A biophysical model for plant cell plate maturation based on the contribution of a spreading force.

Plant cytokinesis, a fundamental process of plant life, involves de novo formation of a "cell plate" partitioning the cytoplasm of dividing cells. Cell plate formation is directed by orchestrated delivery, fusion of cytokinetic vesicles, and membrane maturation to form a nascent cell wall by timely deposition of polysaccharides. During cell plate maturation, the fragile membrane network transitions to a fenestrated sheet and finally a young cell wall. Here, we approximated cell plate sub-structures with testable shapes and adopted the Helfrich-free energy model for membranes, including a stabilizing and spreading force, to understand the transition from a vesicular network to a fenestrated sheet and mature cell plate. Regular cell plate development in the model was possible, with suitable bending modulus, for a two-dimensional late stage spreading force of 2-6 pN/nm, an osmotic pressure difference of 2-10 kPa, and spontaneous curvature between 0 and 0.04 nm-1. With these conditions, stable membrane conformation sizes and morphologies emerged in concordance with stages of cell plate development. To reach a mature cell plate, our model required the late-stage onset of a spreading/stabilizing force coupled with a concurrent loss of spontaneous curvature. Absence of a spreading/stabilizing force predicts failure of maturation. The proposed model provides a framework to interrogate different players in late cytokinesis and potentially other membrane networks that undergo such transitions. Callose, is a polysaccharide that accumulates transiently during cell plate maturation. Callose-related observations were consistent with the proposed model's concept, suggesting that it is one of the factors involved in establishing the spreading force.

Aspergillus niger α-1,3-glucan acts as a virulence factor by inhibiting the insect phenoloxidase system.

Phenoloxidase (PO) is a key enzyme in the melanization process involved in elimination of pathogens in insects. The PO system is rapidly activated in response to pathogen recognition. Inhibition of PO activity can be a way to avoid immune response and increase infection effectiveness. In this study, the effects of inoculation of Galleria mellonella larvae with Aspergillus niger α-1,3-glucan and conidia on PO activity in hemolymph are analyzed in comparison with the effects of ß-1,3/1,6-glucan inoculation. Our results indicate that α-1,3-glucan, a fungal cell wall polysaccharide, can play a role of a virulence factor involved in inhibition of the insect PO system.

Callose-Regulated Symplastic Communication Coordinates Symbiotic Root Nodule Development.

The formation of nitrogen-fixing nodules in legumes involves the initiation of synchronized programs in the root epidermis and cortex to allow rhizobial infection and nodule development. In this study, we provide evidence that symplastic communication, regulated by callose turnover at plasmodesmata (PD), is important for coordinating nodule development and infection in Medicago truncatula. Here, we show that rhizobia promote a reduction in callose levels in inner tissues where nodules initiate. This downregulation coincides with the localized expression of M. truncatula ß-1,3-glucanase 2 (MtBG2), encoding a novel PD-associated callose-degrading enzyme. Spatiotemporal analyses revealed that MtBG2 expression expands from dividing nodule initials to rhizobia-colonized cortical and epidermal tissues. As shown by the transport of fluorescent molecules in vivo, symplastic-connected domains are created in rhizobia-colonized tissues and enhanced in roots constitutively expressing MtBG2. MtBG2-overexpressing roots additionally displayed reduced levels of PD-associated callose. Together, these findings suggest an active role for MtBG2 in callose degradation and in the formation of symplastic domains during sequential nodule developmental stages. Interfering with symplastic connectivity led to drastic nodulation phenotypes. Roots ectopically expressing ß-1,3-glucanases (including MtBG2) exhibited increased nodule number, and those expressing MtBG2 RNAi constructs or a hyperactive callose synthase (under symbiotic promoters) showed defective nodulation phenotypes. Obstructing symplastic connectivity appears to block a signaling pathway required for the expression of NODULE INCEPTION (NIN) and its target NUCLEAR FACTOR-YA1 (NF-YA1) in the cortex. We conclude that symplastic intercellular communication is proactively enhanced by rhizobia, and this is necessary for appropriate coordination of bacterial infection and nodule development.

Unique phagocytic properties of hemocytes of Pacific oyster Crassostrea gigas against yeast and yeast cell-wall derivatives.

For a marine bivalve mollusk such as Pacific oyster Crassostrea gigas, the elimination of foreign particles via hemocyte phagocytosis plays an important role in host defense mechanisms. The hemocytes of C. gigas have a high phagocytic ability for baker's yeast (Saccharomyces cerevisiae) and its cell-wall product zymosan. C. gigas hemocytes might phagocytose yeast cells after binding to polysaccharides on the cell-wall surface, but it is unknown how and what kinds of polysaccharide molecules are recognized. We conducted experiments to determine differences in the phagocytic ability of C. gigas hemocytes against heat-killed yeast (HK yeast), zymosan and zymocel, which are similarly sized and shaped but differ in the polysaccharide composition of their particle surface. We found that both the agranulocytes and granulocytes exerted strong phagocytic ability on all tested particles. The phagocytic index (PI) of granulocytes for zymosan was 9.4 ± 1.7, which significantly differed with that for HK yeast and zymocel (P < 0.05). To evaluate the PI for the three types of particles, and especially to understand the outcome of the much higher PI for zymosan, PI was gauged in increments of 5 (1-5, 6-10, 11-15, and ≥16), and the phagocytic frequencies were compared according to these increments. The results show that a markedly high PI of ≥16 was exhibited by 18.1% of granulocytes for zymosan, significantly higher than 1.7% and 3.9% shown for HK yeast and zymocel, respectively (P < 0.05). These findings indicate that the relatively high PI for zymosan could not be attributed to a situation wherein all phagocytic hemocytes shared a high mean PI, but rather to the ability of some hemocytes to phagocytose a larger portion of zymosan. To determine whether the phagocytosis of these respective particles depended on the recognition of specific polysaccharide receptors on the hemocyte surface, C. gigas hemocytes were pretreated with soluble α-mannan or ß-laminarin and then allowed to phagocytose the three types of the particles. The percentage of phagocytic cells of ß-laminarin-treated granulocytes decreased significantly for zymosan and zymocel, but not for yeast. These results suggest that C. gigas might possess at least two types of hemocytes, and that one type of the hemocytes (granulocytes) is more active for phagocytosis. The granulocytes were found to have multiple subtypes with different phagocytic abilities and multiple phagocytic receptors. Some of the granulocyte subtypes revealed a much stronger phagocytic ability, depending on the presence of ß-glucan receptors for phagocytosis.

[Fungi in the gut - the gut mycobiome]. / Pilze im Darm ­ das Mykobiom des Darms.

Many different fungi, including yeasts and molds, can be found in the intestinal tract of humans constituting the gut mycobiome. In case the bacterial flora is altered, the fungal flora may react inversely. By a so-called fungal diet, however, the composition of the mycobiome can hardly be influenced. Whereas some fungi are only transiently present in the gut after oral uptake, others, such as Candida, Saccharomyces, Rhodotorula, Trichosporon, Geotrichum, amongst others, are members of the residential, autochthonous gut flora. Some of these fungi exert beneficial effects, for example by synthesizing useful materials. Rhodotorula can produce fatty acids and carotenoids. Others are able to metabolize toxic compounds, for example mycotoxins as well as procarcinogenic items in food. Toxins, as well as pathogenic bacteria, can be bound to mannans on the surface of fungi und can consequently be exported. Some fungi are said to exert probiotic activities. Certain fungal constituents, such as glucans, may even stimulate the immune system. On the other hand, some fungi cannot only colonize the gut asymptomatically but can also be noxious under certain conditions when, for example, the bacterial flora is disturbed. By means of their virulence factors, they can damage the gut epithelium and even penetrate into the Mukosa inducing inflammation, They can also aggravate chronic inflammatory processes. Fungi play a role in the development of obesity. Lastly, fungi in the gut represent a reservoir from which they may spread to other sites when the conditions are favorable.

DGE-seq analysis of MUR3-related Arabidopsis mutants provides insight into how dysfunctional xyloglucan affects cell elongation.

Our previous study of the Arabidopsis mur3-3 mutant and mutant plants in which the mur3-3 phenotypes are suppressed (xxt2mur3-3, xxt5mur3-3, xxt1xxt2mur3-3 and 35Spro:XLT2:mur3-3) showed that hypocotyl cell elongation is decreased in plants that synthesize galactose-deficient xyloglucan. To obtain genome-wide insight into the transcriptome changes and regulatory networks that may be involved in this decreased elongation, we performed digital gene expression analyses of the etiolated hypocotyls of wild type (WT), mur3-3 and the four suppressor lines. Numerous differentially expressed genes (DEGs) were detected in comparisons between WT and mur3-3 (1423), xxt2mur3-3 and mur3-3 (675), xxt5mur3-3 and mur3-3 (1272), xxt1xxt2mur3-3 and mur3-3 (1197) and 35Spro:XLT2:mur3-3 vs mur3-3 (121). 550 overlapped DEGs were detected among WT vs mur3-3, xxt2mur3-3 vs mur3-3, xxt5mur3-3 vs mur3-3, and xxt1xxt2mur3-3 vs mur3-3 comparisons. These DEGs include 46 cell wall-related genes, 24 transcription factors, 6 hormone-related genes, 9 protein kinase genes and 9 aquaporin genes. The expression of all of the 550 overlapped genes is restored to near wild-type levels in the four mur3-3 suppressor lines. qRT-PCR of fifteen of these 550 genes showed that their expression levels are consistent with the digital gene expression data. Overexpression of some of these genes (XTH4, XTH30, PME3, EXPA11, MYB88, ROT3, AT5G37790, WAG2 and TIP2;3) that are down-regulated in mur3-3 partially rescued the short hypocotyl phenotype but not the aerial phenotype of mur3-3, indicating that different mechanisms exist between hypocotyl cell elongation and leaf cell elongation.

'Strengthening the fungal cell wall through chitin-glucan cross-links: effects on morphogenesis and cell integrity'.

The cross-linking of polysaccharides to assemble new cell wall in fungi requires transglycosylation mechanisms by which preexisting glycosidic linkages are broken and new linkages are created between the polysaccharides. The molecular mechanisms for these processes, which are essential for fungal cell biology, are only now beginning to be elucidated. Recent development of in vivo and in vitro biochemical approaches has allowed characterization of important aspects about the formation of chitin-glucan covalent cell wall cross-links by cell wall transglycosylases of the CRH family and their biological function. Covalent linkages between chitin and glucan mediated by Crh proteins control morphogenesis and also play important roles in the remodeling of the fungal cell wall as part of the compensatory responses necessary to counterbalance cell wall stress. These enzymes are encoded by multigene families of redundant proteins very well conserved in fungal genomes but absent in mammalian cells. Understanding the molecular basis of fungal adaptation to cell wall stress through these and other cell wall remodeling enzymatic activities offers an opportunity to explore novel antifungal treatments and to identify potential fungal virulence factors.

Transgenic Arabidopsis thaliana plants expressing a ß-1,3-glucanase from sweet sorghum (Sorghum bicolor†L.) show reduced callose deposition and increased tolerance to aluminium toxicity.

Seventy-one cultivars of sweet sorghum (Sorghum bicolor L.) were screened for aluminium (Al) tolerance by measuring relative root growth (RRG). Two contrasting cultivars, ROMA (Al tolerant) and POTCHETSTRM (Al sensitive), were selected to study shorter term responses to Al stress. POTCHETSTRM had higher callose synthase activity, lower ß-1,3-glucanase activity and more callose deposition in the root apices during Al treatment compared with ROMA. We monitored the expression of 12 genes involved in callose synthesis and degradation and found that one of these, SbGlu1 (Sb03g045630.1), which encodes a ß-1,3-glucanase enzyme, best explained the contrasting deposition of callose in ROMA and POTCHETSTRM during Al treatment. Full-length cDNAs of SbGlu1 was prepared from ROMA and POTCHETSTRM and expressed in Arabidopsis thaliana using the constitutive cauliflower mosaic virus (CaMV) 35S promoter. Independent transgenic lines displayed significantly greater Al tolerance than wild-type plants and vector-only controls. This phenotype was associated with greater total ß-1,3-glucanase activity, less Al accumulation and reduced callose deposition in the roots. These results suggest that callose production is not just an early indicator of Al stress in plants but likely to be part of the toxicity pathway that leads to the inhibition of root growth.

Callose is integral to the development of permanent tetrads in the liverwort Sphaerocarpos.

A striking feature of the liverwort Sphaerocarpos is that pairs of male and female spores remain united in permanent tetrads. To identify the nature of this phenomenon and to test the hypothesis that callose is involved, we examined spore wall development in Sphaerocarpos miche lii, with emphasis on the appearance, location and fate of callose vis-à-vis construction of the sculptoderm. All stages of sporogenesis were examined using differential interference contrast optics, and aniline blue fluorescence to locate callose. For precise localization, specimens were immunogold labeled with anti-callose antibody and observed in the transmission electron microscope. Callose plays a role in Sphaerocarpos spore wall development not described in any other plant, including other liverworts. A massive callose matrix forms outside of the sculptured sporocyte plasmalemma that predicts spore wall ornamentation. Consequently, layers of exine form across adjacent spores uniting them. Spore wall development occurs entirely within the callose and involves the production of six layers of prolamellae that give rise to single or stacked tripartite lamellae (TPL). Between spores, an anastomosing network of exine layers forms in lieu of intersporal septum development. As sporopollenin assembles on TPL, callose progressively disappears from the inside outward leaving layers of sporopollenin impregnated exine, the sculptoderm, overlying a thick fibrillar intine. This developmental mechanism provides a direct pathway from callose deposition to sculptured exine that does not involve the intermediary primexine found in pollen wall development. The resulting tetrad, encased in a single wall, provides a simple model for development of permanent dyads and tetrads in the earliest fossil plants.

Intercellular protein movement: deciphering the language of development.

Development in multicellular organisms requires the coordinated production of a large number of specialized cell types through sophisticated signaling mechanisms. Non-cell-autonomous signals are one of the key mechanisms by which organisms coordinate development. In plants, intercellular movement of transcription factors and other mobile signals, such as hormones and peptides, is essential for normal development. Through a combination of different approaches, a large number of non-cell-autonomous signals that control plant development have been identified. We review some of the transcriptional regulators that traffic between cells, as well as how changes in symplasmic continuity affect and are affected by development. We also review current models for how mobile signals move via plasmodesmata and how movement is inhibited. Finally, we consider challenges in and new tools for studying protein movement.

Rapid microwave-assisted synthesis of polydextrose and identification of structure and function.

Microwave irradiation is a rapid and efficient method to synthesize oligomers and can be employed in polysaccharides production. As an artificial polysaccharide, polydextrose is known for its solid performance in food processing and its additional health benefits. This study was aimed at producing polydextrose by microwave irradiation using glucose and sorbitol as substrates; water and phosphoric acid as initiator and catalyst. The actual maximum yield was 99%. Synthetic polydextrose were purified by ethanol elution and Sepherdex G-25 column chromatography. Its purity was demonstrated by the high-performance gel-permeation chromatography as a single symmetrical sharp peak, additionally the average molecular weight was calculated to be 2.131 kDa. FT-IR spectra showed that the synthesized polydextrose has the structural feature similar to Polydextrose-Litesse(®). In vitro fermentation revealed that polydextrose possesses the biological function similar to Polydextrose-Litesse(®) in increasing the concentration of short chain fatty acid and decreasing pH. This research demonstrated the feasibility of a rapid and efficient microwave mediated method to synthesize polydextrose and potentially other value added carbohydrate polymers.

Pre-meiotic endomitosis in the cytokinesis-defective tomato mutant pmcd1 generates tetraploid meiocytes and diploid gametes.

Sexual polyploidization through the formation and functioning of 2n gametes is considered a major route for plant speciation and diversification. The cellular mechanism underlying 2n gamete formation mostly involves a restitution of the meiotic cell cycle, generating dyads and triads instead of tetrad meiotic end-products. As an alternative mechanism, the tomato mutant pmcd1 (for pre-meiotic cytokinesis defect 1), which generates diploid gametes through the ectopic induction of pre-meiotic endomitosis, is presented here. Using cytological approaches, it is demonstrated that male pmcd1 meiocyte initials exhibit clear alterations in cell cycle progression and cell plate formation, and consequently form syncytial cells that display different grades of cellular and/or nuclear fusion. In addition, it was found that other somatic tissue types (e.g. cotyledons and petals) also display occasional defects in cell wall formation and exhibit alterations in callose deposition, indicating that pmcd1 has a general defect in cell plate formation, most probably caused by alterations in callose biosynthesis. In a broader perspective, these findings demonstrate that defects in cytokinesis and cell plate formation may constitute a putative route for diplogamete formation and sexual polyploidization in plants.

Understanding how noncatalytic carbohydrate binding modules can display specificity for xyloglucan.

Plant biomass is central to the carbon cycle and to environmentally sustainable industries exemplified by the biofuel sector. Plant cell wall degrading enzymes generally contain noncatalytic carbohydrate binding modules (CBMs) that fulfil a targeting function, which enhances catalysis. CBMs that bind ß-glucan chains often display broad specificity recognizing ß1,4-glucans (cellulose), ß1,3-ß1,4-mixed linked glucans and xyloglucan, a ß1,4-glucan decorated with α1,6-xylose residues, by targeting structures common to the three polysaccharides. Thus, CBMs that recognize xyloglucan target the ß1,4-glucan backbone and only accommodate the xylose decorations. Here we show that two closely related CBMs, CBM65A and CBM65B, derived from EcCel5A, a Eubacterium cellulosolvens endoglucanase, bind to a range of ß-glucans but, uniquely, display significant preference for xyloglucan. The structures of the two CBMs reveal a ß-sandwich fold. The ligand binding site comprises the ß-sheet that forms the concave surface of the proteins. Binding to the backbone chains of ß-glucans is mediated primarily by five aromatic residues that also make hydrophobic interactions with the xylose side chains of xyloglucan, conferring the distinctive specificity of the CBMs for the decorated polysaccharide. Significantly, and in contrast to other CBMs that recognize ß-glucans, CBM65A utilizes different polar residues to bind cellulose and mixed linked glucans. Thus, Gln(106) is central to cellulose recognition, but is not required for binding to mixed linked glucans. This report reveals the mechanism by which ß-glucan-specific CBMs can distinguish between linear and mixed linked glucans, and show how these CBMs can exploit an extensive hydrophobic platform to target the side chains of decorated ß-glucans.

Ex vivo assessment of contractility, fatigability and alternans in isolated skeletal muscles.

Described here is a method to measure contractility of isolated skeletal muscles. Parameters such as muscle force, muscle power, contractile kinetics, fatigability, and recovery after fatigue can be obtained to assess specific aspects of the excitation-contraction coupling (ECC) process such as excitability, contractile machinery and Ca(2+) handling ability. This method removes the nerve and blood supply and focuses on the isolated skeletal muscle itself. We routinely use this method to identify genetic components that alter the contractile property of skeletal muscle though modulating Ca(2+) signaling pathways. Here, we describe a newly identified skeletal muscle phenotype, i.e., mechanic alternans, as an example of the various and rich information that can be obtained using the in vitro muscle contractility assay. Combination of this assay with single cell assays, genetic approaches and biochemistry assays can provide important insights into the mechanisms of ECC in skeletal muscle.

Behavioral evidence for a glucose polymer taste receptor that is independent of the T1R2+3 heterodimer in a mouse model.

Although it is clear that the heterodimer formed by the T1R2 and T1R3 proteins serves as the primary taste receptor for sweeteners, there is growing evidence that responses to glucose polymers may be mediated by a different taste receptor. Here we report that although T1R2 knock-out (KO) and T1R3 KO mice displayed severely impaired responding to glucose, maltose, and maltotriose in an initial session of a brief-access taste test (5 s trials, 25 min sessions) relative to wild-type (WT) mice, they subsequently increased their licking as a function of concentration for maltose and maltotriose with continued testing, presumably due to associating weak oral cues with positive post-ingestive consequences. Interestingly, these KO mice displayed relatively normal concentration-dependent licking to Polycose, a mixture of glucose polymers, even in the first session. Importantly, the experience-dependent increase in responsiveness to the sugars observed with the T1R2 and T1R3 single KO mice was not statistically significant in the T1R2/3 double KO mice. The double KO mice, however, still displayed significant concentration-dependent responding to Polycose in the first test session, albeit lick rates were slightly lower than those seen for WT mice, perhaps because small amounts of glucose, maltose, and maltotriose found in Polycose were enhancing the signal in WT mice or because T1R2 or T1R3 can possibly heteromerize with another protein to form a fully functional glucose polymer receptor. These findings provide behavioral evidence that glucose polymers, with an optimal chain length greater than three glucose moieties, stimulate a taste receptor independent of the T1R2+3 heterodimer.

Megasporogenesis and programmed cell death in Tillandsia (Bromeliaceae).

The degeneration of three of four meiotic products is a very common process in the female gender of oogamous eukaryotes. In Tillandsia (and many other angiosperms), the surviving megaspore has a callose-free wall in chalazal position while the other three megaspores are completely embedded in callose. Therefore, nutrients and signals can reach more easily the functional megaspore from the nucellus through the chalazal pole with respect to the other megaspores. The abortion of three of four megaspores was already recognized as the result of a programmed cell death (PCD) process. We investigated the process to understand the modality of this specific type of PCD and its relationship to the asymmetric callose deposition around the tetrad. The decision on which of the four megaspores will be the supernumerary megaspores in angiosperms, and hence destined to undergo programmed cell death, appears to be linked to the callose layer deposition around the tetrad. During supernumerary megaspores degeneration, events leading to the deletion of the cells do not appear to belong to a single type of cell death. The first morphological signs are typical of autophagy, including the formation of autophagosomes. The TUNEL positivity and a change in morphology of mitochondria and chloroplasts indicate the passage to an apoptotic-like PCD phase, while the cellular remnants undergo a final process resembling at least partially (ER swelling) necrotic morphological syndromes, eventually leading to a mainly lipidic cell corpse still separated from the functional megaspore by a callose layer.

Production of glucosyltransferase B and glucans by streptococcus mutans strains isolated from caries-free individuals

La glucosiltranferasa B es una enzima producida por Streptococcus mutans, que a partir de la sacarosa, cataliza la síntesis de glucanos insolubles los cuales dan soporte a la biopelícula, siendo uno de los principales factores de virulencia para la generación de la caries dental. Sin embargo, no se ha esclarecido su papel en los individuos libre de caries, portadores delmicroorganismo. El objetivo de este estudio fue determinar la producción de glucosiltransferasa B y la producción de glucanos por cepas de Streptococcus mutans aisladas de biopelícula de 30individuos libres de caries. Las cepas fueron cultivadas en caldo Todd Hewitt y las proteínas extracelulares fueron obtenidas por precipitación con sulfato de amonio las proteínas asociadas amembrana por extracción con urea. La presencia de GtfB fue determinada por peso molecular por SDS–PAGE, confirmada por Western Blot utilizando un anticuerpo específico y la producciónde polisacáridos por separación electroforética, incubación con sacarosa y coloración de Schiff. Los resultados muestran que el 96.7 por ciento de las cepas de Streptococcus mutans producen una banda a la altura del peso molecular correspondiente a las Gtf,de las cuales son reactivas por western blot el 63.4 por ciento El 93.3 por cientode las cepas producen polisacáridos. Conclusiones: la cepas de Streptococcus mutans aisladas de biopelícula de individuos sanos producen factores de virulencia asociados a la caries dental como glucosiltransferasa B y glucanos lo que indica que hay condiciones en la cavidad oral diferentes a estos factores que mantienen al individuo libre de caries dental, los cuales deben ser investigados en la búsqueda de estrategias para controlar la enfermedad.

Chloroplasts divide by contraction of a bundle of nanofilaments consisting of polyglucan.

In chloroplast division, the plastid-dividing (PD) ring is a main structure of the PD machinery and is a universal structure in the plant kingdom. However, the components and formation of the PD ring have been enigmatic. By proteomic analysis of PD machineries isolated from Cyanidioschyzon merolae, we identified the glycosyltransferase protein plastid-dividing ring 1 (PDR1), which constructs the PD ring and is widely conserved from red alga to land plants. Electron microscopy showed that the PDR1 protein forms a ring with carbohydrates at the chloroplast-division site. Fluorometric saccharide ingredient analysis of purified PD ring filaments showed that only glucose was included, and down-regulation of PDR1 impaired chloroplast division. Thus, the chloroplasts are divided by the PD ring, which is a bundle of PDR1-mediated polyglucan filaments.

EPS II-dependent autoaggregation of Sinorhizobium meliloti planktonic cells.

Planktonic cells of Sinorhizobium meliloti, a Gram-negative symbiotic bacterium, display autoaggregation under static conditions. ExpR is a LuxR-type regulator that controls many functions in S. meliloti, including synthesis of two exopolysaccharides, EPS I (succinoglycan) and EPS II (galactoglucan). Since exopolysaccharides are important for bacterial attachment, we studied the involvement of EPS I and II in autoaggregation of S. meliloti. Presence of an intact copy of the expR locus was shown to be necessary for autoaggregation. A mutant incapable of producing EPS I displayed autoaggregation percentage similar to that of parental strain, whereas autoaggregation was significantly lower for a mutant defective in biosynthesis of EPS II. Our findings clearly indicate that EPS II is the essential component involved in autoaggregation of planktonic S. meliloti cells, and that EPS I plays no role in such aggregation.

Sieve tube geometry in relation to phloem flow.

Sieve elements are one of the least understood cell types in plants. Translocation velocities and volume flow to supply sinks with photoassimilates greatly depend on the geometry of the microfluidic sieve tube system and especially on the anatomy of sieve plates and sieve plate pores. Several models for phloem translocation have been developed, but appropriate data on the geometry of pores, plates, sieve elements, and flow parameters are lacking. We developed a method to clear cells from cytoplasmic constituents to image cell walls by scanning electron microscopy. This method allows high-resolution measurements of sieve element and sieve plate geometries. Sieve tube-specific conductivity and its reduction by callose deposition after injury was calculated for green bean (Phaseolus vulgaris), bamboo (Phyllostachys nuda), squash (Cucurbita maxima), castor bean (Ricinus communis), and tomato (Solanum lycopersicum). Phloem sap velocity measurements by magnetic resonance imaging velocimetry indicate that higher conductivity is not accompanied by a higher velocity. Studies on the temporal development of callose show that small sieve plate pores might be occluded by callose within minutes, but plants containing sieve tubes with large pores need additional mechanisms.