H. lacustris cells after 5 days of exposure to 0.2% NaCl. Strikingly, this occurred by acceleration of cell growth, and not by acceleration of aplanospore formation. The level of astaxanthin synthesis was not affected by AZD8055. However, cytological data suggested a role of autophagosomes, lysosomes and Golgi cisternae in cell remodeling during high salt stress.

Leaf Epidermis: The Ambiguous Symplastic Domain.

The ability to develop secondary (post-cytokinetic) plasmodesmata (PD) is an important evolutionary advantage that helps in creating symplastic domains within the plant body. Developmental regulation of secondary PD formation is not completely understood. In flowering plants, secondary PD occur exclusively between cells from different lineages, e.g., at the L1/L2 interface within shoot apices, or between leaf epidermis (L1-derivative), and mesophyll (L2-derivative). However, the highest numbers of secondary PD occur in the minor veins of leaf between bundle sheath cells and phloem companion cells in a group of plant species designated "symplastic" phloem loaders, as opposed to "apoplastic" loaders. This poses a question of whether secondary PD formation is upregulated in general in symplastic loaders. Distribution of PD in leaves and in shoot apices of two symplastic phloem loaders, Alonsoa meridionalis and Asarina barclaiana, was compared with that in two apoplastic loaders, Solanum tuberosum (potato) and Hordeum vulgare (barley), using immunolabeling of the PD-specific proteins and transmission electron microscopy (TEM), respectively. Single-cell sampling was performed to correlate sugar allocation between leaf epidermis and mesophyll to PD abundance. Although the distribution of PD in the leaf lamina (except within the vascular tissues) and in the meristem layers was similar in all species examined, far fewer PD were found at the epidermis/epidermis and mesophyll/epidermis boundaries in apoplastic loaders compared to symplastic loaders. In the latter, the leaf epidermis accumulated sugar, suggesting sugar import from the mesophyll via PD. Thus, leaf epidermis and mesophyll might represent a single symplastic domain in Alonsoa meridionalis and Asarina barclaiana.

Regulation of plasmodesmata in Arabidopsis leaves: ATP, NADPH and chlorophyll b levels matter.

In mature leaves, cell-to-cell transport via plasmodesmata between mesophyll cells links the production of assimilates by photosynthesis with their export to sink organs. This study addresses the question of how signals derived from chloroplasts and photosynthesis influence plasmodesmata permeability. Cell-to-cell transport was analyzed in leaves of the Arabidopsis chlorophyll b-less ch1-3 mutant, the same mutant complemented with a cyanobacterial CAO gene (PhCAO) overaccumulating chlorophyll b, the trxm3 mutant lacking plastidial thioredoxin m3, and the ntrc mutant lacking functional NADPH:thioredoxin reductase C. The regulation of plasmodesmata permeability in these lines could not be traced back to the reduction state of the thioredoxin system or the types and levels of reactive oxygen species produced in chloroplasts; however, it could be related to chloroplast ATP and NADPH production. The results suggest that light enables plasmodesmata closure via an increase in the ATP and NADPH levels produced in photosynthesis, providing a control mechanism for assimilate export based on the rate of photosynthate production in the Calvin-Benson cycle. The level of chlorophyll b influences plasmodesmata permeability via as-yet-unidentified signals. The data also suggest a role of thioredoxin m3 in the regulation of cyclic electron flow around photosystem I.

YABBY Genes in the Development and Evolution of Land Plants.

Mounting evidence from genomic and transcriptomic studies suggests that most genetic networks regulating the morphogenesis of land plant sporophytes were co-opted and modified from those already present in streptophyte algae and gametophytes of bryophytes sensu lato. However, thus far, no candidate genes have been identified that could be responsible for "planation", a conversion from a three-dimensional to a two-dimensional growth pattern. According to the telome theory, "planation" was required for the genesis of the leaf blade in the course of leaf evolution. The key transcription factors responsible for leaf blade development in angiosperms are YABBY proteins, which until recently were thought to be unique for seed plants. Yet, identification of a YABBY homologue in a green alga and the recent findings of YABBY homologues in lycophytes and hornworts suggest that YABBY proteins were already present in the last common ancestor of land plants. Thus, these transcriptional factors could have been involved in "planation", which fosters our understanding of the origin of leaves. Here, we summarise the current data on functions of YABBY proteins in the vegetative and reproductive development of diverse angiosperms and gymnosperms as well as in the development of lycophytes. Furthermore, we discuss a putative role of YABBY proteins in the genesis of multicellular shoot apical meristems and in the evolution of leaves in early divergent terrestrial plants.

Singlet Oxygen in Plants: Generation, Detection, and Signaling Roles.

Singlet oxygen (1O2) refers to the lowest excited electronic state of molecular oxygen. It easily oxidizes biological molecules and, therefore, is cytotoxic. In plant cells, 1O2 is formed mostly in the light in thylakoid membranes by reaction centers of photosystem II. In high concentrations, 1O2 destroys membranes, proteins and DNA, inhibits protein synthesis in chloroplasts leading to photoinhibition of photosynthesis, and can result in cell death. However, 1O2 also acts as a signal relaying information from chloroplasts to the nucleus, regulating expression of nuclear genes. In spite of its extremely short lifetime, 1O2 can diffuse from the chloroplasts into the cytoplasm and the apoplast. As shown by recent studies, 1O2-activated signaling pathways depend not only on the levels but also on the sites of 1O2 production in chloroplasts, and can activate two types of responses, either acclimation to high light or programmed cell death. 1O2 can be produced in high amounts also in root cells during drought stress. This review summarizes recent advances in research on mechanisms and sites of 1O2 generation in plants, on 1O2-activated pathways of retrograde- and cellular signaling, and on the methods to study 1O2 production in plants.

Stomata control is changed in a chlorophyll b-free barley mutant.

The barley (Hordeum vulgare L.) chlorina f2 3613 mutant exhibits low photosynthesis and slow growth. This results from downregulation of the levels of photosynthetic antenna proteins caused by the absence of chl b, the major regulator of photosynthetic antennae in land plants. Here, we demonstrate that, when grown in the field in full sunlight, this mutant displays a changed pattern of stomatal responses compared with the parental wild-type cultivar Donaria. However, stomatal regulation of chlorina f2 3613 plants was restored when plants were placed under a shade cover for several days. The shade cover reduced incident PAR from 2000-2200µmolm-2s-1 to 800-880µmolm-2s-1 as measured at noon. Contents of ABA, the xanthophyll precursors of ABA biosynthesis and minor antenna proteins, as well as reactive oxygen species levels in stomata and the sensitivity of stomata to exogenously supplied ABA, were determined in leaves of wild-type Donaria and chlorina f2 3613 before and after shading. The results support the view that the restoration of stomatal control in barley chlorina f2 3613 is correlated with an increase in the levels of the minor antenna protein Lhcb6, which has recently been implicated in the enhancement of stomatal sensitivity to ABA in Arabidopsis thaliana (L.) Heynh.

The role of ion disequilibrium in induction of root cell death and autophagy by environmental stresses.

Environmental stresses such as salinity, drought, oxidants, heavy metals, hypoxia, extreme temperatures and others can induce autophagy and necrosis-type programmed cell death (PCD) in plant roots. These reactions are accompanied by the generation of reactive oxygen species (ROS) and ion disequilibrium, which is induced by electrolyte/K+ leakage through ROS-activated ion channels, such as the outwardly-rectifying K+ channel GORK and non-selective cation channels. Here, we discuss mechanisms of the stress-induced ion disequilibrium and relate it with ROS generation and onset of morphological, biochemical and genetic symptoms of autophagy and PCD in roots. Based on our own data and that in the literature, we propose a hypothesis on the induction of autophagy and PCD in roots by loss of cytosolic K+. To support this, we present data showing that in conditions of salt stress-induced autophagy, gork1-1 plants lacking root K+ efflux channel have fewer autophagosomes compared with the wild type. Overall, literature analyses and presented data strongly suggest that stress-induced root autophagy and PCD are controlled by the level of cytosolic potassium and ROS.

The levels of peroxisomal catalase protein and activity modulate the onset of cell death in tobacco BY-2 cells via reactive oxygen species levels and autophagy.

In plant cells, peroxisomes participate in the metabolism of reactive oxygen species (ROS). One of the major regulators of cellular ROS levels - catalase (CAT) - occurs exclusively in peroxisomes. CAT activity is required for immunity-triggered autophagic programmed cell death (PCD). Autophagy has been recently demonstrated to represent a route for degradation of peroxisomes in plant cells. In the present study, the dynamics of the cellular peroxisome pool in tobacco BY-2 cell suspension cultures were used to analyse the effects of inhibition of basal autophagy with special attention to CAT activity. Numbers of peroxisomes per cell, levels of CAT protein and activity, cell viability, ROS levels and expression levels of genes encoding components of antioxidant system were analysed upon application of 3-methyladenine (3-MA), an inhibitor of autophagy, and/or aminotriazole (AT), an inhibitor of CAT. When applied separately, 3-MA and AT led to an increase in cell death, but this effect was attenuated by their simultaneous application. The obtained data suggest that both the levels of CAT protein in peroxisomes as well as CAT activity modulate the onset of cell death in tobacco BY-2 cells via ROS levels and autophagy.

The Huperzia selago Shoot Tip Transcriptome Sheds New Light on the Evolution of Leaves.

Lycopodiophyta-consisting of three orders, Lycopodiales, Isoetales and Selaginellales, with different types of shoot apical meristems (SAMs)-form the earliest branch among the extant vascular plants. They represent a sister group to all other vascular plants, from which they differ in that their leaves are microphylls-that is, leaves with a single, unbranched vein, emerging from the protostele without a leaf gap-not megaphylls. All leaves represent determinate organs originating on the flanks of indeterminate SAMs. Thus, leaf formation requires the suppression of indeterminacy, that is, of KNOX transcription factors. In seed plants, this is mediated by different groups of transcription factors including ARP and YABBY.We generated a shoot tip transcriptome of Huperzia selago (Lycopodiales) to examine the genes involved in leaf formation. Our H. selago transcriptome does not contain any ARP homolog, although transcriptomes of Selaginella spp. do. Surprisingly, we discovered a YABBY homolog, although these transcription factors were assumed to have evolved only in seed plants.The existence of a YABBY homolog in H. selago suggests that YABBY evolved already in the common ancestor of the vascular plants, and subsequently was lost in some lineages like Selaginellales, whereas ARP may have been lost in Lycopodiales. The presence of YABBY in the common ancestor of vascular plants would also support the hypothesis that this common ancestor had a simplex SAM. Furthermore, a comparison of the expression patterns of ARP in shoot tips of Selaginella kraussiana (Harrison CJ, etal. 2005. Independent recruitment of a conserved developmental mechanism during leaf evolution. Nature 434(7032):509-514.) and YABBY in shoot tips of H. selago implies that the development of microphylls, unlike megaphylls, does not seem to depend on the combined activities of ARP and YABBY. Altogether, our data show that Lycopodiophyta are a diverse group; so, in order to understand the role of Lycopodiophyta in evolution, representatives of Lycopodiales, Selaginellales, as well as of Isoetales, have to be examined.

The absence of chlorophyll b affects lateral mobility of photosynthetic complexes and lipids in grana membranes of Arabidopsis and barley chlorina mutants.

The lateral mobility of integral components of thylakoid membranes, such as plastoquinone, xanthophylls, and pigment-protein complexes, is critical for the maintenance of efficient light harvesting, high rates of linear electron transport, and successful repair of damaged photosystem II (PSII). The packaging of the photosynthetic pigment-protein complexes in the membrane depends on their size and stereometric parameters which in turn depend on the composition of the complexes. Chlorophyll b (Chlb) is an important regulator of antenna size and composition. In this study, the lateral mobility (the mobile fraction size) of pigment-protein complexes and lipids in grana membranes was analyzed in chlorina mutants of Arabidopsis and barley lacking Chlb. In the Arabidopsis ch1-3 mutant, diffusion of membrane lipids decreased as compared to wild-type plants, but the diffusion of photosynthetic complexes was not affected. In the barley chlorina f2 3613 mutant, the diffusion of pigment-protein complexes significantly decreased, while the diffusion of lipids increased, as compared to wild-type plants. We propose that the size of the mobile fractions of pigment-protein complexes in grana membranes in vivo is higher than reported previously. The data are discussed in the context of the protein composition of antennae, characteristics of the plastoquinone pool, and production of reactive oxygen species in leaves of chlorina mutants.

Chlorophyllide-a-Oxygenase (CAO) deficiency affects the levels of singlet oxygen and formation of plasmodesmata in leaves and shoot apical meristems of barley.

In plants, organogenesis and specification of cell layers and tissues rely on precise symplastic delivery of regulatory molecules via plasmodesmata. Accordingly, abundance and aperture of plasmodesmata at individual cell boundaries should be controlled by the plant. Recently, studies in Arabidopsis established reactive oxygen species as major regulators of plasmodesmata formation and gating. We show that in a barley mutant deficient in the synthesis of chlorophyll b, the numbers of plasmodesmata in leaves and in the shoot apical meristem are significantly higher than in the corresponding wild type, probably due to redox imbalance in the mutant. The resulting disturbance of symplasmic transport is likely to be the reason for the observed delayed floral transition in these mutants.

Plant peroxisomes are degraded by starvation-induced and constitutive autophagy in tobacco BY-2 suspension-cultured cells.

Very recently, autophagy has been recognized as an important degradation pathway for quality control of peroxisomes in Arabidopsis plants. To further characterize the role of autophagy in plant peroxisome degradation, we generated stable transgenic suspension-cultured cell lines of heterotrophic Nicotiana tabacum L. cv. Bright Yellow 2 expressing a peroxisome-targeted version of enhanced yellow fluorescent protein. Indeed, this cell line model system proved advantageous for detailed cytological analyses of autophagy stages and for quantification of cellular peroxisome pools under different culturing conditions and upon inhibitor applications. Complementary biochemical, cytological, and pharmacological analyses provided convincing evidence for peroxisome degradation by bulk autophagy during carbohydrate starvation. This degradation was slowed down by the inhibitor of autophagy, 3-methyladenine (3-MA), but the 3-MA effect ceased at advanced stages of starvation, indicating that another degradation mechanism for peroxisomes might have taken over. 3-MA also caused an increase particularly in peroxisomal proteins and cellular peroxisome numbers when applied under nutrient-rich conditions in the logarithmic growth phase, suggesting a high turnover rate for peroxisomes by basal autophagy under non-stress conditions. Together, our data demonstrate that a great fraction of the peroxisome pool is subject to extensive autophagy-mediated turnover under both nutrient starvation and optimal growth conditions. Our analyses of the cellular pool size of peroxisomes provide a new tool for quantitative investigations of the role of plant peroxisomes in reactive oxygen species metabolism.

Plasmodesmata without callose and calreticulin in higher plants - open channels for fast symplastic transport?

Plasmodesmata (PD) represent membrane-lined channels that link adjacent plant cells across the cell wall. PD of higher plants contain a central tube of endoplasmic reticulum (ER) called desmotubule. Membrane and lumen proteins seem to be able to move through the desmotubule, but most transport processes through PD occur through the cytoplasmic annulus (Brunkard etal., 2013). Calreticulin (CRT), a highly conserved Ca(2+)-binding protein found in all multicellular eukaryotes, predominantly located in the ER, was shown to localize to PD, though not all PD accumulate CRT. In nitrogen-fixing actinorhizal root nodules of the Australian tree Casuarina glauca, the primary walls of infected cells containing the microsymbiont become lignified upon infection. TEM analysis of these nodules showed that during the differentiation of infected cells, PD connecting infected cells, and connecting infected and adjacent uninfected cells, were reduced in number as well as diameter (Schubert etal., 2013). In contrast with PD connecting young infected cells, and most PD connecting mature infected and adjacent uninfected cells, PD connecting mature infected cells did not accumulate CRT. Furthermore, as shown here, these PD were not associated with callose, and based on their diameter, they probably had lost their desmotubules. We speculate that either this is a slow path to PD degradation, or that the loss of callose accumulation and presumably also desmotubules leads to the PD becoming open channels and improves metabolite exchange between cells.

Evolutionary aspects of non-cell-autonomous regulation in vascular plants: structural background and models to study.

Plasmodesmata (PD) serve for the exchange of information in form of miRNA, proteins, and mRNA between adjacent cells in the course of plant development. This fundamental role of PD is well established in angiosperms but has not yet been traced back to the evolutionary ancient plant taxa where functional studies lag behind studies of PD structure and ontogenetic origin. There is convincing evidence that the ability to form secondary (post-cytokinesis) PD, which can connect any adjacent cells, contrary to primary PD which form during cytokinesis and link only cells of the same lineage, appeared in the evolution of higher plants at least twice: in seed plants and in some representatives of the Lycopodiophyta. The (in)ability to form secondary PD is manifested in the symplasmic organization of the shoot apical meristem (SAM) which in most taxa of seedless vascular plants differs dramatically from that in seed plants. Lycopodiophyta appear to be suitable models to analyze the transport of developmental regulators via PD in SAMs with symplasmic organization both different from, as well as analogous to, that in angiosperms, and to understand the evolutionary aspects of the role of this transport in the morphogenesis of vascular plant taxa.

Cytology of the minor-vein phloem in 320 species from the subclass Asteridae suggests a high diversity of phloem-loading modes.

The discovery of abundant plasmodesmata at the bundle sheath/phloem interface in Oleaceae (Gamalei, 1974) and Cucurbitaceae (Turgeon et al., 1975) raised the questions as to whether these plasmodesmata are functional in phloem loading and how widespread symplasmic loading would be. Analysis of over 800 dicot species allowed the definition of "open" and "closed" types of the minor vein phloem depending on the abundance of plasmodesmata between companion cells and bundle sheath (Gamalei, 1989, 1990). These types corresponded to potential symplasmic and apoplasmic phloem loaders, respectively; however, this definition covered a spectrum of diverse structures of phloem endings. Here, a review of detailed cytological analyses of minor veins in 320 species from the subclass Asteridae is presented, including data on companion cell types and their combinations which have not been reported previously. The percentage of Asteridae species with "open" minor vein cytology which also contain sieve-element-companion cell complexes with "closed" cytology, i.e., that show specialization for both symplasmic and apoplasmic phloem loading, was determined. Along with recent data confirming the dissimilar functional specialization of structurally different parts of minor vein phloem in the stachyose-translocating species Alonsoa meridionalis (Voitsekhovskaja et al., 2009), these findings suggest that apoplasmic loading is indispensable in a large group of species previously classified as putative symplasmic loaders. Altogether, this study provides formal classifications of companion cells and of minor veins, respectively, in 24 families of the Asteridae based on their structural features, opening the way to a close investigation of the relationship between structure and function in phloem loading.

Biosynthesis of benzylisoquinoline alkaloids in Corydalis bracteata: compartmentation and seasonal dynamics.

Numerous species of the genus Corydalis (Papaveraceae) produce a large spectrum of benzylisoquinoline alkaloids (BIA), some of which are of potential therapeutic value, but no information on sites of their biosynthesis and compartmentation is available. This study focuses on the biosynthesis, compartmentation and seasonal dynamics of BIA in Corydalis bracteata (Steph. ex Willd) Pers., a geophyte with a very short spring vegetation period, which for the rest of the year is represented by underground tubers with buds. It was found that all organs of C. bracteata contained high levels of BIA, the highest concentrations being detected in underground tuber buds in early autumn. Neither xylem nor phloem sap contained alkaloids throughout the year but BIA were present in the apoplastic wash fluid of the tuber. The absence of long-distance transport of alkaloids was confirmed by the experiment using an isotopically labeled tracer, [ring-(13)C6]-tyramine: when whole plants were fed with the tracer with via the roots, the alkaloids became labeled in the roots only and not in other organs. However, when detached roots, leaves, tubers and stems were exposed to [ring-(13)C6]-tyramine, the label was incorporated into alkaloids in all organs. We conclude that no long-distance translocation of alkaloids occurs between organs of C. bracteata, while in the tuber the cell-to-cell transport of alkaloids could occur via the apoplast. In contrast to other BIA-producing species, every organ of C. bracteata was found to be capable of de novo biosynthesis of the full complement of alkaloids.

Lignification of cell walls of infected cells in Casuarina glauca nodules that depend on symplastic sugar supply is accompanied by reduction of plasmodesmata number and narrowing of plasmodesmata.

The oxygen protection system for the bacterial nitrogen-fixing enzyme complex nitrogenase in actinorhizal nodules of Casuarina glauca resembles that of legume nodules: infected cells contain large amounts of the oxygen-binding protein hemoglobin and are surrounded by an oxygen diffusion barrier. However, while in legume nodules infected cells are located in the central tissue, actinorhizal nodules are composed of modified lateral roots with infected cells in the expanded cortex. Since an oxygen diffusion barrier around the entire cortex would also block oxygen access to the central vascular system where it is required to provide energy for transport processes, here each individual infected cell is surrounded with an oxygen diffusion barrier. In order to assess the effect of these oxygen diffusion barriers on oxygen supply for energy production for transport processes, apoplastic and symplastic sugar transport pathways in C. glauca nodules were examined. The results support the idea that sugar transport to and within the nodule cortex relies to a large extent on the less energy-demanding symplastic mechanism. This is in line with the assumption that oxygen access to the nodule vascular system is substantially restricted. In spite of this dependence on symplastic transport processes to supply sugars to infected cells, plasmodesmal connections between infected cells, and to a lesser degree with uninfected cells, were reduced during the differentiation of infected cells.

Two novel disaccharides, rutinose and methylrutinose, are involved in carbon metabolism in Datisca glomerata.

Datisca glomerata forms nitrogen-fixing root nodules in symbiosis with soil actinomycetes from the genus Frankia. Analysis of sugars in roots, nodules and leaves of D. glomerata revealed the presence of two novel compounds that were identified as alpha-L-rhamnopyranoside-(1 --> 6)-D-glucose (rutinose) and alpha-L-rhamnopyranoside-(1 --> 6)-1-O-beta-D-methylglucose (methylrutinose). Rutinose has been found previously as a/the glycoside part of several flavonoid glycosides, e.g. rutin, also of datiscin, the main flavonoid of Datisca cannabina, but had not been reported as free sugar. Time course analyses suggest that both rutinose and methylrutinose might play a role in transient carbon storage in sink organs and, to a lesser extent, in source leaves. Their concentrations show that they can accumulate in the vacuole. Rutinose, but not methylrutinose, was accepted as a substrate by the tonoplast disaccharide transporter SUT4 from Arabidopsis. In vivo (14)C-labeling and the study of uptake of exogenous sucrose and rutinose from the leaf apoplast showed that neither rutinose nor methylrutinose appreciably participate in phloem translocation of carbon from source to sink organs, despite rutinose being found in the apoplast at significant levels. A model for sugar metabolism in D. glomerata is presented.