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First published online October 2, 2003; 10.1104/pp.103.028142 Plant Physiology 133:1024-1037 (2003) © 2003 American Society of Plant Biologists Vascularization, High-Volume Solution Flow, and Localized Roles for Enzymes of Sucrose Metabolism during Tumorigenesis by Agrobacterium tumefaciens1Institute of Botany, Darmstadt University of Technology, Schnittspahnstrasse 3, 64287 Darmstadt, Germany (R.W., M.L., S.G., A.W., A.K., L.T., K.S., C.I.U.); Department of Plant Sciences, Tel Aviv University, Tel Aviv 69978, Israel (R.A.); Slovak Academy of Sciences, Department of Plant Physiology, Dúbravská cesta 14, 84223 Bratislava, Slovakia (I.M., J.P.); Columbia University, Biosphere 2 Center, P.O. Box 689, Oracle, Arizona 85623 (U.R.); and Department of Horticulture Sciences, University of Florida, P.O. Box 110690, Gainesville, Florida 32611 (K.E.K.)
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.
The significance of this research lies in the identification of functional relationships effecting the redirection of nutrient-bearing water flow and carbohydrate delivery necessary for tumor establishment by Agrobacterium tumefaciens. Early, ethylene-dependent changes in both tumor vascular development and epidermal disruption are shown here linked to key shifts in water flow, sugar gradients, and highly localized roles for enzymes of Suc metabolism. The experimental system used in this work provided three distinct advantages: First, the robust, rapidly growing host species castor bean (Ricinus communis) can support production of unusually large tumors when induced by A. tumefaciens. The size of these stem tumors was invaluable to the functional analyses reported here.
Second, these tumors develop through a series of well-defined events. Initial development is triggered by the expression of the T-DNA-located genes encoding auxin and cytokinin synthesis (iaaM, iaaH, and ipt; Weiler and Schröder, 1987
Third, this system can be readily manipulated by specifically altering ethylene perception, as shown for ethylene-insensitive Never ripe mutant tomato (Lycopersicon esculentum) plants that do not develop tumors when infected with A. tumefaciens (Aloni et al., 1998 Here, treatment of developing tumors with the ethylene synthesis inhibitor aminoethoxyvinyl-Gly (AVG) demonstrated that vascularization and epidermal disruption were part of a critical, ethylene-dependent phase of establishment for tumors induced by A. tumefaciens.
For this reason, focus of the present work was directed toward mechanisms by which vascular development and cuticular rupture could affect tumor establishment. Vascularization is essential for efficient assimilate import from the host plant into the tumor parenchyma cells via symplastic phloem unloading of Suc in the tumor (Malsy et al., 1992 One aim of the present study was to define the relationship between epidermal disruption and nutrient-bearing water flow, particularly in the context of competition for this resource between tumor and host leaves. Toward this end, a method was developed that allowed accurate quantification of the irregular tumor surface and its transpiration rate, and this was done during several weeks of tumor development. Direct evidence was thus obtained for a quantitative comparison of water flow through tumors and leaves and thus the significance of its balance for transpiration for tumor development and growth of the host plant shoot.
A second aim was to determine what changes occurred in effectors of Suc import and osmotic adjustment during vascular development in this distinctive sink for sugars and nutrient-bearing water flow. Enzymes of Suc metabolism and associated shifts in sugar gradients were quantified during development and at specific sites in the tumor to appraise their import and/or osmotic contributions. The Suc-cleaving enzymes, acid cell wall invertase (CWIN), acid vacuolar invertase (VIN), and Suc synthase (SuSy) can play crucial roles in carbohydrate partitioning to sink organs and in this capacity can provide substrates for osmotic adjustment or protection as well as energy expenditure for storage and growth (Koch, 1996 Analysis of this distinctive model system for high-volume solute and Suc transfer has provided several new insights into mechanisms underlying phloem functioning and import into these and other sink organs. First, successful establishment of these tumor sinks involves ethylene-induced transition to high-volume flow of solutes with essential inorganic nutrients, yet this itself does not appear responsible for host dwarfing. Second, sugar balance in actively growing tumors shifts in ratio from hexose to Suc, which would initially favor signals for cell division and expansion and later, those for maturation, storage, and desiccation tolerance. Third, VINs in these tumors appear to mediate this transition in sugar balance as well as symplastic import of Suc. CWINs contributed late in development at the tumor periphery, possibly aiding maintenance of turgor gradients favoring transport to the tumor surface. Finally, data here significantly extend our understanding of respiratory roles for SuSy, which included not only the high-Suc regions at the tumor periphery, but also xylem parenchyma cells ideally positioned for inorganic nutrient uptake, and both companion cells and sieve elements of phloem.
Vascularization and Cuticular Rupture
Vascular development and cuticular rupture appear to be two key steps in establishing solute flow into plant tumors such as those induced by A. tumefaciens (Aloni et al., 1995
Figure 1D shows that tumor development was severely impaired by a 3-week external application of the ethylene biosynthesis inhibitor, AVG, to the infection site. This provides additional evidence supporting the critical role of ethylene in establishment of these sinks and extends earlier work of Aloni et al. (1998
The water vapor conductance (gH2O) of tumors rose rapidly within the 1st week after infection (p.i.) with the strongest increase between the 2nd and 3rd week p.i. (Fig. 2A). The gH2O increased to 54 mmol m–2 s–1 in 6-week-old tumors, whereas uninfected stems had a constant gH2O between 2 and 8 mmol m–2 s–1, independent of their age. Concurrent growth of tumors was sigmoidal (Fig. 2B), with some fresh weights rising above 80 g and some tumors reaching the size of tennis balls (approximately 8 cm in diameter). Changes in the highly irregular tumor surface area were quantified by thorough microscopic analysis (as in Fig. 1B) and were found to increase almost linearly with tumor growth (Fig. 2C). The gH2O thus appears related to the tumor surface area, combined with the timing of rupture and break-up of epidermis and cuticle during the first 3 weeks of gall growth.
Table I compares water-loss parameters of tumors with those of leaves. The gH2O of leaves of uninfected plants was about 360 mmol m–2 s–1 during the light period, i.e. 10 times that of tumors. During the dark period, gH2O of the leaves decreased to 12 mmol m–2 s–1 and that of the tumors only slightly to 28 mmol m–2 s–1 on average. The net transpiration rate (JH2O) of tumors was about seven times lower in light but was twice that of similarly aged control leaves in the dark. When considered on a whole-organ basis, water flow through tumors could thus contribute to a significant amount of water loss, but much less than that of leaves on healthy or infected plants.
The net CO2 emission rate (J'CO2) of uninfected stems varied between 1 and 2 µmol µmol g–1 fresh weight h–1 (Fig. 3) and did not change with exposure of the leaves to light or dark (data not shown). During earlier tumor development, the J'CO2 increased considerably, beginning 2 weeks after initiation and increasing continuously until reaching about 18 µmol CO2 µmol g–1 fresh weight h–1 at 6 weeks p.i. Simultaneously, the dry weight to fresh weight ratio increased from 10% to 15% (data not shown). The mean rate of respiratory loss during the first 8 weeks of tumor growth was thus about 10 mg CO2 g–1 fresh weight d–1, or about 25% to 30% of the estimated carbohydrate demand by these sinks.
ABA distribution, detected in 3-week-old tumors by immunofluorescence with monoclonal antibodies, showed an interesting pattern throughout the tumor. Distinct fluorescence was detected around the vascular bundles (Fig. 1, F and H) and in the very peripheral cell layers (Fig. 1H). At cellular resolution, the ABA-specific fluorescence was localized in parenchyma cells adjacent to vessels (Fig. 1I). In host stems close to developing tumors, the strongest ABA-specific fluorescence was found in phloem companion cells (Fig. 1J) and in xylem parenchyma cells (Fig. 1K). Figure 1L shows the autofluorescence of lignified vessels of the same section as in Figure 1K, however, under 488-nm light. Control cross sections of the host stem treated with 1% (w/v) BSA instead of the primary ABA antibody and with the secondary antibody-Alexa 568 conjugate showed distinct autofluorescence of the lignified vessels only (Fig. 1M).
Suc concentration was highest in the outmost 1 to 2 mm of 6-week-old tumors and clearly decreased toward the center (Fig. 4A). This gradient was minimally evident in 3-week-old tumors, but steepened with tumor age (Fig. 4A). In contrast, an inverse gradient of hexose concentration was found in young tumors, with maximal levels decreasing from the tumor center toward the periphery (Fig. 4B). The gradient decreased with tumor age. Such inverse sugar gradients also appear within primary roots of maize (Zea mays), with steeper gradients in water-stressed seedlings (Sharp et al., 1990
Transpiration increased dramatically during disruption of the epidermis and cuticle of the tumors (Fig. 1, A and C; Table I), and consequent production of compatible solutes could aid protection of tumor parenchyma from desiccation. Three to 4 weeks after tumor induction, the concentrations of Suc and Pro began to rise in the periphery, where they reached respective levels of 20 and 40 times those in the control stem (Fig. 5, A and B). Both Suc and Pro concentrations in the tumor periphery were more than five times those in the tumor center (Fig. 5, A and B). The Pro concentration gradient from its maximum in the periphery toward the tumor center was steeper than that of Suc (Fig. 4, C versus A).
As observed for net CO2 emission, CWIN activity increased after a lag phase and was maximum at 6 to 7 weeks after tumor induction (Fig. 6A). Maximal activity was restricted to the peripheral cell layers at the outermost 1 to 2 mm of the tumor. The CWIN activity was 20 times less in the tumor center and 50 times less in the uninfected stem (Fig. 6A).
Maximum activity of the VIN was twice that of CWIN and peaked much earlier in tumor development (Fig. 6B). Early activity of VIN has also been observed in Helianthus sp. tumors (Kutschera et al., 2000
SuSy activity increased within the 1st week after tumor induction (Fig. 7). In contrast, in rapidly necrotizing tumors of Helianthus sp., SuSy activity was very low and did not exceed that of the control stem (Kutschera et al., 2000
The resolution of immunolocalization of SuSy with polyclonal anti-Sh1 and Sus1-protein antibodies in tissue sections of 2-week-old tumors showed a more distinct gradient and more precise localization in SuSy protein (Fig. 8) than could be achieved by activity measurements upon tissue dissection (Fig. 7). The highest fluorescence label was found in the tumor periphery, a tissue area that lacks vascular bundles in the castor bean system (Fig. 8, A and B). In the individual vascular bundles of the tumor, moderately fluorescent SuSy label was evident in the peripheral parenchyma and companion cells of the phloem immediately inside the encircling xylem (Fig. 8, C and D). At the tumor/host interface, the only xylem cells labeled were parenchyma cells in the tumor vascular bundles, no label was found in the multiseriate rays (Fig. 8, E, F, and top of G). In the control stem, SuSy label appeared in the cytoplasm of the companion cells and the thin cytoplasmic layer in the sieve elements.
The level of resolution possible in this system was exciting and extends earlier work of Nolte and Koch (1993  ) by showing that phloem SuSy includes labeling at or near the plasma membrane of the sieve elements (Fig. 8, G–I). SuSy label (green) was maximally evident in the cytoplasm of the companion cells, and also about 0.27 µm away, where the plasma membrane and/or adjacent cytoplasm of sieve elements was marked by red fluorescence conjugated to monoclonal antibodies specific for plasma membrane H+-ATPase (Figs. 8, G–I, and 9). Quantification of red and green fluorescence confirmed the highly marginal localization of SuSy and plasma membrane H+-ATPase in the sieve elements (Fig. 9).
The dual importance of work presented here lies in its identification of ethylene-dependent mechanisms for both water and Suc delivery to tumors induced by A. tumefaciens. For water, a pivotal disruption of epidermal integrity is shown here to coincide with dramatic increases in fluid flux and capacity for transfer of inorganic nutrients to the tumor. Direct contributions by this flow to concurrent stunting of the host shoot were calculated to be minimal. For Suc, critical changes in vascular development are shown to be linked to (a) a shift from hexose to Suc accumulation, (b) a transition from VIN to CWIN, and (c) previously unidentified sites of SuSy action in specific cells of both xylem and phloem.
Key features of development in the large, A. tumefaciens-induced tumors of castor bean were shown here to be ethylene dependent (Fig. 1, A–E), as observed for Never ripe tomatoes (Aloni et al. 1998
Because tumor proliferation also leads to dwarf size of the host shoot (Fig. 1A), the gall constriction hypothesis was investigated to determine the extent to which water competition by the tumor contributes to these effects. Schurr et al. (1996
In the light, JH2O for a given leaf area of uninfected castor bean plants was about seven times that of tumors on plants of the same age (Table I), whereas leaf transpiration rate dropped to one-half that of tumors in the dark. A further comparison of JH2O for the total transpiring surfaces of a 3-week-old tumor (10 cm2 surface) with a host plant with five leaves (400 cm2 surface each leaf) revealed that, in the light, JH2O of the leaves was about 1,470 times greater than that of the tumor. For a shoot with five leaves, comparative transpiration by the tumor would thus be negligible. However, the transpiration rate for leaves of tumor-infected plants is typically only 10% of that for noninfected plants (Veselov et al., 2003
In conclusion, the amount of water loss through the tumor surface was considerably lower than the sum of water loss through the leaves of uninfected control plants. Ethylene emission from the tumor on ABA induction in the host leaves, leading to drastic stomata closure (Veselov et al., 2003
Substantial demand for Suc import includes high respiratory activity (Fig. 3). The redox potential in plant tumor tissue is relatively low (Beardsley, 1972
In young tumors, the activity of vacuolar, but not CWIN, was consistent with a major role in the direct import of symplastically transported Suc (Pradel et al., 1996
Early tumor growth was also characterized by a close relationship between VIN (Fig. 6B) and accumulation of free auxin and cytokinins in castor bean tumors during their first 2 weeks of development (Veselov et al., 2003
Analysis of later tumor growth revealed distinctive features of CWIN regulation in this system. First, it was not induced during periods of either auxin or cytokinin production by the young tumor. The lack of auxin induction is consistent with the repression of some sugar response elements by auxin (DeWald et al., 1994 These data raise questions about the seemingly enigmatic role of CWIN in a system with an intact symplastic transport path and high levels of Suc accumulation. However, symplastic continuity does not prevent movement of Suc into the cell wall space, and varying degrees of apoplastic transfer can often occur. When it does, a defined fraction of the translocated Suc can be cleaved by CWINs if they are present and active, as indicated for the periphery of maturing tumors (Fig. 6A). Because this extracellular process is isolated from the greater mass of cellular cytoplasm and vacuole, high concentrations of Suc can accumulate in the same cells and tissues. Under these conditions, CWIN can contribute to localized apoplastic import as well as adjustment of osmotically active hexose concentrations immediately outside cells. Both actions could favor establishment of descending turgor gradients so necessary for Suc import. Control of turgor gradients could be invaluable in a rapidly transpiring structure like the castor bean tumors, where water potential and solute flow could vary markedly during a diurnal period and from cell to cell.
Finally, a key developmental role may be played by the CWIN at the tumor periphery, because even symplastical intact systems such as carrot (Daucus carota) plantlets, can require CWIN for normal separation of expanding leaves (Sturm et al., 1995
SuSy distribution in the tumor is compatible with three new aspects of its function, two of these with special implications for the developing castor bean tumor. The first of these was the extent of its labeling along a gradient toward the periphery (Fig. 8A). SuSy action in these cells would have been invaluable to a dual role in facilitating both Suc import and high-volume solution flow. Cytoplasmic action of SuSy would complement that of CWIN in maintaining low turgor at the terminal end of the transport path by rapid carbohydrate metabolization. This, plus the osmoprotective advantage of an endogenous, high-Suc environment in the cytoplasm of these cells (Figs. 4 and 5), would be compatible with a SuSy-based path for respiration (Huber and Akazawa, 1986 The second new aspect of SuSy localization was its identification in the distinctive xylem parenchyma cells of the tumor (Fig. 8, C and G). They are strategically positioned adjacent to large vessel elements, where they can readily function in mineral nutrient uptake. Furthermore, they lie at sites where they can mediate xylem-to-phloem transfer of nutrients. Immunolocalization data for these key cells in the present system support a mechanistic role for SuSy in acquisition and handling of the inorganic nutrients so critical to tumor establishment.
Finally, the clarity of SuSy localization achieved in this system allowed a resolution of its role in phloem function, regardless of position in tumor or control tissue (Figs. 8I and 9). Previous work had established that SuSy promoters are active in phloem (Yang and Russell, 1990
This SuSy localization in sieve elements is an important extension of earlier research in several ways. First, data now indicate that the previously envisioned role of SuSy in contributing UDP-Glc to biosynthesis of callose for protective plugging of phloem (Nolte and Koch, 1993 In conclusion, vascularization and cuticular rupture, both central to ethylene-dependent establishment of tumors, were appraised here relative to their functional significance for water and sugar transfer. Data support the following advances.
For water flow: (a) A striking relationship was revealed between the timing of cuticular rupture in young tumors and dramatic increases in the water flow essential for delivery of inorganic nutrients. (b) Assessment of the gall constriction hypothesis (Aloni et al., 1995 For sugar utilization and import: (a) Successful tumors accumulated first hexoses and then Suc in a change that often alters sugar signals from those favoring cell division to those for maturation and desiccation tolerance. Direct effects of this shift would also aid initial tumor expansion, and later gradients in turgor and osmoprotection. (b) Tumor development also involved a transition from VIN, as a mediator of symplastic import and hexose concentration in young tumors, to CWIN, positioned to maintain descending turgor gradients in the periphery of older tumors. Correlation of both VIN and CWIN with ABA concentration gradients is in line with recent evidence for common paths for sugar and ABA signals. (c) Three new roles for SuSy were implicated, including: (i) import and respiration in the osmoprotective, high-Suc cytoplasm at the tumor periphery, (ii) mineral nutrient uptake and transfer by specific xylem parenchyma cells present in successfully established tumors, and (iii) functioning of phloem, not only in companion cells, but also in the unique interior of sieve elements.
Plant Material and Tumor Induction
Castor bean (Ricinus communis L. var gibsonii cv Carmencita; Walz Samen, Stuttgart, Germany) was grown and infected with Agrobacterium tumefaciens C58 as described in detail previously (Pradel et al., 1996
To compare the transpiration rate of tumors with that of leaves, the highly irregular surface of the tumors (Fig. 1, A and B) had to be determined. A plastic polymer (Xantopren, Haraeus Kulzer, Dormagen, Germany) was spread over the tumor surface. The mixture of the activator (8 drops) and Xantopren (1 g) became elastic after a few minutes and was removed, giving the shape of the irregular surface. The slightly sticky impression was covered with a layer of glass balls (0.06–0.46 mm in diameter; Sodaklarglas, Wenzel, Heidelberg). The ratio of the difference in weight of the Xantropren-impression with and without glass balls to the weight of the glass ball layer of a known surface yielded the tumor surface. The surface of the leaves was scanned and calculated by the NIH Image software (developed at the United States National Institutes of Health and available on the Internet at http://rsb.info.nih.gov/nih-image), taking into account that castor bean leaves are amphistomatic.
Cuticles of tumor cross sections were stained red for 10 s in Sudan III solution (0.2 g dissolved in 100 mL of isopropanol and 100 mL of distilled water). The sections were viewed in glycerol with an Aristoplan epifluorescence microscope (Leica, Bensheim, Germany). Micrographs were reproduced from color slides taken with an Orthomat E camera system (Leica) on Kodak Ektachrome Elite 100 (Eastman Kodak, Rochester, NY).
To investigate the influence of ethylene on tumor growth and vascularization, the ethylene synthesis inhibitor AVG was applied at the site of infection 1 d after inoculation with bacteria. A Terostat funnel (Teroson, Heidelberg) was maintained filled with 10 µM aqueous AVG solution or distilled water as control. After 3 weeks, cross sections from tumor and host stem tissue were stained with toluidine blue (0.05%) and were viewed with a stereo photomacroscope (M 400, Wild, Heerbrugg, Switzerland). The micrographs were reproduced from Kodak Ektachrome 64T color slides.
Net CO2 emission of uninfected stems and tumors was measured with a minicuvette system (Walz, Effeltrich, Germany) as described previously (Rascher et al., 1998
Either randomized samples of the whole tumor or 2-mm-thick tangential tumor sections and samples of the control stem (100–500 mg) were harvested, rapidly frozen in liquid nitrogen, and homogenized in Teflon capsules with a micro-dismembrator (Braun, Melsungen, Germany). For the determination of acid CWIN and soluble VIN, the frozen powder was suspended in 5 mL of buffer containing 100 mM MOPS, 250 mM sorbitol, 10 mM KCl, 10 mM MgCl2, 1 mM phenylmethylsulfonyl fluoride, 3% (w/v) polyvinylpyrrolidone (for CWIN) or 5% (w/w) polyvinylpolypyrrolidone (for VIN), and 2 mM dithiothreitol (DTT), at pH 6. After centrifugation, the pellet was resuspended in the buffer without polyvinylpyrrolidone. After washing three times with distilled water, the pellet was resuspended for the CWIN assay in hydrolyzing buffer (20 mM triethanolamine hydrochloride, 6.7 mM citric acid, and 20 mM Suc, pH 4.6) and incubated at 37°C for 30 min; the supernatant was used for the VIN assay. The reaction was stopped by heating at 100°C for 5 min. After centrifugation, the released Glc was determined in the supernatant by the dianisidin method (Fermognost, Feinchemie Sebnitz, Germany).
SuSy was determined according to Dancer et al. (1990
The metabolites were determined after methanol extraction (80%). Suc was determined colorimetrically at 490 nm, hexoses at 535 nm according to Büttner et al. (1985
For immunolocalization 2- and 3-week-old tumor samples were fixed for 4 h at 22°C with 4% (w/v) paraformaldehyde or N(3-dimethylethylaminopropyl)-N-ethylcarbodiimide-HCl in 0.1x phosphate-buffered saline (PBS) containing 0.1% (v/v) Triton X-100 (0.1x PBS: 13.7 mM NaCl, 0.15 mM KH2PO4, 0.79 mM Na2HPO4, and 0.27 mM KCl, pH 7). After dehydration with a graded series of ethanol at 22°C, the tissue was imbedded overnight in Steedman's wax, a polyester with a low melting point (PEG 400 distearate in hexadecanol 9:1 [w/w]). Cross sections of 12 µm thickness were prepared with a cryomicrotome (Cryocut CM 3050, Leica) and collected on poly-L-Lys coated slides. The sections were dewaxed with decreasing ethanol concentrations, rinsed with 0.1x PBS, incubated with buffer for 30 min, and with 100% (v/v) methanol for 10 min at –20°C. For immunolabeling, the sections were incubated overnight with the polyclonal rabbit anti-SuSy serum, raised against a combination of Sh1 and Sus1 gene products extracted from whole maize (Zea mays) kernels (W64A x 182E) 22 d after pollination (Koch et al., 1992
Upon request, all novel materials described in this publication will be made available in a timely manner for noncommercial research purposes, subject to the requisite permission from any third-party owners of all parts of the material. Obtaining any permission will be the responsibility of the requestor.
We thank Sylvia Lenz, Ingrid Scheel (Darmstadt University of Technology) and Heike Weiner (University of Heidelberg) for valuable technical assistance and Prof. Ulrich Lüttge (Darmstadt University of Technology) for support and critical discussions. Received June 5, 2003; returned for revision July 14, 2003; accepted July 27, 2003.
Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.103.028142.
1 This work was supported by the Deutsch Forschungsgemeinschaft/Sonderforschungsbereich (grant no. 199 to C.I.U.). * Corresponding author; e-mail uleb{at}bio.tu-darmstadt.de; fax 49–6151–164630.
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ABSTRACT
RESULTS
ka, unpublished data). 
-aminocapronic acid, 2 mM DTT, 0.1% [v/v] Triton X-100, 1 mM phenylmethylsulfonyl fluoride, and 20 mg of polyvinylpolypyrrolidone). Extracts were purified over a Sephadex G-25 column in desalting buffer (50 mM HEPES, 12 mM MgCl2, and 2 mM DTT, pH 7.4). The incubation buffer contained 20 mM HEPES, pH 7.0, 100 mM Suc, and 4 mM UDP. The reaction was carried out in buffer containing 165 mM Gly, 4 mM MgCl2, 0.25 mM NAD, pH 8.7, and 0.07 units of UDP-Glc dehydrogenase. 
-fructosidase in carrot. Planta 195: 601–610
