| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
|
First published online March 14, 2008; 10.1104/pp.108.115956 Plant Physiology 147:1603-1618 (2008) © 2008 American Society of Plant Biologists
Sucrose Synthase Is Associated with the Cell Wall of Tobacco Pollen Tubes1,[W]Dipartimento Scienze Ambientali "G. Sarfatti," Università di Siena, 53100 Siena, Italy
Sucrose synthase (Sus; EC 2.4.1.13) is a key enzyme of sucrose metabolism in plant cells, providing carbon for respiration and for the synthesis of cell wall polymers and starch. Since Sus is important for plant cell growth, insights into its structure, localization, and features are useful for defining the relationships between nutrients, growth, and cell morphogenesis. We used the pollen tube of tobacco (Nicotiana tabacum) as a cell model to characterize the main features of Sus with regard to cell growth and cell wall synthesis. Apart from its role during sexual reproduction, the pollen tube is a typical tip-growing cell, and the proper construction of its cell wall is essential for correct shaping and direction of growth. The outer cell wall layer of pollen tubes consists of pectins, but the inner layer is composed of cellulose and callose; both polymers require metabolic precursors in the form of UDP-glucose, which is synthesized by Sus. We identified an 88-kD polypeptide in the soluble, plasma membrane and Golgi fraction of pollen tubes. The protein was also found in association with the cell wall. After purification, the protein showed an enzyme activity similar to that of maize (Zea mays) Sus. Distribution of Sus was affected by brefeldin A and depended on the nutrition status of the pollen tube, because an absence of metabolic sugars in the growth medium caused Sus to distribute differently during tube elongation. Analysis by bidimensional electrophoresis indicated that Sus exists as two isoforms, one of which is phosphorylated and more abundant in the cytoplasm and cell wall and the other of which is not phosphorylated and is specific to the plasma membrane. Results indicate that the protein has a role in the construction of the extracellular matrix and thus in the morphogenesis of pollen tubes.
Sucrose synthase (Sus; EC 2.4.1.13) is a key enzyme of Suc metabolism and catalyzes the reversible transformation of Suc into Fru and UDP-Glc. Although its common name suggests synthetic activity, Suc in phloem seems to be produced mainly by other enzyme complexes, such as Suc-P synthase and Suc-P hydrolase. The main activity of Sus, therefore, is restricted to tissues that metabolize Suc (Baroja-Fernandez et al., 2003  ; Konishi et al., 2004), and monitoring of its activity provides useful information about the transport and consumption of carbohydrates during the development of plant cells and tissues (Wittich and Willemse, 1999). Because of its critical function, Sus is localized in different cell structures, such as the cytoskeleton (Winter et al., 1998), cell membranes (Matic et al., 2004), and the tonoplast (Etxeberria and Gonzalez, 2003). The protein is present in two distinct forms. The soluble form of Sus is generally involved in the respiration process. The second form of Sus is associated with plasma membranes or cell walls and is generally involved in the synthesis of cell wall components, providing metabolites (usually UDP-Glc) for callose synthase and cellulose synthase (Amor et al., 1995). The association of Sus with cell membranes may be regulated by phosphorylation/dephosphorylation processes (Anguenot et al., 2006). This model is supported by evidence obtained with cotton fibers (Ruan et al., 1997), in which mutants deficient in fiber synthesis showed altered levels of Sus (Ruan and Chourey, 1998) and suppression of its gene activity inhibited fiber growth (Ruan et al., 2003). The enzyme has also been localized in association with thickenings of the secondary cell wall (Salnikov et al., 2003). Another example is Zinnia elegans, in which Sus is present at higher concentrations close to the plasma membrane and microtubules, just below the secondary wall thickenings (Salnikov et al., 2001). In maize (Zea mays), three distinct Sus genes have been identified. Two (Sh1 and Sus1) coded for two isoenzymes (SUS-SH1 and SUS1) that have a critical role in the synthesis of cellulose. The third gene (Sus2) encoded a protein necessary for the synthesis of starch precursors (Chourey et al., 1998). In roots of Triticum aestivum under hypoxic conditions, the enzyme activity of Sus increased considerably in the apical and central regions of roots and overlapped the deposition pattern of cellulose (Albrecht and Mustroph, 2003).
The pollen tube is a fundamental cell during sexual reproduction in higher plants and is characterized by fast apical growth that requires much energy. Pectins, cellulose, and callose are the main components of cell walls and are produced continuously to sustain pollen tube growth. During its development in the anther, pollen accumulates large quantities of carbohydrates, which constitute a large part of its dry weight (Pacini et al., 2006
The anisotropic development of pollen tubes is reflected in the nonuniform distribution of the cell wall components (Geitmann and Steer, 2006
Pollen tubes convert most of the energy stored in internal and external carbohydrates to generate intracytoplasmic movement and to construct the cell wall matrix. Intracellular movement promotes the progressive accumulation of organelles and molecules in pollen tubes. Proper construction of cell walls is fundamental for morphogenesis and directional growth. In an attempt to associate carbohydrate metabolism with the process of cell wall synthesis, we tested the hypothesis that Sus has a critical role in this relationship. Since Sus plays different roles in carbon metabolism and is a critical intersection for directing carbon to different sinks (Ruan et al., 1997
Control of Immunological Cross-Reactivity The cross-reactivity of anti-Sus was initially tested against different protein extracts. As the three antibodies (K2, K3, and K4) had almost the same pattern of cross-reactivity, Figure 1A only shows the cross-reactivity of K2. The antibody was tested on the soluble (S) and the membrane protein (M) fractions of whole flowering plants of Arabidopsis (lanes 1 and 2) and of leaves of maize (lanes 3 and 4), rice (Oryza sativa; lanes 5 and 6), and tobacco (lanes 7 and 8). In all cases, the antibody cross-reacted with an approximately 90-kD polypeptide in the soluble and membrane fractions.
The three antibodies were then assessed on the soluble and postnuclear supernatant (PNS) proteins of tobacco pollen tubes (Fig. 1B). Lanes 2 and 3 show the electrophoretic profiles of the two samples, while lanes 4 to 9 show the immunoblots with antibodies K2, K3, and K4. In all cases, we observed strong specific cross-reactivity against an 88-kD polypeptide.
To demonstrate that the cross-reacting 88-kD polypeptide had the typical biochemical properties of Sus, we first isolated the protein from tobacco pollen tubes. As a control, Sus was also isolated from maize kernels (Fig. 2 ). Maize Sus (Fig. 2A) was isolated through sequential chromatography on anion exchange chromatography (AEC), gel permeation chromatography (GPC), and hydrophobic interaction chromatography (HIC) columns. After fractionation by AEC, the K2 antibody identified two distinct pools, eluting at 10% and 50% elution buffer, respectively (approximately 100 and 500 mM KCl). The two pools, designated pool1-AEC and pool2-AEC (lanes 4 and 5 in the top panel), were processed separately by GPC. The resulting pools (pool1-GPC and pool2-GPC) eluted with an apparent molecular mass of 400 kD (lanes 6 and 7). The two protein pools were subsequently fractionated by HIC, which produced two final pools (pool1-HIC and pool2-HIC, lanes 8 and 9, respectively), both eluting at approximately 250 mM (NH4)2SO4. The purity of the two samples was estimated at approximately 85% and 90%, respectively, using Quantity One software. The bottom panel shows the corresponding immunoblot with K2 antibody on the fractions in the top panel.
The protocol used for maize kernels was adapted for Sus isolation from pollen tubes (Fig. 2B). In this case, we detected only a single cross-reacting protein. The top panel shows gel analysis of the main fractions. The fraction pool after AEC (pool-AEC, lane 3) eluted around 150 mM KCl and was further fractionated by GPC. Pool-GPC (lane 4) eluted at approximately 200 kD and was further fractionated by HIC, which yielded pool-HIC (lane 5). Unlike for the isolation of maize Sus, we performed an additional purification step by AEC on a Mini-Q column. The final pool (pool-Mini-Q) contained a single band at 88 kD, the purity of which was more than 95%. The bottom panel shows the corresponding immunoblot with K2 antibody on the fractions shown in the top panel.
The enzyme activity of Sus from pollen tubes was analyzed in the cleavage and synthesis pathways by quantifying Suc and Fru (Table I ). In the test, maize Sus was assayed to validate experimental conditions and compare enzyme properties. In the case of Suc cleavage, we found that no products were obtained in the absence of pollen Sus or when denatured enzyme was used. When active pollen Sus was used, Suc was degraded to almost 50% of its molar concentration. In contrast, the two forms of maize Sus were more active in the cleavage pathway. When pollen Sus was assayed in the synthesis direction, it only converted one-third of the initial Fru into Suc. The two forms of maize Sus catalyzed the conversion of greater amounts of Fru into Suc.
Subcellular Distribution of Pollen Sus The immunoblot analysis of Figure 1 revealed a cross-reacting band associated with the membrane fraction of pollen tubes. The subcellular distribution of Sus was consequently investigated by fractionating the pool of pollen tube organelles along a 15% to 65% linear Suc gradient and by probing each fraction with Sus antibodies. The 20 fractions showed different protein concentrations, with the highest protein contents usually recovered around fractions 12 to 13 (42% Suc; Fig. 3A ). Immunoblot analysis revealed that the 88-kD polypeptide elutes in fractions 8 to 11, corresponding to 34% to 42% Suc concentration (Fig. 3B), and a weaker signal was also found in fractions 4 to 6 (24%–28% Suc). These fractions were also analyzed for specific organelle markers (Fig. 3C). IDPase activity (a marker of Golgi apparatus and vesicles) was mostly detected in fractions 4 to 6, strictly corresponding to the smaller peak of the 88-kD polypeptide. A marker of rough endoplasmic reticulum (cytochrome c reductase) was found mainly in fractions 8 to 10, whereas vanadate-sensitive ATPase activity (a marker of plasma membrane) was detected in fractions 8 to 11, almost overlapping the second major peak of Sus. The cytochrome c oxidase activity (a marker of mitochondria) peaked around fractions 14 to 17.
Detection of Sus in Plasma Membrane and Cell Wall Sus is reported to be associated with the plasma membrane of plant cells. We investigated the association of pollen Sus with the plasma membrane by isolating a plasma membrane fraction by the two-aqueous-phase partitioning technique (Fig. 4A ). The Sus antibodies failed to detect any signal (apart from a faint band; dot) in purified plasma membrane (lane 3), whereas the antibodies identified the 88-kD polypeptide in the soluble (lane 1) and PNS (lane 2) samples. As a control, the same fractions were probed with the 6C6 antibody (which cross-reacts with a 77-kD plasma membrane protein of pollen tubes); the antibody recognized the protein in the plasma membrane fraction (lane 6) as well as in the PNS fraction (lane 5) but not among soluble proteins (lane 4).
We also evaluated the association of Sus with the pool of cell wall proteins from tobacco pollen tubes (Fig. 4B). Proteins were extracted from the cell wall by differential extraction with Triton X-100, NaCl, and SDS. The 88-kD polypeptide was found in the cell wall protein pool after extraction with Triton X-100 (lane 1). A clear signal was also detected in NaCl-extracted proteins (lane 2), and the strongest signal was found in the SDS-extracted protein pool (lane 3).
The previous results indicated that pollen Sus is associated with plasma membrane and Golgi compartments and with the cell wall. The enzyme assays indicated that the protein shares typical biochemical properties of the Sus family. To assess the variability of Sus isoforms with regard to the cellular compartments, we analyzed the total protein pool, the soluble fraction, PNS, and SDS-extracted cell wall proteins by bidimensional electrophoresis and immunoblot. The total protein extract (Fig. 5A ) showed two cross-reacting spots at 88 kD with a pI of 5.5 to 6.0 (arrow and arrowhead). The blot was overloaded to detect all possible isoforms. The cytoplasmic fraction contained an abundant protein pool in which the K2 antibody again recognized the two protein spots (Fig. 5B). Cross-reactivity was almost identical in the PNS pool (Fig. 5C). When the SDS-extracted cell wall proteins were analyzed, the antibody cross-reacted with two protein spots that were identical in molecular mass and pI (Fig. 5D). The relative intensity of the left spot (the more acidic one; arrowhead) was similar in all cases and was always less than that of the basic spot on the right (arrow). Unlike the acidic spot, the basic spot showed a relative intensity 10 times greater in the soluble fraction, being relatively similar in the PNS and cell wall pools. Control experiments were done by mixing different samples. Immunoblot of those combinations always provided the same spot pattern, suggesting that spot position was identical in all samples.
To investigate the phosphorylation status of Sus in different pollen tube fractions, we performed double two-dimensional (2-D) immunoblot analyses using a monoclonal anti-phospho-Ser antibody in combination with the polyclonal anti-Sus. Simultaneous visualization of the two immunological reactions was achieved using the DyeChrome Double Western Blot Stain Kit (see Supplemental Fig. S1 for analysis on one-dimensional (1-D) gels and phosphoprotein standards). When tested on cytosolic proteins (Fig. 5E), two spots were detected by anti-Sus (top panel) but only one spot was also labeled by anti-phospho-Ser (middle panel), as shown by the overlap of the two pseudocolored images (bottom panel). The vesicle sample (Fig. 5F, corresponding to fractions 4–6 in Fig. 3) also showed a couple of spots when labeled by anti-Sus (top); again, only one spot was also labeled by anti-phospho-Ser (middle), as confirmed by the overlap (bottom). The SDS-extracted cell wall proteins (Fig. 5G) showed a similar pattern, as only one of the two spots identified by anti-Sus (top) was also labeled by anti-phospho-Ser (middle, with overlap in the bottom panel). The membrane sample from fractions 8 to 11 of Figure 3 showed a dissimilar pattern (Fig. 5H); anti-Sus labeled a single spot (top) that was not labeled by anti-phospho-Ser (middle), as confirmed by overlapping pseudocolored images (bottom). The big red dot in Figure 8H (middle and bottom panels) is a phosphorylated plasma membrane protein that was not recognized by anti-Sus (as shown in the top panel); this means that the protein spot was not Sus but an unrelated protein that migrates very close to Sus.
In control experiments, samples were pretreated with 20 units of alkaline phosphatase, which removed almost all signals detected by anti-phospho-Ser. The two Sus spots were not labeled after staining with monoclonal anti-phospho-Thr and anti-phospho-Tyr (data not shown).
The distribution of Sus in tobacco pollen tubes was analyzed by immunofluorescence microscopy with K2, K3, and K4 antibodies. Results were extremely similar for all three antibodies. In germinating pollen grains (Fig. 6A1 ), labeling was localized mainly in the tube apex (arrow). A confocal analysis in the central focal plane of similar tubes confirmed that labeling was not only cortical but also detected in the tip domain (Fig. 6A2, arrow); in this specific pollen tube, labeling started to accumulate at the cell periphery (arrowhead). A pollen tube at the start of germination is shown in Supplemental Figure S2A. After elongation (tube length, 50–100 µm), the protein was distributed along the cell border in the apical domain (Fig. 6B, arrow); the signal also appeared and increased progressively in the basal domain (arrowhead). At 100 to 150 µm of pollen tube length, the signal at the tube border was stronger and extended toward the pollen grain (Fig. 6C, arrow). The staining pattern along the cell border was continuous (neither spotted nor clustered), and the pollen tube cytoplasm always showed weak diffuse staining. After 3 h of growth (Fig. 6D; length, approximately 200 µm), peripheral apical staining was still intense and remained constant even after longer elongation times. Fluorescence intensity was measured along the curvature of the pollen tube from the extreme tip down to the cell border in 200-µm-long pollen tubes (n = 20; Fig. 6E, thick black line); anti-Sus labeling was greater in the first 20 µm of the tubes, decreasing toward the base. As a control, preimmune serum provided no staining (data not shown).
Measurement of anti-Sus labeling in 200-µm-long tubes after brefeldin A treatment (Fig. 6E, thin black line; see next paragraph for details) showed loss of high-intensity staining in the apical domain. The two major cell wall polymers (callose and cellulose) were also measured in similar tubes after labeling with Aniline Blue and Calcofluor White, respectively. Callose (Fig. 6E, thick dark gray line) showed a lower intensity at the tip and increased progressively 10 to 20 µm from the tube apex, reaching a maximum of approximately 30 µm. Cellulose (thick light gray line) was detected in the apical domain and showed a similar pattern to callose except for a lower rate of increase.
More information on the distribution of pollen Sus was obtained by immunoelectron microscopy. In this case, Sus was localized in the cytoplasm and more consistently between the cytoplasm and the cell wall, presumably in the plasma membrane (Fig. 7A
, arrowhead), reminiscent of its pattern in cotton fiber (Ruan, 2007
When we assessed the distribution of Sus in pollen tubes treated with the protein transport inhibitor brefeldin A (Nebenfuhr et al., 2002 ), the control pollen tubes showed typical cortical staining (Fig. 8A1
), whereas those treated with brefeldin A for 20 min (Fig. 8A2) showed a reduction in apical staining and a series of circular structures near the cell border (arrowheads). Typical cortical staining was partly maintained (arrow). In pollen tubes treated with brefeldin A for 30 min (Fig. 8A3), we observed the appearance of indefinite structures (possibly of membranous origin) in the cytoplasm (arrows). When analyzed by immunogold electron microscopy with anti-Sus antibody, those tubes showed that Sus was associated with large vesicular structures measuring approximately 350 nm (Fig. 8B1). Comparison of our results with the literature (Geitmann et al., 1996) suggested that the unidentified structures may arise from dictyosomes or from the fusion of secretory vesicles. At the same time, association of Sus with the cell wall was reduced drastically (Fig. 8, B2 and B3).
When we tested the effect of different osmotica on the distribution of Sus, the growth of pollen tubes germinating in medium containing 0.39 M glycerol or 0.1 M polyethylene glycol (PEG)-400 (O'Kelley, 1955
Since glycerol in the growth medium altered the distribution of Sus in pollen tubes, we assessed the difference by quantitative immunoblot analysis of proteins from different cell compartments: cytoplasmic, PNS, and SDS-extracted cell wall proteins (Fig. 9A ). The assay was performed on pollen tubes germinated in glycerol- or Suc-based medium for different times (0, 30, 60, 90, 180, and 360 min). In the case of glycerol, the germination times were only 0, 30, 60, and 90 min because the tubes showed distorted growth after 90 min. When the immunoblot results were evaluated quantitatively (Fig. 9B), we found that the quantity of cytoplasmic Sus (top panel) showed a peak around 30 min in Suc-based medium (BK-S, black bars) and then decreased gradually during tube growth. When pollen tubes were grown in glycerol-based medium (BK-G, white bars), cytoplasmic Sus increased visibly with germination time. PNS-associated Sus (middle panel) was higher before germination in Suc-based medium, then remained almost steady at a lower level. On the contrary, in glycerol medium, the membrane-associated enzyme increased over 90 min. Cell wall-associated Sus (bottom panel) increased progressively with germination time in Suc medium, while in glycerol medium it remained at significantly lower levels. These results indicate that the concentration of cell wall-associated Sus increased progressively in Suc-based medium, while the amount of membrane-associated protein decreased correspondingly; in the case of glycerol-based medium, the quantity of cell wall-associated enzyme remained low while Sus appeared to accumulate in the membrane and soluble fractions.
In this study, we attempted to characterize Sus in tobacco pollen tubes using different methodological approaches (biochemical, cytological, immunological, and proteomic). Tobacco pollen tubes were found to contain a Sus-like protein of 88 kD. The polypeptide shares several features with Sus of other species and is mainly associated with the pollen tube cell wall. Identification of Sus was based on cross-reactivity with antibodies against Sus of other species; however, the specific pattern of cross-reactivity coupled with enzyme assays indicated that the pollen protein belonged to the Sus family.
The first approach to the characterization of Sus in tobacco pollen tubes was immunological. Analysis of leaf extracts from different plants confirmed the wide cross-reactivity of the antibodies K2, K3, and K4, which identified bands with similar molecular mass in flowering plants of Arabidopsis and in leaves of maize, rice, and tobacco. This finding is not surprising, because the amino acid sequence of Sus is highly conserved. A comparison among Sus from the four species indicated a similarity of approximately 70% to 80%. The immunological result was weaker in Arabidopsis; however, this finding is in agreement with the literature, because in some cases (like in rosette leaves of Arabidopsis) Sus is very low (Bieniawska et al., 2007
The native molecular mass of the protein was calculated by GPC on Sephacryl S-300 columns. Comparison with mass standards (β-amylase, 210 kD; bovine serum albumin, 66 kD; cytochrome c, 12 kD) indicated a molecular mass of 200 kD for pollen tube Sus, suggesting that the protein forms a dimer of two 88-kD subunits. The literature indicates that Sus usually exists as a tetramer with a native molecular mass between 540 kD (Gross and Pharr, 1982
The 88-kD band resolved into two distinct spots by proteomic assay. Plants generally express different isoforms of Sus. For example, the genome of Arabidopsis contains six genes that are expressed differentially in tissues and during plant development (Bieniawska et al., 2007
Since the two isoforms are not separated by chromatographic techniques, the enzyme activity of Sus was tested on a mixture of both. The results indicate that pollen Sus functions in both directions (Suc cleavage and synthesis) as in pea (Pisum sativum; Dejardin et al., 1997
Pollen Sus was localized in two distinct protein pools: the soluble and membrane pools. These results are in agreement with the literature. Soluble Sus has been described extensively in different plants (Winter and Huber, 2000 We are convinced that Sus located in the cell wall was not contaminated by plasma membrane-associated Sus. The cell wall protein fraction was the result of extensive washing necessary to remove contaminating proteins. Additional Triton washing also separated proteins that bind to cell wall-associated membranes. In addition, most cell wall-associated Sus is recovered after washing with SDS (indicating strong binding affinity with the cell wall). The association of Sus with the plasma membrane (or generally with membranes) was supported by the immunolocalization experiments. A partial sequence of a 1,265-bp mRNA coding for the putative Sus of tobacco pollen tubes was obtained in our laboratory (accession no. EU148354). The sequence has 96% identity with tobacco mRNA for Sus (partial coding sequence) and 89% identity with tomato and potato mRNA for Sus.
Our results indicate that pollen Sus is also associated with the Golgi membranes. Association of Sus with the Golgi fraction has already been described in maize (Buckeridge et al., 1999
Phosphorylation analysis indicated that the phosphorylated Sus isoform is relatively more abundant in the cytoplasm and in cell walls, while the nonphosphorylated isoform is associated with the plasma membrane. This evidence suggests that the protein is not subject to extensive posttranslational modifications during transfer to the cell wall, whereas dephosphorylation of Ser residues is necessary for the association of Sus with the plasma membrane. The literature does not contain any indications about posttranslational modification of cell wall-associated Sus, only about the phosphorylation mechanisms presumed to regulate the association of Sus with the plasma membrane (Komina et al., 2002
The different distribution of Sus between pollen tubes grown in Suc- and glycerol/PEG-based media suggests that the type of osmoticum influences the expression of Sus or its distribution within the cell. Glycerol and PEG cannot be used by the pollen tube as a carbon source, and pollen tube elongation ceases after 1.5 to 2 h of growth in these media. This is indirect confirmation that pollen tubes mainly utilize internal sugars in initial growth stages and external carbohydrates (in growth media or in the style) during later growth stages (Ylstra et al., 1998
Comparing our results with the literature allows us to formulate some hypotheses on Sus activity in pollen tubes. The association of Sus with the plasma membrane and cell wall suggests that the enzyme is involved in the construction of cellulose microfibrils and callose. This is largely supported by the current literature (Chourey et al., 1998
Another interesting possibility is that Sus has a role in the synthesis of xyloglucans. Sus has already been localized in Golgi membranes (Buckeridge et al., 1999
On the basis of our observations, we propose a model in which external Suc is converted into Glc and Fru through the activity of a cell wall invertase (Ylstra et al., 1998
Reagents
Tobacco (Nicotiana tabacum) plants and other species used in this research were grown in the Botanical Gardens of Siena University. Three different batches (K2, K3, and K4) of Sus antiserum were kindly provided by Dr. Wolfgang Werr (University of Köln, Institut für Entwicklungsbiologie); the characteristics of the antibody are described in the literature (Heinlein and Starlinger, 1989
Tobacco pollen was progressively thawed from storage at –20°C and hydrated overnight in a humid chamber. It was germinated in BK medium [1.62 mM H3BO3, 1.25 mM Ca(NO3)2·4H2O, 2.97 mM KNO3, and 1.65 mM MgSO4·7H2O; Brewbaker and Kwack, 1963
After germination for 3 h, the pollen was collected by centrifugation at 1,000g for 5 min at 25°C and was then washed twice with BRB25 buffer (25 mM HEPES, pH 7.5, 2 mM EGTA, and 2 mM MgCl2) + 15% Suc. The pollen was resuspended in lysis buffer (BRB25 containing 2 mM dithiothreitol [DTT], 1 mM phenylmethylsulfonyl fluoride [PMSF], 10 µL/mL protease inhibitors, 1 mM NaN3, and 10% mannitol) and then lysed on ice using a motor-driven Potter-Elvehjem homogenizer. After lysis, samples were centrifuged at 1,000g for 10 min at 4°C, and the supernatant was centrifuged again at 100,000g for 45 min at 4°C over a 20% (w/v) Suc cushion. The supernatant (soluble fraction) was collected. The pellet, containing the membrane fraction of pollen tubes (PNS), was resuspended in resuspension buffer (BRB25 plus 10% mannitol, 1 mM DTT, and 1 mM PMSF) for subsequent procedures.
Proteins were extracted from leaves of maize (Zea mays), rice (Oryza sativa), and tobacco and from whole flowering plants of Arabidopsis (Arabidopsis thaliana). Approximately 1 to 3 g of leaves (or plants in the case of Arabidopsis) was ground in liquid nitrogen, and the powder was mixed 1:2 with extraction buffer (0.1 M Tris-HCl, pH 8, 10 mM MgCl2, 2 mM EGTA, 1% mannitol, 1 mM PMSF, 10 µL/mL protease inhibitors, 1 mM DTT, and 1% polyvinylpolypyrrolidone). Samples were incubated on ice for 15 min and centrifuged at 1,000g for 10 min at 4°C. The pellet was discarded, and the supernatant was centrifuged at 100,000g for 1 h at 4°C. The resulting supernatant (containing soluble proteins) and pellet (containing membrane-bound organelles) were resuspended directly in sample buffer for 1-D electrophoresis (Laemmli, 1970
Sus was isolated from maize kernels using a combination of chromatography techniques performed with an AKTA Purifier system (GE HealthCare). The method is based on protocols used for the isolation of Sus from different plant species (Sebkova et al., 1995 Sus was isolated from 3-h-old tobacco pollen tubes using the method described above for maize Sus but with a final additional separation step on a Mini-Q column (GE HealthCare). Briefly, after HIC, the fraction pool was desalted using a PC3.2/10 Fast Desalting column (GE HealthCare), then loaded on the Mini-Q column, which was eluted at 1 mL/min with 20 column volumes of the same buffers as for the AEC on Resource-Q. Fractions positive to immunoblot were pooled and dialyzed overnight against storage buffer.
The synthesis direction of Sus is enhanced by alkaline pH (>8; Romer et al., 2004
The synthesis pathway was assayed as outlined in the literature (Dejardin et al., 1997 Determination of Suc and Fru in all samples was performed by HPLC analysis on a Waters LC1 system using a Waters 2410 refractometric detector. The column was a Supelco C-611 (300 x 7.8 mm, 10-µm pore size) at a constant temperature of 60°C. The mobile phase consisted of 10–4 M NaOH at a flow rate of 0.5 mL/min. The volume of injected samples was 20 µL. The instrument was calibrated with known amounts of Suc and Fru, separately or together. The amount of sugars produced in the assays was calculated by integrating the corresponding peak areas of the chromatograms.
Organelles (the PNS) from 3-h-old pollen tubes were fractionated on 15% to 65% linear Suc gradients in gradient buffer (50 mM imidazole, pH 7.5, 2 mM EDTA, 1 mM PMSF, and 1 mM DTT) using a Hoefer SG30 gradient former (GE HealthCare). The PNS was loaded on top of 10-mL Suc gradients prepared in 13-mL Beckman tubes (Beckman Coulter). Samples were centrifuged in a Beckman Optima L-80 ultracentrifuge at 100,000g for 8 h (4°C) and separated into 20 fractions of 0.5 mL each. Fractions were assayed for protein concentration, Sus, and specific organelle markers.
The plasma membrane fraction of tobacco pollen tubes grown for 3 h was isolated according to known methods (Carlson and Chourey, 1996
Cell wall proteins from 3-h-old tobacco pollen tubes were isolated as described in the literature (Li et al., 1983
Indirect immunofluorescence microscopy was performed according to Del Casino et al. (1993)
Immunogold labeling on tobacco pollen tubes was performed according to the protocol described by Li et al. (1995)
The relative quantity of Sus was estimated in three different cell compartments during pollen tube growth. We considered the cytoplasmic (soluble) fraction, the PNS fraction, and the SDS-extracted cell wall proteins of pollen tubes grown for fixed times (0, 30, 60, 90, 180, and 360 min) in both Suc- and glycerol-based media. These fractions were collected as described previously, and then equal amounts of proteins were immunoblotted with Sus antibody. Signals from at least three different experiments were quantified using Quantity One software.
1-D electrophoresis was carried out as described (Laemmli, 1970 For 2-D electrophoresis, samples were prepared using the ReadyPrep 2-D Cleanup Kit from Bio-Rad. Total proteins from germinated pollen tubes were extracted using the Total Protein Extraction Kit (Bio-Rad) and the ReadyPrep 2-D Cleanup Kit. Proteins were separated in the first dimension by isoelectric focusing using a Multiphor II apparatus (GE HealthCare) according to the manufacturer's instructions. Immobiline DryStrip gels (7 cm long; GE HealthCare) contained a pH gradient of 3 to 10 (for the first screening) or 4 to 7 (for sharper separation). Gels were run at 200 V (2 mA, 5 W) for 1 min, 3,500 V for 1.5 h, and 3,500 V for a further 1.5 h. Samples were included in the rehydration buffer. After running, gels were equilibrated in equilibration buffer (prepared as indicated by the manufacturer) for 15 min or, alternatively, frozen and stored. Proteins were separated in the second dimension by SDS gel electrophoresis on a Bio-Rad Mini-Protean II using 1.0-mm-thick 10% acrylamide gels. At least three gels were run for each protein fraction. Parallel unstained gels were blotted onto nitrocellulose membranes and probed with antibodies.
Immunoblotting was performed as described (Towbin et al., 1979
For visualizing the phosphorylation status of Sus, we performed double immunoblot experiments using the DyeChrome Double Western Blot Stain Kit (Invitrogen). For these experiments, extraction of protein samples was performed as described previously with the addition of 1 mM Na3VO4 in all buffers. Proteins were blotted on polyvinylidene difluoride membranes and labeled as described in the Double Western Blot Kit manual. Proteins were simultaneously labeled with the Sus antibody and with a monoclonal antibody against phospho-Ser (Sigma-Aldrich). In control experiments, samples were prepared without 1 mM Na3VO4 but were pretreated with 20 units of alkaline phosphatase (Sigma-Aldrich; Tanase et al., 2002
Images of Coomassie Brilliant Blue-stained gels were captured using the Fluor-S MultiImager from Bio-Rad. The molecular mass of proteins was evaluated using Quantity One software (Bio-Rad). The immunoblot membranes were developed directly in the Fluor-S MultiImager using Quantity One software; images of blots were captured at different times and then superimposed on images of prestained standards obtained using the same membrane. Protein content was analyzed in each blot (from three different experiments) using the Volume Analysis Report of Quantity One; the volumes of reactive bands were calculated as Adjusted Volumes (volume minus the background volume of each blot). Analysis of 2-D gels was done with PDQuest software (Bio-Rad) after storing gel images with the Fluor-S MultiImager, while pI and molecular mass were estimated by comparison with 2-D standards (Bio-Rad). At least three different blots from independent experiments were analyzed. Visualization of DyeChrome double staining was achieved with the Fluor-S MultiImager using UV epifluorescence illumination of membranes and 530BP and 610LP filters. Images were compared using the MultiViewer command of PD Quest software.
Protein concentration was evaluated using the colorimetric method of Bradford (1976) The GenBank accession number for the partial 1,265-bp mRNA for Sus of tobacco pollen tubes is EU148354.
The following materials are available in the online version of this article.
We express our gratitude to Dr. Yong-Ling Ruan (Commonwealth Scientific and Industrial Research Organization Plant Industry, Canberra, Australia) for critically revising and commenting on the manuscript and to Dr. Massimo Nepi, Mr. Massimo Guarnieri, and Dr. Daniele Artesi (Dipartimento Scienze Ambientali, University of Siena) for the analysis of sugars by HPLC. We also thank Prof. Wolfgang Werr (Institut für Entwicklungsbiologie, Koln University, Koln, Germany) for kindly providing the antibodies against Sus. We are grateful to the gardeners of the Dipartimento di Scienze Ambientali of Siena University for growing plants and for pollen collection. Received January 7, 2008; accepted March 9, 2008; published March 14, 2008.
1 This work was supported by grants under the Human Frontier Science Program (http://www.hfsp.org/) and the Research Athenaeum Program of Siena University. 
2 Present address: Plant Cytology and Morphology, Wageningen University, Arboretumlaan 4, 6703 BD Wageningen, The Netherlands. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Giampiero Cai (cai{at}unisi.it).
[W] The online version of this article contains Web-only data. www.plantphysiol.org/cgi/doi/10.1104/pp.108.115956 * Corresponding author; e-mail cai{at}unisi.it.
Albrecht G, Mustroph A (2003) Localization of sucrose synthase in wheat roots: Increased in situ activity of sucrose synthase correlates with cell wall thickening by cellulose deposition under hypoxia. Planta 217: 252–260[Web of Science][Medline] Amor Y, Haigler CH, Johnson S, Wainscott M, Delmer DP (1995) A membrane-associated form of sucrose synthase and its potential role in synthesis of cellulose and callose in plants. Proc Natl Acad Sci USA 92: 9353–9357 Anderson JR, Barnes WS, Bedinger P (2002) 2,6-Dichlorobenzonitrile, a cellulose biosynthesis inhibitor, affects morphology and structural integrity of petunia and lily pollen tubes. J Plant Physiol 159: 61–67[CrossRef][Web of Science] Anguenot R, Nguyen-Quoc B, Yelle S, Michaud D (2006) Protein phosphorylation and membrane association of sucrose synthase in developing tomato fruit. Plant Physiol Biochem 44: 294–300[CrossRef][Web of Science][Medline] Aoki N, Hirose T, Scofield GN, Whitfeld PR, Furbank RT (2003) The sucrose transporter gene family in rice. Plant Cell Physiol 44: 223–232 Baroja-Fernandez E, Munoz FJ, Saikusa T, Rodriguez-Lopez M, Akazawa T, Pozueta-Romero J (2003) Sucrose synthase catalyzes the de novo production of ADPglucose linked to starch biosynthesis in heterotrophic tissues of plants. Plant Cell Physiol 44: 500–509 Bieniawska Z, Paul Barratt DH, Garlick AP, Thole V, Kruger NJ, Martin C, Zrenner R, Smith AM (2007) Analysis of the sucrose synthase gene family in Arabidopsis. Plant J 49: 810–828[CrossRef][Web of Science][Medline] Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248–254[CrossRef][Web of Science][Medline] Brewbaker JL, Kwack BH (1963) The essential role of calcium ion in pollen germination and pollen tube growth. Am J Bot 50: 859–865[CrossRef][Web of Science] Brownfield L, Ford K, Doblin MS, Newbigin E, Read S, Bacic A (2007) Proteomic and biochemical evidence links the callose synthase in Nicotiana alata pollen tubes to the product of the NaGSL1 gene. Plant J 52: 147–156[CrossRef][Web of Science][Medline] Buckeridge MS, Vergara CE, Carpita NC (1999) The mechanism of synthesis of a mixed-linkage (1 Cai G, Moscatelli A, Del Casino C, Chevrier V, Mazzi M, Tiezzi A, Cresti M (1996) The anti-centrosome mAb 6C6 reacts with a plasma membrane-associated polypeptide of 77-kDa from the Nicotiana tabacum pollen tubes. Protoplasma 190: 68–78[CrossRef][Web of Science] Cai G, Ovidi E, Romagnoli S, Vantard M, Cresti M, Tiezzi A (2005) Identification and characterization of plasma membrane proteins that bind to microtubules in pollen tubes and generative cells of tobacco. Plant Cell Physiol 46: 563–578 Cardenas L, McKenna ST, Kunkel JG, Hepler PK (2006) NAD(P)H oscillates in pollen tubes and is correlated with tip growth. Plant Physiol 142: 1460–1468 Carlson SJ, Chourey PS (1996) Evidence for plasma membrane-associated forms of sucrose synthase in maize. Mol Gen Genet 252: 303–310[Web of Science][Medline] Chourey PS, Taliercio EW, Carlson SJ, Ruan YL (1998) Genetic evidence that the two isozymes of sucrose synthase present in developing maize endosperm are critical, one for cell wall integrity and the other for starch biosynthesis. Mol Gen Genet 259: 88–96[CrossRef][Web of Science][Medline] Cresti M, VanWent JL (1976) Callose deposition and plug formation in Petunia pollen tubes in situ. Planta 133: 35–40[CrossRef][Web of Science] Dejardin A, Rochat C, Maugenest S, Boutin JP (1997) Purification, characterization and physiological role of sucrose synthase in the pea seed coat (Pisum sativum L.). Planta 201: 128–137[CrossRef][Web of Science][Medline] Del Casino C, Li Y, Moscatelli A, Scali M, Tiezzi A, Cresti M (1993) Distribution of microtubules during the growth of tobacco pollen tubes. Biol Cell 79: 125–132[CrossRef][Web of Science] Derksen J, Knuiman B, Hoedemaekers K, Guyon A, Bonhomme S, Pierson ES (2002) Growth and cellular organization of Arabidopsis pollen tubes in vitro. Sex Plant Reprod 15: 133–139[CrossRef] Derksen J, Li YQ, Knuiman B, Geurts H (1999) The wall of Pinus sylvestris L. pollen tubes. Protoplasma 208: 26–36 Duncan KA, Huber SC (2007) Sucrose synthase oligomerization and F-actin association are regulated by sucrose concentration and phosphorylation. Plant Cell Physiol 48: 1612–1623 Echt CS, Chourey PS (1985) A comparison of two sucrose synthatase isozyme from normal and shrunken-1 maize. Plant Physiol 79: 530–536 Etxeberria E, Gonzalez P (2003) Evidence for a tonoplast-associated form of sucrose synthase and its potential involvement in sucrose mobilization from the vacuole. J Exp Bot 54: 1407–1414 Ferguson C, Teeri TT, Siika-aho M, Read SM, Bacic A (1998) Location of cellulose and callose in pollen tubes and grains of Nicotiana tabacum. Planta 206: 452–460[CrossRef][Web of Science] Geitmann A, Steer M (2006) The architecture and properties of the pollen tube cell wall. In R Malho, ed, The Pollen Tube. Springer-Verlag, Berlin, pp 177–200 Geitmann A, Wojciechowicz K, Cresti M (1996) Inhibition of intracellular pectin transport in pollen tubes by monensin, brefeldin A and cytochalasin D. Bot Acta 109: 373–381[Web of Science] Gross KC, Pharr DM (1982) Cucumber fruit sucrose synthetase isozymes. Phytochemistry 21: 1241–1244[CrossRef][Web of Science] Haigler CH, Ivanova-Datcheva M, Hogan PS, Salnikov VV, Hwang S, Martin K, Delmer DP (2001) Carbon partitioning to cellulose synthesis. Plant Mol Biol 47: 29–51[CrossRef][Web of Science][Medline] Hardin SC, Duncan KA, Huber SC (2006) Determination of structural requirements and probable regulatory effectors for membrane association of maize sucrose synthase 1. Plant Physiol 141: 1106–1119 Hardin SC, Winter H, Huber SC (2004) Phosphorylation of the amino terminus of maize sucrose synthase in relation to membrane association and enzyme activity. Plant Physiol 134: 1427–1438 Heinlein M, Starlinger P (1989) Tissue- and cell-specific expression of the two sucrose synthase isoenzymes in developing maize kernels. Mol Gen Genet 215: 441–446[CrossRef][Web of Science] Herrero M, Dickinson HG (1980) Pollen tube growth following compatible and incompatible intraspecific pollinations in Petunia hybrida. Planta 148: 217–221[CrossRef][Web of Science] Hong Z, Zhang Z, Olson JM, Verma DP (2001) A novel UDP-glucose transferase is part of the callose synthase complex and interacts with phragmoplastin at the forming cell plate. Plant Cell 13: 769–780 Huang DY, Wang AY (1998) Purification and characterization of sucrose synthase isozymes from etiolated rice seedlings. Biochem Mol Biol Int 46: 107–113[Web of Science][Medline] Johnson MA, von Besser K, Zhou Q, Smith E, Aux G, Patton D, Levin JZ, Preuss D (2004) Arabidopsis hapless mutations define essential gametophytic functions. Genetics 168: 971–982 Karrer EE, Rodriguez RL (1992) Metabolic regulation of rice alpha-amylase and sucrose synthase genes in planta. Plant J 2: 517–523[Web of Science][Medline] Komina O, Zhou Y, Sarath G, Chollet R (2002) In vivo and in vitro phosphorylation of membrane and soluble forms of soybean nodule sucrose synthase. Plant Physiol 129: 1664–1673 Konishi T, Ohmiya Y, Hayashi T (2004) Evidence that sucrose loaded into the phloem of a poplar leaf is used directly by sucrose synthase associated with various beta-glucan synthases in the stem. Plant Physiol 134: 1146–1152 Kropf DL, Bisgrove SR, Hable WE (1998) Cytoskeletal control of polar growth in plant cells. Curr Opin Cell Biol 10: 117–122[CrossRef][Web of Science][Medline] Labarca C, Loewus F (1972) The nutritional role of pistil exudate in pollen tube wall formation in Lilium longiflorum. I. Utilization of injected stigmatic exudate. Plant Physiol 50: 7–14 Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685[CrossRef][Web of Science][Medline] Lennon KA, Lord EM (2000) In vivo pollen tube cell of Arabidopsis thaliana. I. Tube cell cytoplasm and wall. Protoplasma 214: 45–56[CrossRef][Web of Science] Li H, Bacic A, Read SM (1999) Role of a callose synthase zymogen in regulating wall deposition in pollen tubes of Nicotiana alata Link et Otto. Planta 208: 528–538[CrossRef][Web of Science] Li YQ, Croes AF, Linskens HF (1983) Cell-wall proteins in pollen and roots of Lilium longiflorum: extraction and partial characterization. Planta 158: 422–427[CrossRef][Web of Science] Li YQ, Faleri C, Geitmann A, Zhang HQ, Cresti M (1995) Immunogold localization of arabinogalactan proteins, unesterified and esterified pectins in pollen grains and pollen tubes of Nicotiana tabacum L. Protoplasma 189: 26–36[CrossRef][Web of Science] Lovy-Wheeler A, Cardenas L, Kunkel JG, Hepler PK (2006) Differential organelle movement on the actin cytoskeleton in lily pollen tubes. Cell Motil Cytoskeleton 64: 217–232[CrossRef][Web of Science] Matic S, Akerlund HE, Everitt E, Widell S (2004) Sucrose synthase isoforms in cultured tobacco cells. Plant Physiol Biochem 42: 299–306[CrossRef][Web of Science][Medline] Munoz P, Norambuena L, Orellana A (1996) Evidence for a UDP-glucose transporter in Golgi apparatus-derived vesicles from pea and its possible role in polysaccharide biosynthesis. Plant Physiol 112: 1585–1594[Abstract] Nebenfuhr A, Ritzenthaler C, Robinson DG (2002) Brefeldin A: deciphering an enigmatic inhibitor of secretion. Plant Physiol 130: 1102–1108 O'Kelley JC (1955) External carbohydrates in growth and respiration of pollen tubes in vitro. Am J Bot 42: 322–327[CrossRef][Web of Science] Pacini E, Guarnieri M, Nepi M (2006) Pollen carbohydrates and water content during development, presentation, and dispersal: a short review. Protoplasma 228: 73–77[CrossRef][Web of Science][Medline] Parre E, Geitmann A (2005) More than a leak sealant: the mechanical properties of callose in pollen tubes. Plant Physiol 137: 274–286 Parton RM, Fischer-Parton S, Watahiki MK, Trewavas AJ (2001) Dynamics of the apical vesicle accumulation and the rate of growth are related in individual pollen tubes. J Cell Sci 114: 2685–2695[Web of Science][Medline] Robinson DG, Hinz G (2001) Organelle isolation. In C Hawes, B Satiat-Jeunemaitre, eds, Plant Cell Biology. Oxford University Press, Oxford, pp 295–323 Romer U, Schrader H, Gunther N, Nettelstroth N, Frommer WB, Elling L (2004) Expression, purification and characterization of recombinant sucrose synthase 1 from Solanum tuberosum L. for carbohydrate engineering. J Biotechnol 107: 135–149[CrossRef][Web of Science][Medline] Ross HA, Davies HV (1992) Purification and characterization of sucrose synthase from the cotyledons of Vicia faba L. Plant Physiol 100: 1008–1013 Ruan Y (2007) Rapid cell expansion and cellulose synthesis regulated by plasmodesmata and sugar: insights from the single-celled cotton fibre. Funct Plant Biol 34: 1–10[CrossRef] Ruan YL, Chourey PS (1998) A fiberless seed mutation in cotton is associated with lack of fiber cell initiation in ovule epidermis and alterations in sucrose synthase expression and carbon partitioning in developing seeds. Plant Physiol 118: 399–406 Ruan YL, Chourey PS, Delmer DP, Perez-Grau L (1997) The differential expression of sucrose synthase in relation to diverse patterns of carbon partitioning in developing cotton seed. Plant Physiol 115: 375–385[Abstract] Ruan YL, Llewellyn DJ, Furbank RT (2003) Suppression of sucrose synthase gene expression represses cotton fiber cell initiation, elongation, and seed development. Plant Cell 15: 952–964 Rutten TL, Knuiman B (1993) Brefeldin A effects on tobacco pollen tubes. Eur J Cell Biol 61: 247–255[Web of Science][Medline] Salnikov VV, Grimson MJ, Delmer DP, Haigler CH (2001) Sucrose synthase localizes to cellulose synthesis sites in tracheary elements. Phytochemistry 57: 823–833[CrossRef][Web of Science][Medline] Salnikov VV, Grimson MJ, Seagull RW, Haigler CH (2003) Localization of sucrose synthase and callose in freeze-substituted secondary-wall-stage cotton fibers. Protoplasma 221: 175–184[CrossRef][Web of Science][Medline] Schafer WE, Rohwer JM, Botha FC (2005) Partial purification and characterisation of sucrose synthase in sugarcane. J Plant Physiol 162: 11–20[CrossRef][Web of Science][Medline] Schlupmann H, Bacic A, Read SM (1994) Uridine diphosphate glucose metabolism and callose synthesis in cultured pollen tubes of Nicotiana alata Link et Otto. Plant Physiol 105: 659–670[Abstract] Sebkova V, Unger C, Hardegger M, Sturm A (1995) Biochemical, physiological, and molecular characterization of sucrose synthase from Daucus carota. Plant Physiol 108: 75–83[Abstract] Tanase K, Shiratake K, Mori H, Yamaki S (2002) Changes in the phosphorylation state of sucrose synthase during development of Japanese pear fruit. Physiol Plant 114: 21–26[CrossRef][Medline] Tanase K, Yamaki S (2000) Purification and characterization of two sucrose synthase isoforms from Japanese pear fruit. Plant Cell Physiol 41: 408–414 Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76: 4350–4354 Turner A, Bacic A, Harris PJ, Read SM (1998) Membrane fractionation and enrichment of callose synthase from pollen tubes of Nicotiana alata Link et Otto. Planta 205: 380–388[CrossRef][Web of Science][Medline] Winter H, Huber JL, Huber SC (1997) Membrane association of sucrose synthase: changes during the graviresponse and possible control by protein phosphorylation. FEBS Lett 420: 151–155[CrossRef][Web of Science][Medline] Winter H, Huber JL, Huber SC (1998) Identification of sucrose synthase as an actin-binding protein. FEBS Lett 430: 205–208[CrossRef][Web of Science][Medline] Winter H, Huber SC (2000) Regulation of sucrose metabolism in higher plants: localization and regulation of activity of key enzymes. Crit Rev Biochem Mol Biol 35: 253–289[Web of Science][Medline] Wittich PE, Willemse MTM (1999) Sucrose utilization during ovule and seed development of Gasteria verrucosa (Mill.) H. Duval as monitored by sucrose synthase and invertase localization. Protoplasma 208: 136–148[CrossRef][Web of Science] Yen SF, Su JC, Sung HY (1994) Purification and characterization of rice sucrose synthase isozymes. Biochem Mol Biol Int 34: 613–620[Web of Science][Medline] Ylstra B, Garrido D, Busscher J, van Tunen AJ (1998) Hexose transport in growing Petunia pollen tubes and characterization of a pollen-specific, putative monosaccharide transporter. Plant Physiol 118: 297–304 Zonia L, Muller M, Munnik T (2006) Hydrodynamics and cell volume oscillations in the pollen tube apical region are integral components of the biomechanics of Nicotiana tabacum pollen tube growth. Cell Biochem Biophys 46: 209–232[CrossRef][Medline] This article has been cited by other articles:
|
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| ASPB Publications | PLANT PHYSIOLOGY® | THE PLANT CELL | |
|---|---|---|---|
TOP
ABSTRACT
RESULTS
Particle (Threshold option). In controls, the primary antibodies were omitted or preimmune serum was used. 
