Vascularization, high-volume solution flow, and localized roles for enzymes of sucrose metabolism during tumorigenesis by Agrobacterium tumefaciens

Vascular differentiation and epidermal disruption are associated with establishment of tumors induced by Agrobacterium tumefaciens. Here, we address the relationship of these processes to the redirection of nutrient-bearing water flow and carbohydrate delivery for tumor growth within the castor bean (Ricinus communis) host. Treatment with aminoethoxyvinyl-glycine showed that vascular differentiation and epidermal disruption were central to ethylene-dependent tumor establishment. CO2 release paralleled tumor growth, but water flow increased dramatically during the first 3 weeks. However, tumor water loss contributed little to water flow to host shoots. Tumor water loss was followed by accumulation of the osmoprotectants, sucrose (Suc) and proline, in the tumor periphery, shifting hexose-to-Suc balance in favor of sugar signals for maturation and desiccation tolerance. Concurrent activities and sites of action for enzymes of Suc metabolism changed: Vacuolar invertase predominated during initial import of Suc into the symplastic continuum, corresponding to hexose concentrations in expanding tumors. Later, Suc synthase (SuSy) and cell wall invertase rose in the tumor periphery to modulate both Suc accumulation and descending turgor for import by metabolization. Sites of abscisic acid immunolocalization correlated with both central vacuolar invertase and peripheral cell wall invertase. Vascular roles were indicated by SuSy immunolocalization in xylem parenchyma for inorganic nutrient uptake and in phloem, where resolution allowed SuSy identification in sieve elements and companion cells, which has widespread implications for SuSy function in transport. Together, data indicate key roles for ethylene-dependent vascularization and cuticular disruption in the redirection of water flow and carbohydrate transport for successful tumor establishment.     

Role of cytokinin in the regulation of root gravitropism

The models explaining root gravitropism propose that the growth response of plants to gravity is regulated by asymmetric distribution of auxin (indole-3-acetic acid, IAA). Since cytokinin has a negative regulatory role in root growth, we suspected that it might function as an inhibitor of tropic root elongation during gravity response. Therefore, we examined the free-bioactive-cytokinin-dependent ARR5::GUS expression pattern in root tips of transformants of Arabidopsis thaliana (L.) Heynh., visualized high cytokinin concentrations in the root cap with specific monoclonal antibodies, and complemented the analyses by external application of cytokinin. Our findings show that mainly the statocytes of the cap produce cytokinin, which may contribute to the regulation of root gravitropism. The homogenous symmetric expression of the cytokinin-responsive promoter in vertical root caps rapidly changed within less than 30 min of gravistimulation into an asymmetrical activation pattern, visualized as a lateral, distinctly stained, concentrated spot on the new lower root side of the cap cells. This asymmetric cytokinin distribution obviously caused initiation of a downward curvature near the root apex during the early rapid phase of gravity response, by inhibiting elongation at the lower side and promoting growth at the upper side of the distal elongation zone closely behind the root cap. Exogenous cytokinin applied to vertical roots induced root bending towards the application site, confirming the suspected inhibitory effect of cytokinin in root gravitropism. Our results suggest that the early root graviresponse is controlled by cytokinin. We conclude that both cytokinin and auxin are key hormones that regulate root gravitropism.Electronic Supplementary Material Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s00425-004-1381-8     

Binding of a photoaffinity analogue of glutathione to MRP1 (ABCC1) within two cytoplasmic regions (L0 and L1) as well as transmembrane domains 10-11 and 16-17

MRP1 (or ABCC1) is an ABC membrane protein that transports a wide range of natural products as well as glutathione (GSH)-, glucuronate-, and sulfate-conjugated metabolites. In addition, free GSH is required for MRP1 to transport several chemotherapeutic drugs. However, the mechanisms regulating the influence of GSH on MRP1 is poorly understood, and the location of GSH binding site(s) within MRP1 have yet to be determined. To address these issues, we have synthesized a [(125)I] labeled azido-derivative of GSH (IAAGSH) to photoaffinity label MRP1. Our results revealed that IAAGSH labeled MRP1 with high specificity, and binding was inhibited by MRP1 substrates leukotriene C(4) and MK571. Interestingly, verapamil and vincristine enhanced IAAGSH photolabeling of MRP1, in agreement with observations that both drugs enhance GSH transport. We observed GSH to be the best inhibitor of photoaffinity labeling, as compared to oxidized glutathione (GSSG) and four different GSH alkyl derivatives. These observations indicate that IAAGSH interacted with MRP1 in a similar manner as unmodified GSH. Moreover, using eight MRP1-HA variants, each containing hemagglutinin A (HA) epitopes inserted at different sites in MRP1, we mapped the GSH binding sites in MRP1. Our GSH analogue photoaffinity labeled four MRP1 polypeptides that were located within two cytoplasmic domains in linker sequences (L0 and L1) as well as transmembrane domains 10-11 and 16-17. The photoaffinity labeling of polypeptides within L0 and L1 domains is further confirmed using two MRP1-specific monoclonal antibodies (MRPr1 and QCRL1) with epitopes within the linker domains. Taken together, this study provides the most precise information to date on the location of GSH binding sites in MRP1.     

Lignocellulose affects Mn2+ regulation of peroxidase transcript levels in solid-state cultures of Pleurotus ostreatus

The effect of Mn2+ amendment on peroxidase gene expression was studied during Pleurotus ostreatus growth on cotton stalks. Four peroxidase-encoding genes were expressed differentially and in a manner different from that observed in defined media. Mn2+ affects mnp3 expression even 2 h after its addition to the cultures, suggesting a direct effect of the metal ion on expression.     

Indirect evidence for bulk water flow in root cortical cell walls of three dicotyledonous species

Water moves radially through the root in response to the tension generated by the transpiration stream. This movement occurs through both the cell walls and the protoplasts of the cells intervening between the soil solution and the lumena of the tracheary elements. The mechanism of movement is commonly believed to be diffusion in both these compartments. In the present study, we applied the apoplastic, fluorescent tracer, berberine, to roots of three dicotyledonous (Helianthus annuus L. cv. Mammoth Russian, Phaseolus vulgaris L. cv. Kinghorn wax, and Phaseolus aureus Roxb.) and four monocotyledonous species (Triticum aestivum L., Hordeum vulgare L., Zea mays L. cv. Seneca Chief, and Allium cepa L. cv. Ebeneezer). The tracer was precipitated in place by potassium thiocyanate. The entry of berberine into the main roots of the monocotyledonous species was limited, and no conclusions could be drawn about its movement. Tracer entered more readily into the main roots of dicotyledonous species and its movement by diffusion (in excised roots) was characterized by an evenly advancing diffusion ring in the cortex. However, when short treatment times were used for transpiring plants, some berberine was moved across the cortex by solvent drag, resulting in the formation of isolated crystals near the endodermis in advance of the diffusion ring. The phenomenon of solvent drag, in turn, is indirect evidence for movement of water by bulk flow in the cortical cell walls. Whether or not bulk flow also occurred in lateral roots could not be determined since the narrow width of the cortex and the high permeability of the walls to berberine resulted in very fast progression of the diffusion ring. Received: 11 March 1998 / Accepted: 18 May 1998     

Bark structure and mode of canopy regeneration in trees of Melia azedarach L.

Summary The bark texture of Melia azedarach L. changes from smooth to furrowed as trees age. In trees that were cut down, those with smooth bark sprouted below the cut from suppressed buds; trees with thick, furrowed bark sprouted at the edge of the cut surface from adventitious buds. The trees that had thin, furrowed bark sprouted mainly at the edge of the cut from adventitious buds, but sometimes also from suppressed buds in cracks. The relationship between sprouting pattern and tree architecture are discussed.     

A galling aphid furnishes its home with a built-in pipeline to the host food supply

Galling insects modify the developmental pathways of their host plants and create a protected and favourable microhabitat for their offspring. Galling aphids reproduce in their galls and the resulting clones often comprise hundreds of individuals. We followed the histological changes in the host Pistacia palaestina (Anacardiaceae) induced by the galling aphid Geoica wertheimae (Aphidoidea, Pemphigidae: Fordinae). We showed that the leaf tissues are altered in the gall in a way that gives the aphids easy access to the vascular system from which they obtain their nourishment. Specifically, the cuticle that lines the normal leaf epidermal cells is absent on the surface of the inner gall cavity, and the normal palisade cells are replaced by parenchymatous tissue with numerous wide latex ducts. Associated with these ducts, many new sieve tubes (phloem elements) are formed just a few cell layers below the inner gall surface. This arrangement enables the hundreds of aphids to feed simultaneously and continuously in the gall throughout the summer.     

VEGFA is necessary for chondrocyte survival during bone development

To directly examine the role of vascular endothelial growth factor (VEGFA) in cartilage development, we conditionally knocked out Vegfa in chondrocytes, using the Col2a1 promoter to drive expression of Cre recombinase. Our study of Vegfa conditional knockout (CKO) mice provides new in-vivo evidence for two important functions of VEGFA in bone formation. First, VEGFA plays a significant role in both early and late stages of cartilage vascularization, since Vegfa CKO mice showed delayed invasion of blood vessels into primary ossification centers and delayed removal of terminal hypertrophic chondrocytes. Second, VEGFA is crucial for chondrocyte survival, since massive cell death was seen in joint and epiphyseal regions of Vegfa CKO endochondral bones. Chondrocytes in these regions were found to upregulate expression of Vegfa in wild-type mice at the time when massive cell death occurred in the Vegfa CKO mice. The expression of the VEGFA receptors Npr1 and Npr2 in epiphyseal chondrocytes and lack of blood vessel reduction in the vicinity of the cartilaginous elements in the Vegfa CKO mice raise the possibility that the observed cell death is the result of a direct involvement of VEGFA in chondrocyte survival. Interestingly, the extensive cell death seen in Vegfa CKO null bones had a striking similarity to the cell death phenotype observed when hypoxia-inducible factor 1 alpha (Hif1a) expression was abolished in developing cartilage. This similarity of cell death phenotypes and the deficient VEGFA production in Hif1a null epiphyseal chondrocytes demonstrate that HIF1 alpha and VEGFA are components of a key pathway to support chondrocyte survival during embryonic bone development.