Oligomerization, Membrane Association, and in Vivo Phosphorylation of Sugarcane UDP-glucose Pyrophosphorylase

Background: UDP-glucose pyrophosphorylase (UGPase) is a key enzyme in the biosynthesis of sucrose and the cell wall.

Results: UGPase was phosphorylated in and associated with the membrane in vivo. Redox modification modulated UGPase activity by changing its oligomeric state.

Conclusion: Phosphorylation, redox modification, and oligomerization regulate UGPase.

Significance: Our data broaden the understanding of biomass biosynthesis in the bioenergy crop sugarcane.

Abstract

Sugarcane is a monocot plant that accumulates sucrose to levels of up to 50% of dry weight in the stalk. The mechanisms that are involved in sucrose accumulation in sugarcane are not well understood, and little is known with regard to factors that control the extent of sucrose storage in the stalks. UDP-glucose pyrophosphorylase (UGPase; EC 2.7.7.9) is an enzyme that produces UDP-glucose, a key precursor for sucrose metabolism and cell wall biosynthesis. The objective of this work was to gain insights into the ScUGPase-1 expression pattern and regulatory mechanisms that control protein activity. ScUGPase-1 expression was negatively correlated with the sucrose content in the internodes during development, and only slight differences in the expression patterns were observed between two cultivars that differ in sucrose content. The intracellular localization of ScUGPase-1 indicated partial membrane association of this soluble protein in both the leaves and internodes. Using a phospho-specific antibody, we observed that ScUGPase-1 was phosphorylated in vivo at the Ser-419 site in the soluble and membrane fractions from the leaves but not from the internodes. The purified recombinant enzyme was kinetically characterized in the direction of UDP-glucose formation, and the enzyme activity was affected by redox modification. Preincubation with H₂O₂ strongly inhibited this activity, which could be reversed by DTT. Small angle x-ray scattering analysis indicated that the dimer interface is located at the C terminus and provided the first structural model of the dimer of sugarcane UGPase in solution.

Introduction

Most of the ethanol that is produced worldwide derives from plant juice containing sucrose from sugarcane in Brazil and starch from corn in the United States (1). The development of high yielding sugarcane cultivars that are adapted to different conditions remains a major challenge for breeders (1). The current sugarcane cultivars are based on crosses between Saccharum officinarum, a domesticated sugar producing species, and Saccharum spontaneum, a wild species. The Saccharum hybrid of these two species was back-crossed to S. officinarum to generate sugarcane with a complex genome that incorporates the higher sugar content of S. officinarum with the disease and stress resistance traits of S. spontaneum (2, 3).

Sucrose (α-d-glucopyranosyl-1,2-d-fructofuranose) is the world's most abundant disaccharide. Sucrose is produced primarily in leaf mesophyll cells and is then transported throughout the phloem. In many plant species, sucrose is exported to the apoplast prior to being loaded into the phloem. As a result, the concentration of sucrose in the leaf apoplast increases as photosynthesis occurs. Sugarcane is a C₄ plant that accumulates sucrose to levels of up to 50% of dry weight in the stalk (4).

UDP-glucose pyrophosphorylase (UGPase⁴; EC 2.7.7.9) is an important enzyme for sucrose synthesis and cell wall formation and is essential for plant survival because a deficiency in UGPase activity causes male sterility (5). This enzyme is responsible for the production of uridine diphosphate glucose (UDP-glucose) using glucose-1-phosphate (Glc-1-P) and uridine-5′-triphosphate (UTP) in source tissues. In sink tissues, the enzyme sucrose synthase can form UDP-glucose by cleaving sucrose, which is then utilized by UGPase to form glucose 1-phosphate. UDP-glucose is a substrate for cellulose and callose biosynthesis at the plasmalemma (6–10).

The mechanisms that are involved in sucrose accumulation in sugarcane are not well understood, and little is known with regard to the factors that control the extent of sucrose storage in the stalks and the production of cellulose, including the mechanisms that regulate UGPase activity. The UGPase activity in the UDP-glucose synthesis direction of the reaction occurs predominantly in photosynthetic tissues, directing carbon flux toward sucrose synthesis (10). Protein oligomerization is supposed to be a key regulatory mechanism controlling UGPase activity, possibly also controlling the entire pathway of sucrose synthesis (11). In addition, protein phosphorylation and the binding of 14-3-3 proteins could be other posttranslational modifications that are involved in regulating UGPase activity, subcellular localization, and protein turnover. Barley UGPase binds to 14-3-3 proteins in vivo (12), and yeast UGPase phosphorylation does not affect its activity but leads to a decreased glycogen content and an increased cell wall glucan content (13).

The objective of this work was to gain insight into the ScUGPase-1 expression pattern and regulatory mechanisms of protein activity. Combining gene expression and immunoblotting analyses, ScUGPase-1 was mainly distributed in the stems of sugarcane. There was a decrease in ScUGPase-1 gene expression in the stems as the maturation progressed between two sugarcane cultivars differing in their ability to accumulate sucrose. ScUGPase-1 was detected in the cytoplasm, in agreement with its role in sucrose synthesis. Interestingly, ScUGPase-1 was also found to be associated with the plasma membrane, which could indicate a role in cellulose synthesis. In addition, using a phospho-specific antibody, we observed that ScUGPase-1 was phosphorylated in vivo at the Ser-419 site in the soluble and membrane fractions of the leaves but not in those of the internodes. Finally, kinetics and small angle x-ray scattering data provided evidence of a possible redox and oligomeric modulation of ScUGPase-1 in vitro.

EXPERIMENTAL PROCEDURES

Plant Material

Sugarcane cultivars RB855156 (High Brix) and RB935744 (Low Brix) were grown in the field for 8 months at the Centro de Ciências Agrárias, UFSCAR (Araras, SP, Brazil). Samples of mature leaf (leaf +1) and the first, fifth, and ninth internodes were collected from sugarcane cultivars with different agronomical traits concerning sucrose content. The tissues were sectioned, frozen in liquid nitrogen, and stored at −80 °C.

RNA Isolation and Quantitative PCR

RNA was isolated from a mature leaf and the first, fifth, and ninth internodes using the RNeasy Plant minikit (Qiagen) following the manufacturer's protocol. First strand cDNAs were synthesized using 2.5 μg of DNA-I-treated RNA (500 ng/μl), oligo(dT)₁₇ primer, and 10 mm dNTP mix. The RNA samples were denatured at 65 °C for 10 min and placed on ice for 2 min, after which 5× First Strand Buffer, 0.1 m DTT, and Superscript II RT enzyme (200 units/μl) were added (Invitrogen). The reaction was performed at 42 °C for 50 min and at 70 °C for 15 min in a thermocycler. Quantitative PCR (qPCR) was performed in the Applied Biosystems 7500 real-time PCR system (Invitrogen). To each cDNA sample, SYBR Green PCR Master Mix (Invitrogen), 10 μm forward primer, 10 μm reverse primer, and water were added. The C-terminal region of the coding sequence of the ScUGPase-1 gene was used as a template to design the primers (Table 1). The reactions were incubated at 95 °C for 10 min, followed by 40 cycles at 95 °C for 15 s and 60 °C for 1 min as described by Varkonyi-Gasic et al. (14). The sugarcane polyubiquitin gene (CA179923) was used as a reference sample (15) using the primers that are described in Table 1. The reactions were conducted with three biological replicates, each in triplicate. The displayed qPCR result values are relative to those of a mature leaf in each cultivar. To calculate the -fold change, we used the Web-based qPCR system considering the primer reaction efficiencies (16).

TABLE 1.

Specific primers that were used for the experiments

Primer name	Sequence (5′–3′)	Nucleotides	Tm
			°C
ScUGPase-1_Fw	CAC CGC CGC CGC TGC C	16	59
ScUGPase-1_Rv	TTA AAG ATC CTC AGG GCC A	19	53
pRT104-ScUGPase_Fw	CAT GGA GGC CGA ATT CAT GGC CGC CGC TG	29	68
pRT104-ScUGPase_Rv	GCA GGT CGA CGG ATC CAA GAT CCT CAG GGC CA	32	65
RT-ScUGPase_Fw	TCC AGC TAA CCC TTC GAT TGA G	22	56
RT-ScUGPase_Rv	GAG CAA GGA AAT TGG CAA CCT	21	56.2
PUB-Fw	CCG GTC CTT TAA ACC AAC TCA GT	23	57
PUB-Rv	CCC TCT GGT GTA CCT CCA TTT G	22	57.3

Membrane Extraction

Leaf and internode membranes were ground in an extraction buffer containing 20 mm Tris-HCl, pH 8.8, 150 mm NaCl, 1 mm EDTA, 1 mm PMSF, 5 mm benzamidine, and 4% (v/v) glycerol. The extract was spun at 6,000 × g for 15 min (4 °C), and the resultant supernatant was further centrifuged at 100,000 × g for 2 h (4 °C). The pellet was resuspended in 150 μl of resuspension buffer containing 10 mm Tris-HCl, pH 7.3, 150 mm NaCl, 1 mm EDTA, 1 mm PMSF, 1 mm benzamidine, 1 mm MgCl₂, 1% (v/v) glycerol, and 0.1% (v/v) Triton-X 100. The sample was clarified and centrifuged at 100,000 × g for 1 h (4 °C). The total membrane protein in the supernatant was quantified by the Bradford method. The membrane protein was adjusted to 0.5–1.0 mg/ml.

Immunoblotting

For the immunoblot detection, the protein samples were separated on 7 or 10% polyacrylamide-SDS gels and transferred to specific polyvinylidene fluoride fluorescence (PVDF) membranes (Millipore). The membranes were blocked in a 2% (v/v) fish gelatin solution in PBS buffer before being incubated with primary antibodies at a 1:1000 dilution in PBST buffer. The washes were performed in PBT, and an anti-rabbit Alexa Fluor 680-conjugated secondary antibody was used at a 1:10,000 dilution in PBST. Barley anti-UGPase (anti-HvUGPase) and phospho-specific (anti-pS419) primary antibodies used were polyclonal and were raised in rabbits. Immunoblots were generated using LI-COR Odyssey, and the signal was detected based on fluorescence.

Subcellular Localization and Confocal Microscopy

The sequence that encodes ScUGPase-1 was amplified by PCR using specific primers as described in Table 1 and cloned as an NdeI/BamHI fragment into a modified pRT104 vector (17), generating the construct pRT104::ScUGPase-1-GFP. The cauliflower mosaic virus 35 S promoter drove the expression of the fusion protein. The in-frame fusion of GFP-ScUGPase-1 was confirmed by nucleotide sequence analysis. Particle bombardment was used to introduce the plasmid into onion inner epidermal cells with a biolistic helium particle accelerator system (Biomics) under a pressure of 1000 p.s.i. The plasmid was precipitated using tungsten M10 particles (Bio-Rad) in 50 μl of 2.5 mm CaCl₂ and 20 μl of 100 mm spermidine. The target distance between the stop screen and onion piece was set at 9 cm. After bombardment, the onion pieces were kept in darkness at 25 °C for 24 h. Onion epidermal cells were examined at INFABIC (National Institute of Science and Technology on Photonics Applied to Cell Biology) at the University of Campinas (UNICAMP) using a Zeiss LSM780-NLO confocal microscope (Carl Zeiss). Single optical sections were scanned as resulting images for each transient expression.

Determination of Hydrogen Peroxide

The levels of hydrogen peroxide in the sugarcane internode and leaf extract were determined by the ferrous ammonium sulfate/xylenol orange method as described previously (18). The samples were measured at 560-nm absorbance and compared with standard samples of hydrogen peroxide. The measurements were conducted with three biological replicates.

Cloning, Expression, and Protein Purification

The pSPORT vector containing the sequence that encodes the ScUGPase-1 protein was obtained from the SUCEST database, Sugarcane Assembled Sequence number SCQGLR1062D04.g. The nucleotide sequence for full-length ScUGPase-1 was amplified by PCR and cloned into the pENTR-D/TOPO vector, generating the construct pTOPO::ScUGPase-1. The PCR was performed using specific primers that are described in Table 1. Taq AccuPrime DNA Polymerase (Invitrogen) was used to amplify the PCR products, which were then separated by gel electrophoresis and purified. The clones were created by in vivo recombination using the gateway system of pTOPO::ScUGPase-1 with the pET160-DEST destination vector (Invitrogen) in chemically competent Escherichia coli TOP10 cells. The recombinant plasmid pET160::ScUGPase-1 was transferred into E. coli DH5α cells and then sequenced. The recombinant plasmid pET160::ScUGPase-1 was transformed into the competent E. coli BL21(DE3) strain by thermal shock for protein expression. The cells were grown in 2 ml of LB medium containing ampicillin (100 mg/ml) at 37 °C, 250 rpm, for 16 h. The culture was used to inoculate 2 liters of the same medium under the same culture conditions until the A₆₀₀ reached 0.6–0.8. The expression of the recombinant ScUGPase-1 was induced by the addition of isopropyl β-d-1-thiogalactopyranoside to a final concentration of 1 mm and incubated for 4 h. The culture was harvested by 10 min of centrifugation at 6,000 × g, 4 °C, and stored for further use at −20 °C. The bacterial pellet that was obtained from 2 liters of liquid culture was suspended in 40 ml of binding buffer containing 50 mm sodium phosphate, 100 mm NaCl, and 5% glycerol, pH 7.4. The protease inhibitor PMSF (1 mm) was added, along with lysozyme (1 mg/ml). The cells were lysed by sonication on ice in an ultrasonic cell disruptor (Cole-Parmer Instrument, Chicago, IL). The lysate obtained was centrifuged at 27,000 × g for 30 min at 4 °C. The supernatant containing the soluble cellular material (total volume of 80 ml) was used for purification using immobilized metal ion affinity chromatography. A column containing 1.5 ml of Ni-NTA-agarose resin (Qiagen) was equilibrated with 10 column volumes of binding buffer. The supernatant was loaded onto the column by gravity. The column was washed with 10 column volumes of wash buffer containing 50 mm sodium phosphate, 100 mm NaCl, 5% glycerol, and 10 mm imidazole, pH 7.3. The recombinant His₆-tagged ScUGPase-1 was eluted with binding buffer containing increasing imidazole concentration (10, 20, 50, 100, and 200 mm). Fractions containing the full-length His₆-tagged ScUGPase-1 protein were pooled and analyzed by 12% SDS-PAGE to determine the molecular mass and purity of the protein. The total protein concentration was determined by A₂₈₀ quantification. At this point, one fraction of the purified protein was collected for gel filtration chromatography, whereas the other fraction was submitted to His₆ tag cleavage. Following immobilized metal ion affinity chromatography of the His₆-tagged ScUGPase-1, the purified protein was pooled, and recombinant tobacco etch virus (rTEV) His₆-tagged protease was added in a 1:10 rTEV/protein ratio. Following the incubation, the protein sample was loaded onto a gravity flow Ni-NTA column pre-equilibrated in buffer containing 50 mm sodium phosphate and 100 mm NaCl, pH 7.4, for a second affinity chromatography to separate the His₆ tag from ScUGPase-1. The flow-through containing the protein was collected, dialyzed against buffer containing 50 mm sodium phosphate, pH 7.4, 100 mm NaCl, and 5% glycerol, and finally concentrated to 1–10 mg/ml using the Vivaspin 10,000 molecular weight cut-off Ultrafiltration spin column (GE Healthcare).

Enzyme Activity and Kinetic Characterization

The activity of ScUGPase-1 was assayed in the direction of synthesis of UDP-glucose as described previously (19). The assay was performed at 25 °C. The reaction was conducted in a buffer containing 20 mm MOPS, pH 7.5, 1 mm DTT, 5 mm MgCl₂, 1.5 mm Glc-1-P, 1 mm UTP, 0.015 units/μl pyrophosphatase, 0.2 mg/ml BSA, and the purified protein at a 1:100,000 dilution in a total volume of 60 μl. Malachite green color reagent was added to finish the reaction. The samples were read at 650 nm with a spectrophotometer. The kinetic data were plotted as specific activity (μmol·min⁻¹·mg⁻¹) versus substrate concentration. Kinetic constants were acquired by fitting the data to the Hill equation with a non-linear least-squares formula using the program Origin version 7.0. Hill plots were used to calculate the Hill coefficient and the kinetic constants that correspond to the maximal velocity (V_max), catalytic constant (k_cat), and the concentration of the substrate that leads to the half-maximal velocity (K_m). The kinetic constants are means of at least three independent set of data that were reproducible within ±10%.

Redox Modification

For the oxidation assay, the purified ScUGPase-1 was incubated in a buffer containing 20 mm MOPS, pH 7.5, at 25 °C for 30 min in the presence of different concentrations of hydrogen peroxide (H₂O₂). After incubation, aliquots were withdrawn and assayed for ScUGPase-1 activity. For the reduction assay, the oxidized enzyme was incubated in the same buffer as above in the presence of different concentrations of DTT. After 30 min of incubation, aliquots were withdrawn and assayed for ScUGPase-1 activity. The reactions were performed as described previously (19).

Gel Filtration

The His₆-tagged ScUGPase-1 was incubated in a buffer containing 20 mm MOPS, pH 7.5, 150 mm NaCl, 1 mm EDTA, and 1 mm hydrogen peroxide (H₂O₂) for 30 min, and 200 μl of the oxidized ScUGPase-1 was applied to a Superdex 200 10/300 GL column (GE Healthcare) on an AKTA FPLC system (GE Healthcare). A 20 mm MOPS, pH 7.5, 150 mm NaCl, and 1 mm EDTA mixture was used as the elution buffer. The flow rate was 0.5 ml min⁻¹. Fractions of 1 ml were collected. The fractions corresponding to a dimer of the His₆-tagged ScUGPase-1 were pooled and concentrated to 0.684 mg/ml using an Amicon Ultra-4 10,000 molecular weight cut-off centrifugal filter (Millipore).

Small Angle X-ray Scattering (SAXS) Data Collection and Reduction

The SAXS data for the monomeric form of ScUGPase-1 in buffer containing 50 mm sodium phosphate, pH 7.2, 5% glycerol, and 100 mm NaCl, were collected at the wavelength λ = 1.49 Å using a MAR CCD 165 detector on the SAXS-2 beamline of the Brazilian National Synchrotron Light Laboratory (LNLS/Brazil) (20) with a sample-to-detector distance of 890.26 mm over a range of 0.0159 < q < 0.3751 Å⁻¹ (q = 4πsinθ/λ, where 2θ is the scattering angle). The scattering patterns were measured with a 300-s exposure time at 12 °C for samples with concentrations in the range of 1.0–10.5 mg/ml. CCD dark current and natural background correction were performed on the raw two-dimensional images as appropriate. A subsequent data collection was performed at LNLS on the beamline SAXS-1 with a wavelength of 1.55 Å using a Dectris-Pilatus (300 K, 84 × 107 mm) two-dimensional detector. On this occasion, samples of a construct of ScUGPase-1 with an additional N terminus of 32 amino acid residues, including six histidines (MHHHHHHGAGGCCPGCCGGGENLYFQGIITSL), in a buffer containing 20 mm MOPS, pH 7.5, 150 mm NaCl, and 1 mm EDTA were analyzed at concentrations of 0.64, 2.09, and 2.32 mg/ml, the first of which was later found to correspond to a protein dimer in solution. X-ray scattering data were collected at 17 °C, and the sample-to-detector distance was 1603.43 mm, covering a momentum transfer interval of 0.0067 < q < 0.2780 Å⁻¹. The protein samples that were initially kept on ice were centrifuged for ∼5 min at 14,100 × g and room temperature, prior to injection in the sample holder. To reduce potential radiation damage, for each sample, successive frames were recorded with exposure times ranging from 3 to 400 s. The corresponding buffer solutions were measured for 100 s. For the data that were collected at the beamline SAXS-1, absolute calibration of the scattering intensity was performed using water as a secondary standard (21, 22). Correction by sample attenuation, normalization of the collected images to the intensity of the transmitted beam, buffer scattering subtraction, and radial integration were performed with FIT2 (23).

SAXS Profile Analysis and Overall Parameters

The scattering-intensity profiles were analyzed with ATSAS 2.5.1.1 (24) running under Slackware Linux. The zero-angle scattering intensity, I(0) and the radius of gyration, R_g, were calculated from data at very low q values (qR_g ≤ ∼1.3) using the Guinier approximation (25), I(q) = I(0)exp(−q²R_g²/3). The pair-distance distribution function, P(r), was calculated by the indirect Fourier transform method using GNOM (26), from which additional estimates for R_g and I(0) were also obtained. The Kratky plot (27–29) was used to assess the conformational state of the protein in solution. The molecular masses were estimated by the excluded particle volume that was calculated from the scattering curves using GNOM and DATPOROD (24) as well as by the procedure that was described by Fischer and colleagues (30). These methods are independent of sample concentration, which was not determined with enough accuracy in either case, the monomer or dimer (His₆-tagged construct).

Ab Initio Envelope Reconstruction

Ab initio low resolution envelopes for the scattering particles in solution were determined from the scattering curves as follows. The curve corresponding to the ScUGPase-1 monomer included scattering up to q = 0.3751 Å⁻¹, which prompted us to use GASBOR (31) for shape recovery, a software that implements a simulated annealing algorithm based on a chainlike assembly of dummy residues that considers the internal structure of the protein. The GASBOR models were recovered from the scattering curve using default parameters and a total number of residues equal to 476. For the protein dimer, whose final curve was limited to the lower q_max = 0.2 Å⁻¹, DAMMIF (32) was used instead. For better results, the annealing parameters were initially fine-tuned by running DAMMIF in the interactive mode. Convergence was assured by using prolate particle anisometry, as indicated by the asymmetric profile of the dimer P(r), along with the expected point group P2 symmetry as inferred from available homolog crystal structures. The looseness penalty was adjusted to a value of 0.2 to reject unlike models, and an atomic radius of 1.5 was employed, leading to a higher number of beads and finer details on the volume surface. In both cases, to verify the reliability and reproducibility of the adopted procedure, 40 models were obtained in independent runs and were subsequently analyzed using DAMAVER (33). The cross-correlation table based on the normalized spatial discrepancy confirmed the stability and reliability of the ab initio shape determination. From the 40 models that were initially obtained for the monomer, five outliers were automatically discharged. The normalized spatial discrepancy for the remaining 35 models was 0.96 ± 0.04, indicating a very narrow spatial distribution of the GASBOR models. The same procedure led to a normalized spatial discrepancy of 0.55 ± 0.12 for the dimer after the exclusion of a single outlier, which assures a highly significant similarity between the solutions that were found by DAMMIF. In the dimer case, DAMAVER version 3.2 was used to correctly handle the model symmetry. To maintain the original features of the most typical GASBOR model, the model with the lowest average normalized spatial discrepancy compared with the others was reported. For the dimer, the final model that was obtained with DAMFILT after averaging is presented.

Molecular Modeling

Despite numerous attempts, the crystallization of ScUGPase-1 was not successful. Thus, the envelopes that were recovered from the SAXS experiments were interpreted based on a computational model that was built using the I-TASSER server (34). An approach that combines threading and the SAXS profile for fold recognition as implemented in the SAXSTER server (35) was also used, with no significant improvement compared with the previous approach, most probably due to the globular protein shape and the high sequence identity of the available homolog models. Therefore, the I-TASSER model with the highest confidence score (C-score = 1.14) was chosen to represent the three-dimensional atomic structure of the ScUGPase-1 monomer. To interpret the SAXS envelope for the ScUGPase-1 dimer in solution, a template was selected based on a large scale assessment of possible dimers that were obtained from available homolog crystal structures by applying crystallographic symmetry operators. For each possible dimer, a theoretical scattering curve was calculated and fitted to the ScUGPase-1 experimental data with CRYSOL (36). The best agreement was obtained for a dimer that was constructed by applying crystal symmetry operations to the contents of the asymmetric unit that was reported for the homolog UGPase from Arabidopsis thaliana (PDB entry 1Z90) (37). This arrangement was observed in various other homolog crystal structures, as discussed below. The final model for the ScUGPase-1 dimer was obtained by individually superposing the ScUGPase-1 monomer onto chains A and B of the referred structure and combining the resulting monomer chains. Throughout the processing, C shell scripts were used to automate computational jobs. Superpositions were performed with SUPCOMB (38). Data plotting was conducted with GNUPLOT, and model visualization and graphical analyses were performed with RASMOL (39) and PyMOL (PyMOL Molecular Graphics System, version 1.4.1, Schrödinger, LCC).

RESULTS

ScUGPase-1 Expression in Sugarcane

In plants, UGPase genes are expressed in all tissues, including the roots, tubers, leaves, stems, and young seeds (40). To evaluate the expression of the ScUGPase-1 gene (GenBank^TM accession number KF278717), we investigated its expression in the internode and mature leaf tissues of sugarcane cultivars that differ in their ability to accumulate sugar (Fig. 1). In sugarcane, the youngest internode (number 1) is the one at the top of the plant, close to the leaves, whereas the oldest internodes with a higher sugar content are those closest to the ground. ScUGPase-1 expression decreased with internode maturation. The gene was also expressed in the leaves, although at lower levels, even when compared with mature internode number 9.

FIGURE 1.

Relative expression of ScUGPase-1 as determined by RT-qPCR from two sugarcane cultivars contrasting in sucrose content. A, real-time PCR results for the sugarcane cultivar RB855156 with a high sucrose content. B, real-time PCR results for the sugarcane cultivar RB 935744 with a low sucrose content. The reactions were performed in triplicate. *, p < 0.05. Error bars, S.D.

Protein Localization and Phosphorylation

UDP-glucose is a precursor for cell wall biosynthesis in photosynthetic tissues (8), where ScUGPase-1 exists predominantly as a soluble cytosolic protein (41). In barley, high UGPase activities were found in the membrane fraction (42). To evaluate the ScUGPase-1 expression in the sugarcane internode and leaf, the soluble and membrane fractions were extracted and analyzed by immunoblotting using the anti-HvUGPase antibody. In vivo, ScUGPase-1 also cross-reacted with the anti-HvUGPase antibody and was present in the soluble and membrane fractions of the internode (Fig. 2A) and leaf (Fig. 2B) tissue. Interestingly, in Fig. 2B, we noticed the presence of a ∼100 kDa band in both of the leaf tissue fractions. The size is consistent with a putative homodimer of the protein ScUGPase-1. Despite this unexpected result, several authors have observed that proteins with different oligomeric forms present some degree of resistance to the conditions used in the SDS-PAGE (43–46). Strategically located disulfide bonds, salt bridges, and hydrophobic residues at the surface of the proteins have been suggested as potential characteristics that could explain the resistance of some proteins to reducing agents (46). In addition, posttranslational modification may regulate the protein activity. The amino acid sequence of ScUGPase-1 is strongly conserved among other plant UGPases (Fig. 3) and contains a serine at position 419 that is predicted to be a phosphorylation site and a 14-3-3 binding site (12). To evaluate ScUGPase-1 in relation to its phosphorylation status in vivo, we once again extracted the soluble and membrane fractions from sugarcane internode and leaf and immunoblotted the crude extract using the anti-pS419 antibody. ScUGPase-1 phosphorylation was not detected in the internode (Fig. 4A), whereas in the soluble and membrane fractions from the leaves, a clear phosphorylation signal was observed (Fig. 4B). To complement the results in Fig. 2, a GFP-ScUGPase-1 fusion protein was transiently expressed in the onion epidermis, indicating not only cytosolic localization but also an association with the cell membrane (Fig. 5); these findings are consistent with the fractionation and immunoblotting analyses.

FIGURE 2.

Immunoblotting analysis of the membrane and soluble fraction of ScUGPase-1 in the internodes and leaves. Shown are immunoblots of the internodes (A) and leaves (B) using an anti-HvUGPase antibody. The numbers indicate the proteins of the microsomal fraction (1), the proteins of the soluble fraction (2), and the ScUGPase-1 recombinant protein (3). M, molecular mass standard.

FIGURE 3.

Multiple-sequence alignment of UGPases from different plant species. Shown are accession number KF278717 (Saccharum spp. UGPase), accession number ACG32096 (Zea mays UGPase), accession number ACA50487 (Oryza sativa UGPase), accession number CAA62689 (Hordeum vulgare UGPase), accession number XP_002453185 (Sorghum bicolor UGPase), and accession number P19595 (A. thaliana UGPase). The blue triangle indicates the serine that is predicted to be phosphorylated at the 14-3-3 binding site.

FIGURE 4.

Phosphorylation analysis of the membrane and soluble fraction of ScUGPase-1 in the internodes and leaves. Shown are immunoblots of the internodes (A) and leaves (B) using an anti-pS419 antibody. The numbers indicate the proteins of the microsomal fraction (1), the proteins of the soluble fraction (2), and the ScUGPase-1 recombinant protein (3). M, molecular mass standard.

FIGURE 5.

Subcellular localization of GFP-ScUGPase-1. A, pRT104::ScUGPase-1-GFP construction map. B, confocal microscopy of an onion epidermis cell expressing the GFP-ScUGPase-1. Bars, 100 μm.

Levels of Hydrogen Peroxide in the Internode and Leaves

Fig. 2B indicates the presence of a putative ScUGPase-1 dimer in sugarcane leaves. In addition, oxidizing agents regulate the quaternary state of UGPase (47). Therefore, we evaluated the range of levels of H₂O₂ internode and leaf tissues of sugarcane. In the internodes, the presence of H₂O₂ was almost undetectable, whereas in the leaves, we observed a range between 1 and 2.5 μmol/g fresh weight, which is equivalent to 20 and 50 mm H₂O₂, respectively (data not shown). These values are compatible with the levels of H₂O₂ in field-grown plants in a study that suggested a normal range between 1 and 5 μmol/g fresh weight (48). These results indicating higher levels of H₂O₂ in sugarcane leaves corroborate the notion that H₂O₂ may influence the oligomeric state of ScUGPase-1 in vivo.

Cloning, Expression, and Protein Purification

To produce the recombinant ScUGPase-1, we cloned the entire coding sequence into the pET160-DEST vector. The protein was expressed in BL21(DE3) cells and purified by affinity chromatography onto an Ni-NTA-agarose resin. The His₆-tagged ScUGPase-1 was obtained with high purity as determined by SDS-PAGE (Fig. 6, lane 1). We also produced the recombinant protein without the His tag by treating the His₆-tagged ScUGPase-1 with rTEV protease. After a new immobilized metal ion affinity chromatography purification to remove the rTEV and the His tag, the cleavage protein yielded a polypeptide with a molecular mass of ∼52 kDa as determined by SDS-PAGE (Fig. 6, lane 2), in which two elution fractions corresponding to the oligomers and the dimer of His₆-tagged ScUGPase-1 for the SAXS experiment were collected (see below). Then the His₆-tagged ScUGPase-1 was also submitted to affinity chromatography on an Ni-NTA-agarose resin, followed by protease cleavage. After cleavage with the rTEV protease, the purification yielded a protein with a high level of purity and a molecular mass of ∼52 kDa as determined by SDS-PAGE (Fig. 2).

FIGURE 6.

SDS-PAGE of the recombinant ScUGPase-1. Shown are the His₆-tagged ScUGPase-1 (1) and recombinant ScUGPase-1 (2) after cleavage with the rTEV protease. M, molecular mass standard. After electrophoresis, the gel was stained with Coomassie Blue.

Characterization of ScUGPase-1 Activity

To calculate the kinetic parameters of the recombinant ScUGPase-1, we evaluated the enzyme activity at different concentrations of substrates in the direction of UDP-Glc synthesis. Fig. 7 presents the hyperbolic saturation curves for UTP (Fig. 7A), Glc-1-P (Fig. 7B), and Mg²⁺ (Fig. 7C) that were used to calculate the kinetic parameters (Table 2). The K_m values for Glc-1-P and UTP were 0.20 and 0.16 mm, respectively. The V_max for UDP-glucose synthesis was 2,151 ± 67 μmol·min⁻¹·mg⁻¹, corresponding to a k_cat number of 1.8 × 10⁸ s⁻¹. The ScUGPase-1 activity is ∼6-fold higher than that determined for the recombinant UGPases from A. thaliana. However, the substrate affinities were similar to those found for the recombinant UGPase1 and UGPase2 from Arabidopsis (49).

FIGURE 7.

Saturation curves for the recombinant ScUGPase-1. A, Glc-1-P concentration ranging from 0 to 2.5 mm. B, UTP concentration ranging from 0 to 3.5 mm. C, Mg²⁺ concentration ranging from 0 to 20 mm. When each substrate was analyzed, the other two substrates were maintained at a fixed saturation concentration.

TABLE 2.

Kinetic parameters of the recombinant ScUGPase-1

Substrate	Km	kcat	kcat/Km
	mm	s−1	s−1·mm−1
Glc-1-P	0.20 ± 0.01		9 × 108
UTP	0.16 ± 0.01	1.8 × 108	1.1 × 109
Mg2+	1.26 ± 0.06

Redox Modulation

According to Martínez et al. (47), UGPase activity from Entamoeba histolytica is regulated by redox mechanisms affecting the activity of the enzyme involving oxidative and reducing agents that are generally found in vivo. To evaluate the effects of redox modification on ScUGPase-1 activity, the recombinant protein was incubated with H₂O₂ and DTT. ScUGPase-1 activity was reduced by almost 80% when incubated with 20 mm H₂O₂ (Fig. 8A) and was restored after incubation with DTT (Fig. 8B). The reversible effect on the enzyme activity indicates a probable regulation of the protein in vivo according to the redox status of the cell. In addition, to evaluate the effects of redox modification on the oligomeric state of the protein, gel filtration chromatography using a Superdex 200 10/300 GL column was performed. The oxidation also affected the quaternary structure of the protein. In fact, the recombinant oxidized His₆-tagged ScUGPase-1 eluted as dimers and oligomers (data not shown). For a deeper structural characterization, the fraction that was recovered as a dimer was used for a SAXS experiment.

FIGURE 8.

Redox regulation of the recombinant ScUGPase-1 activity. A, inactivation of ScUGPase-1 by different concentrations of H₂O₂. B, effect of the reducing agent DTT on oxidized ScUGPase-1. Error bars, S.E.

SAXS Overall Parameters and Molecular Envelopes

To obtain a structural model of ScUGPase-1 in solution, we performed a small angle x-ray scattering experiment. The final SAXS scattering curves that were obtained from two different constructs of ScUGPase-1, referred to as monomer and dimer, the latter including a His₆-tagged N terminus, are shown in Fig. 9, A and B, respectively. The linearity of Guinier plots was used to indicate data quality and monodispersity. The contribution of small amounts of potential higher order oligomers that were detected at very low q values was reduced by carefully limiting the lowest q value in the scattering curves. For the monomer, the best results were obtained for the sample at a protein concentration of 10.5 mg/ml.

FIGURE 9.

SAXS analysis of the ScUGPase-1 constructs in solution. A, final monomer scattering curve (open squares) with an inset showing the linear fitting in the Guinier region. The solid line corresponds to the fitting of the theoretical curve that was calculated for the ScUGPase-1 monomer that was obtained by computational methods. A χ value of 2.55 was obtained with CRYSOL (36) up to a q_max = 0.25, as shown in the plot. Values as low as 1.44 were obtained, limiting the range to 0.20 Å⁻¹ and indicating a correspondence of the gross low resolution features of the scattering particle in solution and the monomer rigid model. B, solution scattering profile (open circles) of the ScUGPase-1 dimer in the q range that was limited after data processing. Inset, linear fitting that was obtained from the Guinier analysis. The solid line corresponds to the fitting of the theoretical curve as calculated for the computational dimer model in the same range that was automatically refined by PRIMUS/AUTOGNOM (24) during the indirect Fourier transform procedure. The discrepancy between the experimental data and the theoretical curve was calculated by CRYSOL, resulting in a χ value of 1.45. C, Kratky plot that was derived from the experimental curves that were normalized for I(0) = 1 for proper visualization. After buffer scattering subtraction, a few negative intensity points were obtained for the dimer curve at higher q values due to the very low sample concentration. The plot is limited to the region Iq² > 0. D, distance distribution function as calculated from the experimental curve that was used for the ab initio envelope reconstruction. The monomer P(r) (open squares) exhibits a nearly centered peak, indicating a globular scattering particle. For the second sample, a typical profile with two clearly distinct maxima indicates the presence of a dimer in solution, as anticipated by the molecular mass estimates; the higher peak to the left of the center of the range indicates that the dimer exhibits a prolate shape.

In the case of the dimer curve (Fig. 9B), a cut-off at higher angles was also applied by discharging extremely noisy data that were observed for q > 0.2 Å⁻¹, which were a result of the low sample concentration (0.64 mg/ml). The analysis of the samples of this construct that were prepared at higher concentrations indicated the presence of higher order oligomers, preventing further data processing. A sequential series of scattering patterns with increasing exposure time was measured for the lowest concentration sample. Visible changes in the sequential scattering profiles were clearly correlated with the cumulative exposure time. Thus, to prevent possible artifacts decurrent from radiation damage, only the first curve (Fig. 9B) was used for subsequent analyses.

The Kratky plots (Fig. 9C) exhibited a maximum that is typical for compact, correctly enovelated globular proteins. The R_g estimates that were calculated from the linear regression in the Guinier region (Fig. 8, A and B, inset) were 27.5 and 50.8 Å for the monomer and dimer samples, respectively.

More accurate values for R_g were obtained from the indirect Fourier transform that was performed with GNOM (26), which considers the full-range scattering curve to calculate the distance distribution functions that were shown in Fig. 9D. For the monomer, the maximum intramolecular distance was 85.0 Å, resulting in an R_g that was also equal to 27.5 Å. The corresponding excluded volume that was calculated with AUTOPOROD (24) was V_p = 85.9 nm³, allowing us to estimate the molecular mass of the protein in solution as 53.0 kDa, in excellent agreement with the value of 56.1 kDa that was calculated by the method that was described by Fischer et al. (30), with the linear and angular coefficients that were determined for q_max = 0.30 Å⁻¹. These results are consistent with the presence of a ScUGPase-1 monomer in solution, whose molecular mass, as calculated from the amino acid sequence, was 53.3 kDa.

The dimer P(r) has a maximum intramolecular distance of 152.7 Å, from which R_g was estimated at 45.7 Å, in good agreement (within error) with the value that was predicted from the Guinier analysis. The two maxima that were noted in the P(r) profile are typical for protein dimers in solution. The molecular mass of the scattering particle in solution resulted was 105.4 kDa, as calculated from an excluded volume of V_p = 170.7 nm³ (24) and 114.4 kDa (for q_max = 0.15) (30). In addition, despite the inherent limitations of this technique, the molecular mass estimated is consistent with the expected value of 2 × 55.6 = 111.2 kDa, which was calculated taking into account the His₆ tag in the construct, which unequivocally indicates the presence of an ScUGPase-1 dimer in solution.

The SAXS envelopes that were recovered from the scattering curves are shown in Fig. 10. The excellent fitting of the theoretical curves that were computed with CRYSOL with the ScUGPase-1 monomer and dimer models that were obtained by computational methods (Fig. 9, A and B, respectively) can be visually confirmed by the corresponding superpositions that are shown in Fig. 10.

FIGURE 10.

PyMOL surface representations of the monomer (top row) and dimer (bottom row) as recovered from the experimental SAXS data that were superposed onto the respective computational models, represented as a schematic drawing and viewed nearly along the point group P2 dimer symmetry. The solvent layer sparsely distributed over the protein surface, as calculated by GASBOR (31), is shown as spheres. For the dimer, each monomer chain is shown in different hues of yellow; the N terminus containing the His₆ tag, disordered in solution as indicated by the computational analysis, was omitted for clarity. The scale and perspectives are the same in both rows. The middle and right columns are rotated clockwise by 90° around the y and x axes, respectively. The figures were prepared with PyMOL and edited using GIMP under Slackware Linux.

DISCUSSION

In plants, UGPase genes are expressed in all tissues, including the roots, tubers, leaves, stems, and young seeds (40). In the banana, UGPase genes are up-regulated by ethylene (50), whereas UGPase genes are up-regulated by phosphate deficiency, light exposure, and sucrose supplementation in the potato (51, 52). In this study, we evaluated the correlation between the sucrose content and ScUGPase-1 gene expression in the sugarcane cultivars RB855156 (with a higher sucrose content) and RB935744 (with a lower sucrose content), but the ScUGPase-1 profiles were very similar in these cultivars. Sucrose accumulation is under a complex regulatory network, and ScUGPase-1 most likely acts in concert with the expression of genes encoding other key enzymes that are involved in sucrose production, such as sucrose synthase, sucrose phosphate synthase, and invertases (53, 54). Interestingly, in both of the cultivars, ScUGPase-1 expression in the internodes was modulated during the maturation process, with higher expression during the early stages of internode maturation, suggesting a possible role of ScUGPase-1 in cell wall biosynthesis. In addition, the elongation of sugarcane internodes is followed by increasing sucrose concentration (53). The accumulation of UGPase products decreases the protein activity (10). In addition, there is evidence of regulation at the gene expression level (eventually having an effect on the UGPase protein content/activity), suggesting tight posttranscriptional/translation control (55, 56).

The ScUGPase-1 localization and phosphorylation state were also studied. ScUGPase-1 is predominantly found in the cytoplasm (10), but rice and tobacco cells also contain high UGPase activity in microsomes (57), and barley presents UGPase in plasmalemma membranes (42). Our results, which are a combination of the transient expression of GFP-ScUGPase-1 in the onion epidermis and plant protein extract immunoblots, indicated that the protein is located in both the soluble and membrane fractions. The presence of a membrane-bound form of ScUGPase-1 producing UDP-glucose would facilitate an efficient transfer of the glucose molecule in channeling carbon into cell wall glucans. In sink tissues, UDP-glucose is mainly derived from UGPase and sucrose synthase reactions (58). Sucrose synthase content is low in mature leaves but is active and prominent in sink tissues, breaking down sucrose into UDP-glucose and fructose. Phosphorylation modifies sucrose synthase activity (promoting UDP-Glc production) and protein localization (favoring membrane dissociation) (59). We speculate that both of the enzymes work in concert to produce UDP-Glc.

Protein phosphorylation has also been described as a regulatory mechanism in UGPases from A. thaliana (49) and Saccharomyces cerevisiae (60). ScUGPase-1 contains the sequence RFKS⁴¹⁹IPSI, which is a crucial motif for the phosphorylation and binding of 14-3-3 proteins (61), including a characteristic phosphoserine residue at position 419. Interestingly, ScUGPase-1 was phosphorylated in vivo in the cytoplasm and membranes from leaf tissues but not in those from internodes. In source tissues, SPS is considered the major enzyme that is involved in the conversion of photoassimilate to sucrose and uses the UDP-Glc that is provided by UGPase. SPS activity is regulated by covalent modification via phosphorylation/dephosphorylation (61) and via allosteric effectors (62). Sucrose accumulation increases under light, whereas dark conditions lead to starch degradation (56). During the day, SPS is active, and during the night or when the rate of sucrose synthesis exceeds the capacity of the leaf to export or store sucrose, SPS is phosphorylated and binds to 14-3-3, which leads to the inhibition of SPS activity and sucrose synthesis (61). Based on our results, ScUGPase-1 was phosphorylated only in leaf tissues. The lack of phosphorylation in the plasmalemma from internode tissues suggests that, in sink tissues, the ScUGPase-1 reaction is linked to the sucrose synthase reaction to provide substrate for cellulose biosynthesis. This assumption has already been addressed by Kleczkowski et al. (58), who suggested that UDP-glucose comes from the UGPase reaction because Arabidopsis sus mutants (63) presented the same phenotype as that of the wild type with respect to growth and development. In addition, in a recent study, UGPase was demonstrated to be involved in plant height growth and biomass accumulation (9). We speculate that in source tissues, ScUGPase-1 activity is regulated by the phosphorylation of a serine at the 14-3-3 binding site. It is possible that this regulation acts in coordination with SPS, but further study is required to establish the effect of ScUGPase-1 phosphorylation and to identify its physiological significance.

Further characterization of the ScUGPase-1 enzymatic activity and its regulatory mechanisms demonstrated that the kinetic properties of the recombinant enzyme are similar to those that have been reported previously for other UGPases, even when the enzymatic activity was ∼6-fold higher than that described for the Arabidopsis recombinant enzymes. Furthermore, when the catalytic efficiency is estimated by the ratio k_cat/K_m (s⁻¹·mm⁻¹), it is clear that the recombinant ScUGPase-1 is ∼2 times more efficient than are recombinant A. thaliana UGPases (49).

The activity of ScUGPase-1 was also modulated by redox compounds. The redox assay showed that the oxidized agent H₂O₂ inactivated ScUGPase-1, and this inhibition was completely reversed by the addition of the reducing agent DTT. This result agrees with the previous report demonstrating that the activity of UGPase from E. histolytica is modulated by redox mechanisms (47). Reactive oxygen species, which include hydrogen peroxide (H₂O₂), play crucial roles in modulating many physiologic and pathologic processes in vivo. Abiotic stresses, such as excess light, drought, and salt stress, enhance the production of reactive oxygen species, and proteomics analysis demonstrated that UGPase activity is reduced by salt stress (64). In addition, higher photosynthetic activity induces both the generation of reactive oxygen species and the accumulation of soluble sugars (65). In agreement with previous reports, our data indicated higher levels of H₂O₂ in sugarcane leaves and led us to speculate that the H₂O₂ levels in sugarcane could modulate ScUGPase-1 activity in response to changes in the redox state of the cell. The inactivation of ScUGPase-1 by redox modification could be explained through S-glutathionylation because H₂O₂ may inactivate enzymes by oxidizing their thiol groups (66). Under stress conditions, the generation of H₂O₂ can modify the ScUGPase-1 thiol group, resulting in the formation of mixed disulfides between the thiols of ScUGPase-1 and glutathione (GSH). This assumption is supported by evidence demonstrating that crude protein extracts that were prepared from Arabidopsis plants and cell cultures were thiolated in vitro, and UGPase was one of the proteins that underwent thiolation with oxidized glutathione (GSSG) linked to biotin (67). In this study, it was also demonstrated that by gel filtration chromatography of the oxidized protein, it was possible to purify the protein as a dimer. It was previously shown that the accumulation of UDP-Glc decreases UGPase activity by promoting the dimeric association of the protein; instead, the presence of either single or both substrates shifts from UGPase dimers toward monomers, the active form of the enzyme (10). Here, we found that the oligomeric state was also modified in response to changes in the redox conditions, consequently modulating the enzyme activity. Moreover, the immunoblotting of the proteins of the soluble fraction from the leaves using a barley anti-UGPase antibody detected the monomer and a putative homodimer of the ScUGPase-1 in vivo. Although the conditions for the formation of the dimer have not been identified, there is still evidence of the occurrence of different oligomeric states of the sugarcane protein.

The ScUGPase-1 sequence was compared with the four other closely related agronomically important grasses (maize, rice, barley, and sorghum) and a model plant, represented by A. thaliana. All of the sequences that were analyzed by a multiple-sequence alignment showed significant identity with ScUGPase-1 and are likely to share details of their tertiary and quaternary structures. UGPase crystallographic structures in the PDB indicated that homologous proteins from A. thaliana (PDB codes 1Z90, 2ICX, 2ICY, and 2Q4J), S. cerevisiae (PDB code 2IK5), and Trypanosoma brucei (PDB code 3GUE) contain two molecules in the asymmetric unit, representing a dimer. This enzyme exists as a mixture of oligomers (monomers, dimers, tetramers, etc.) in solution, with the enzyme in the monomeric state being by far the most catalytically active species (68). According to our small angle x-ray scattering analysis, the ScUGPase-1 monomeric form has a molecular envelope in solution similar to that of the corresponding monomer of the crystal structure of UGPase from A. thaliana (PDB code 1Z90) (37). A remarkable fact, however, is the unexpected dimer interface that was observed for ScUGPase-1 in solution, which occurs via the C-terminal domain, indicating a configuration that is similar to the end-to-end interaction of different UGPases (human UGPase, PDB code 3R2W (69); S. cerevisiae, PDB code 2IK5 (70); A. thaliana, PDB code 1Z90 (37), and Leishmania major, PDB code 2OEG (71)). In addition, as Fig. 10 suggests, the dimer envelope does not correspond to a simple juxtaposition of two identical copies of the monomer envelope, a striking indication that conformational changes most likely occur upon dimerization. Moreover, the static computational model that is shown in Fig. 10 (top row), although fitting with the SAXS data (which are subject to a low resolution limit), may be composed by domains in different orientations with respect to the A. thaliana homolog, as indicated by a portion of the envelope that was not perfectly filled by the C terminus. Altogether, these results seem to be an indication that ScUGPase-1 undergoes conformational changes upon dimerization that require domain movements involving the C terminus of the protein. This hypothesis is further supported by a recent study using a deletion of the last 8 amino acids in the C-terminal region of UGPase from barley. As a result, the mutant protein had a higher activity compared with that of the wild type and was observed only in its monomeric form (11).

Altogether, the results from this study provide insight into the expression profile of the gene that encodes ScUGPase-1 and demonstrate that phosphorylation, membrane association, and redox modulation might play roles as regulatory mechanisms of the sucrose metabolic pathway and cell wall biosynthesis.