Carbon partitioning to cellulose synthesis

This article discusses the importance and implications of regulating carbon partitioning to cellulose synthesis, the characteristics of cells that serve as major sinks for cellulose deposition, and enzymes that participate in the conversion of supplied carbon to cellulose. Cotton fibers, which deposit almost pure cellulose into their secondary cell walls, are referred to as a primary model system. For sucrose synthase, we discuss its proposed role in channeling UDP-Glc to cellulose synthase during secondary wall deposition, its gene family, its manipulation in transgenic plants, and mechanisms that may regulate its association with sites of polysaccharide synthesis. For cellulose synthase, we discuss the organization of the gene family and how protein diversity could relate to control of carbon partitioning to cellulose synthesis. Other enzymes emphasized include UDP-Glc pyrophosphorylase and sucrose phosphate synthase. New data are included on phosphorylation of cotton fiber sucrose synthase, possible regulation by Ca²⁺ of sucrose synthase localization, electron microscopic immunolocalization of sucrose synthase in cotton fibers, and phylogenetic relationships between cellulose synthase proteins, including three new ones identified in differentiating tracheary elements of Zinnia elegans. We develop a model for metabolism related to cellulose synthesis that implicates the changing intracellular localization of sucrose synthase as a molecular switch between survival metabolism and growth and/or differentiation processes involving cellulose synthesis. Abbreviations: CesA, cellulose synthase; Csl, cellulose-like synthase (genes); DCB, dichlobenil; DPA, days after anthesis; SPS, sucrose phosphate synthase; SuSy, sucrose synthase; P-SuSy, particulate SuSy; S-SuSy, soluble SuSy     

Sucrose synthase localizes to cellulose synthesis sites in tracheary elements.

The synthesis of crystalline cellulose microfibrils in plants is a highly coordinated process that occurs at the interface of the cortex, plasma membrane, and cell wall. There is evidence that cellulose biogenesis is facilitated by the interaction of several proteins, but the details are just beginning to be understood. In particular, sucrose synthase, microtubules, and actin have been proposed to possibly associate with cellulose synthases (microfibril terminal complexes) in the plasma membrane. Differentiating tracheary elements of Zinnia elegans L. were used as a model system to determine the localization of sucrose synthase and actin in relation to the plasma membrane and its underlying microtubules during the deposition of patterned, cellulose-rich secondary walls. Cortical actin occurs with similar density both between and under secondary wall thickenings. In contrast, sucrose synthase is highly enriched near the plasma membrane and the microtubules under the secondary wall thickenings. Both actin and sucrose synthase lie closer to the plasma membrane than the microtubules. These results show that the preferential localization of sucrose synthase at sites of high-rate cellulose synthesis can be generalized beyond cotton fibers, and they establish a spatial context for further work on a multi-protein complex that may facilitate secondary wall cellulose synthesis.     

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

Sucrose (Suc) synthase (SuSy) is believed to function in channeling UDP-Glc from Suc to various beta-glucan synthases. We produced transgenic poplars (Populus alba) overexpressing a mutant form (S11E) of mung bean (Vigna radiata) SuSy, which appeared in part in the microsomal membranes of the stems. Expression of SuSy in these membranes enhanced the incorporation of radioactive Suc into cellulose, together with the metabolic recycling of fructose (Fru), when dual-labeled Suc was fed directly into the phloem of the leaf. This overexpression also enhanced the direct incorporation of the glucosyl moiety of Suc into the glucan backbone of xyloglucan and increased recycling of Fru, although the Fru recycling system for cellulose synthesis at the plasma membrane might differ from that for xyloglucan synthesis in the Golgi network. These findings suggest that some of the Suc loaded into the phloem of a poplar leaf is used directly by SuSys associated with xyloglucan and cellulose synthases in the stem. This may be a key function of SuSy because the high-energy bond between the Glc and Fru moieties of Suc is conserved and used for polysaccharide syntheses in this sink tissue.     

The Differential Expression of Sucrose Synthase in Relation to Diverse Patterns of Carbon Partitioning in Developing Cotton Seed

Developing cotton (Gossypium hirsutum L.) seed exhibits complex patterns of carbon allocation in which incoming sucrose (Suc) is partitioned to three major sinks: the fibers, seed coat, and cotyledons, which synthesize cellulose, starch, and storage proteins or oils, respectively. In this study we investigated the role of Suc synthase (SuSy) in the mobilization of Suc into such sinks. Assessments of SuSy gene expression at various levels led to the surprising conclusion that, in contrast to that found for other plants, SuSy does not appear to play a role in starch synthesis in the cotton seed. However, our demonstration of functional symplastic connections between the phloem-unloading area and the fiber cells, as well as the SuSy expression pattern in fibers, indicates a major role of SuSy in partitioning carbon to fiber cellulose synthesis. SuSy expression is also high in transfer cells of the seed coat facing the cotyledons. Such high levels of SuSy could contribute to the synthesis of the thickened cell walls and to the energy generation for Suc efflux to the seed apoplast. The expression of SuSy in cotyledons also suggests a role in protein and lipid synthesis. In summary, the developing cotton seed provides an excellent example of the diverse roles played by SuSy in carbon metabolism.     

Physical and Kinetic Evidence for an Association between Sucrose-Phosphate Synthase and Sucrose-Phosphate Phosphatase

The possible formation of a multienzyme complex between sucrose (Suc)-phosphate synthase (SPS) and Suc-phosphate phosphatase (SPP) was examined by measuring the rates of Suc-6-phosphate (Suc-6-P) synthesis and hydrolysis in mixing experiments with partially purified enzymes from spinach (Spinacia oleracea) and rice (Oryza sativa) leaves. The addition of SPP to SPS stimulated the rate of Suc-6-P synthesis. SPS inhibited the hydrolysis of exogenous Suc-6-P by SPP when added in the absence of its substrate (i.e. UDP-glucose) but stimulated SPP activity when the SPS substrates were present and used to generate Suc-6-P directly in the reaction. Results from isotope-dilution experiments suggest that Suc-6-P was channeled between SPS and SPP. A portion of the SPS activity comigrated with SPP during native polyacrylamide gel electrophoresis, providing physical evidence for an enzyme-enzyme interaction. Taken together, these results strongly suggest that SPS and SPP associate to form a multienzyme complex.     

Transgenic cotton over-producing spinach sucrose phosphate synthase showed enhanced leaf sucrose synthesis and improved fiber quality under controlled environmental conditions

Prior data indicated that enhanced availability of sucrose, a major product of photosynthesis in source leaves and the carbon source for secondary wall cellulose synthesis in fiber sinks, might improve fiber quality under abiotic stress conditions. To test this hypothesis, a family of transgenic cotton plants (Gossypium hirsutum cv. Coker 312 elite) was produced that over-expressed spinach sucrose-phosphate synthase (SPS) because of its role in regulation of sucrose synthesis in photosynthetic and heterotrophic tissues. A family of 12 independent transgenic lines was characterized in terms of foreign gene insertion, expression of spinach SPS, production of spinach SPS protein, and development of enhanced extractable V _max SPS activity in leaf and fiber. Lines with the highest V _max SPS activity were further characterized in terms of carbon partitioning and fiber quality compared to wild-type and transgenic null controls. Leaves of transgenic SPS over-expressing lines showed higher sucrose:starch ratio and partitioning of ¹⁴C to sucrose in preference to starch. In two growth chamber experiments with cool nights, ambient CO₂ concentration, and limited light below the canopy, the transgenic line with the highest SPS activity in leaf and fiber had higher fiber micronaire and maturity ratio associated with greater thickness of the cellulosic secondary wall.     

Transient transformation and RNA silencing in Zinnia tracheary element differentiating cell cultures

The Zinnia elegans cell culture system is a robust and physiologically relevant in vitro system for the study of xylem formation. Freshly isolated mesophyll cells of Zinnia can be hormonally induced to semisynchronously transdifferentiate into tracheary elements (TEs). Although the system has proven to be valuable, its utility is diminished by the lack of an efficient transformation protocol. We herein present a novel method to introduce DNA/RNA efficiently into Zinnia cells by electroporation-based transient transformation. Using reporter gene plasmids, we optimized the system for efficiency of transformation and ability for the transformed cells to transdifferentiate into TEs. Optimal conditions included a partial digestion of the cell walls by pectolyase, a low voltage and high capacitance electrical pulse and an optimal medium to maintain cell viability during transformation. Beyond the simple expression of a reporter protein in Zinnia cells, we extended our protocol to subcellular protein targeting, simultaneous co-expression of several reporter proteins and promoter-activity monitoring during TE differentiation. Most importantly, we tested the system for double-stranded RNA (dsRNA)-induced RNA silencing. By introducing in vitro -synthesized dsRNAs, we were able to phenocopy the Arabidopsis cellulose synthase (CesA) mutants that had defects in secondary cell-wall synthesis. Suppressing the expression of Zinnia CesA homologues resulted in an increase of abnormal TEs with aberrant secondary walls. Our electroporation-based transient transformation protocol provides the suite of tools long required for functional analysis and developmental studies at single cell levels.     

Elevated Levels of Both Sucrose-Phosphate Synthase and Sucrose Synthase in Vicia Guard Cells Indicate Cell-Specific Carbohydrate Interconversions

A long series of reports correlate larger stomatal aperture size with elevated concentration of sucrose (Suc) in guard cells. To assess the role and autonomy of guard cells with respect to these changes, we have determined quantitatively the cellular distribution of the synthetic enzyme, Suc-phosphate synthase (SPS) and the degradative enzyme Suc synthase (SS) in Vicia leaflet. As expected for Suc-exporting cells, the photosynthetic parenchyma had a high SPS:SS ratio of approximately 45. Also as expected, in epidermal cells, which had only few and rudimentary plastids, the SPS:SS ratio was low (0.4). Of all cells and tissues measured, those that had the highest specific activity of SPS (about 4.8 [mu]mol mg-1 of protein h-1) were guard cells. Guard cells also had a very high relative specific activity of SS.     

The proteins involved in sucrose synthesis in the marine cyanobacterium Synechococcus sp. PCC 7002 are encoded by two genes transcribed from a gene cluster

It has been reported that higher plants and cyanobacteria synthesize sucrose (Suc) by a similar sequential action of sucrose-phosphate synthase (SPS) and sucrose-phosphate phosphatase (SPP). In the genome of the marine unicellular cyanobacterium Synechococcus sp. PCC 7002 there is a sequence that was not annotated as a putative SPP encoding gene (sppA), although the sequence was available. In this study, we functionally characterize the sppA gene of that strain and demonstrate that it is cotranscribed with spsA, the SPS encoding gene. This is the first report on the coordination of Suc synthesis gene expression in an oxygenic-photosynthetic organism.     

Characterization of a novel cellulose synthesis inhibitor

The physiological effects of an experimental herbicide and cellulose synthesis inhibitor, N2-(1-ethyl-3-phenylpropyl)-6-(1-fluoro-1-methylethyl)-1,3,5-triazine-2,4-diamine, called AE F150944, are described. In the aminotriazine molecular class, AE F150944 is structurally distinct from other known cellulose synthesis inhibitors. It specifically inhibits crystalline cellulose synthesis in plants without affecting other processes that were tested. The effects of AE F150944 on dicotyledonous plants were tested on cultured mesophyll cells of Zinnia elegans L. cv. Envy, which can be selectively induced to expand via primary wall synthesis or to differentiate into tracheary elements via secondary wall synthesis. The IC₅₀ values during primary and secondary wall synthesis in Z. elegans were 3.91×10^–8 M and 3.67×10^–9 M, respectively. The IC₅₀ in suspension cultures of the monocot Sorghum halapense (L.) Pers., which were dividing and synthesizing primary walls, was 1.67×10^–10 M. At maximally inhibitory concentrations, 18–33% residual crystalline cellulose synthesis activity remained, with the most residual activity observed during primary wall synthesis in Z. elegans. Addition to Z. elegans cells of two other cellulose synthesis inhibitors, 1 M 2,6-dichlorobenzonitrile and isoxaben, along with AE F150944 did not eliminate the residual cellulose synthesis, indicating little synergy between the three inhibitors. In differentiating tracheary elements, AE F150944 inhibited the deposition of detectable cellulose into patterned secondary wall thickenings, which was correlated with delocalization of lignin as described previously for 2, 6-dichlorobenzonitrile. Freeze-fracture electron microscopy showed that the plasma membrane below the patterned thickenings of AE F150944-treated tracheary elements was depleted of cellulose-synthase-containing rosettes, which appeared to be inserted intact into the plasma membrane followed by their rapid disaggregation. AE F150944 also inhibited cellulose-dependent growth in the rosette-containing alga, Spirogyra pratensis, but it did not inhibit cellulose synthesis in Acetobacter xylinum or Dictyostelium discoideum, both of which synthesize cellulose via linear terminal complexes. Therefore, AE F150944 may inhibit crystalline cellulose synthesis by destabilizing plasma membrane rosettes.Abbreviations AE F150944 N2-(1-ethyl-3-phenylpropyl)-6-(1-fluoro-1-methylethyl)-1,3,5-triazine-2,4-diamine - CBI cellulose biosynthesis inhibiting - CGA CGA 325615, 1-cyclohexyl-5-(2,3,4,5,6-pentafluorophenoxy)-14,2,4,6-thiatriazin-3-amine - DCB 2,6-dichlorobenzonitrile - TE tracheary element     

Sucrose synthase isoforms in cultured tobacco cells.

The plant enzyme sucrose synthase (SuSy; EC 2.4.1.13) catalyzes the reversible conversion of sucrose and UDP into UDP-glucose (UDP-Glc) and fructose. The enzyme exists in different isoforms and is both located in the cytosol, membrane-bound and associated to the actin cytoskeleton. We here investigate sucrose synthase from tobacco (Nicotiana tabacum L.) BY-2 heterotrophic cell suspensions. Two different isoforms of sucrose synthase SuSy1 and SuSy2, could be purified from cytosolic extracts of these cells using a combination of poly(ethylene glycol) (PEG) precipitation, gel filtration, ion-exchange chromatography and affinity chromatography. They were clearly distinct, both with regard to the binding to the ion-exchange column and with regard to their kinetic and regulatory properties. SuSy1, the more abundant species, showed lower V(max) and K(m) for sucrose and UDP compared to the less abundant SuSy2. The activity of SuSy2 in the breakdown direction was stimulated by 60% by actin, in contrast to that of SuSy1, which showed a 17% inhibition. An indication of interaction between SuSy1 and actin was obtained by partitioning in aqueous Dextran-PEG two-phase systems. Furthermore, fructose 2,6-bisphosphate (F26BP) at micromolar concentrations stimulated SuSy2 in the presence of actin while SuSy1 was strongly inhibited by fructose. Possible roles of these two isoforms in the sucrose turnover in BY-2 cells are discussed.     

Characterization of the Biosynthetic Pathway of Cellulose from Glucose and Fructose in Acetobacter xylinum

For characterization of the biosynthetic pathway of cellulose in a cellulose-producing Acetobacter xylinum strain BPR2001, the activities of several enzymes were measured. The activity of phosphoglucose isomerase catalyzing the conversion of fructose-6- phosphate into glucose-6-phosphate was greatly increased by fructose in the medium. The UDP-glucose pyrophosphorylase activity catalyzing the synthesis of UDP-glucose was very high in strain BPR2001, consistent with the idea that this is the key enzyme in cellulose biosynthesis. Strain BPR2001 was found to have a fructose-specific phosphoenolpyruvate-dependent phosphotransferase system (PTS).     

Diurnal Changes in Maize Leaf Photosynthesis : II. Levels of Metabolic Intermediates of Sucrose Synthesis and the Regulatory Metabolite Fructose 2,6-Bisphosphate

Diurnal changes in the regulatory metabolite, fructose-2,6-bisphosphate (F26BP), and key metabolic intermediates of sucrose biosynthesis were studied in maize (Zea mays L. cv Pioneer 3184) during a day-night cycle. Whole leaf concentrations of dihydroxyacetonephosphate (DHAP) and fructose 1,6-bisphosphate changed markedly during the photoperiod. DHAP concentration was correlated positively with the rate of sucrose formation in vivo (assimilate export plus sucrose accumulation) and extractable activity of sucrose phosphate synthase (SPS). The changes closely followed net photosynthetic rate, which tracked irradiance. The other metabolic intermediates measured (glucose 6-phosphate, fructose 6-phosphate, and UDP-glucose) were either relatively constant over the 24 hour period or changed in a different pattern. Diurnal changes in leaf F26BP concentrations were pronounced, and fundamentally different than the pattern reported with other species. F26BP concentration decreased at the beginning of the day and remained low and constant; a 3- to 4-fold increase occurred with darkness, and slowly declined thereafter. In general, leaf F26BP concentration was negatively correlated with net photosynthetic rate, and also leaf DHAP concentration. Consequently, co-ordination of the regulation of cytosolic fructose 1,6-bisphosphatase and SPS was apparent. The results support the postulate that in maize leaves the activation state of SPS may be dependent on availability of DHAP and possibly other metabolites.     

Carbon allocation in developing spruce needles. Enzymes and intermediates of sucrose metabolism

In lyophilized needles of Norway spruce ( Picea abies [L.] Karsten) and starting from bud break, we determined enzyme activities (sucrose phosphate synthase [SPS; EC 2.4,1.14]. sucrose synthase [SS; EC 2.4,1.13]. acid invertase [AI; EC 3.2,1.26]) and intermediates (starch, sucrose, glucose, fructose; fructose 6-phosphate, fructose 2.6-bisphosphate [F26BP]) of carbohydrate metabolism together with needle weight, shoot length, chlorophyll and protein. For up to 110 days after bud break, samples were taken twice a week from about 25-year-old trees under field conditions. At least three periods can be distinguished during needle maturation. During the first period (up to 45 days after bud break) Al showed the highest extractable activity. This coincided with very high levels of F26BP (up to 11 pmol [mg dry weight]⁻¹) and a transient increase of starch in parallel to a decrease of sucrose. The interval between 45 and 70 days after bud break was characterized by high SS activity (ratio of fructose/glucose >1), much decreased levels of F26BP (down to below 1 pmol [mg dry weight]⁻¹), and a pronounced increase in the dry weight/fresh weight ratio. In parallel, starch declined and soluble carbohydrates increased. Finally, needle maturation was characterized by decreasing SS and continuously increasing SPS activities, so that the ratio of SPS/SS increased more than 6-fold. AI. however, did not decline with maturation. Changes in pool sizes of metabolites and enzyme activities (AI. SPS) are consistent with current concepts on sink/source transition. SS is obviously important with regard to the synthesis of structural polysaccharides.     

Antisense repression of sucrose synthase in carrot (Daucus carota L.) affects growth rather than sucrose partitioning

To unravel the roles of sucrose synthase in carrot, we reduced its activity in transgenic carrot plants by an antisense approach. For this purpose, the cDNA for the main form of carrot sucrose synthase was expressed in antisense orientation behind the 35S promoter of cauliflower mosaic virus. In independent antisense plant lines grown in soil, sucrose synthase activity was reduced in tap roots but not in leaves. In the sink organs, sucrose utilization was markedly decreased and higher levels of sucrose but lower levels of UDP-glucose, glucose, fructose, starch and cellulose were found. The phenotype of the antisense plants clearly differed from that of control plants. Both leaves and roots were markedly smaller, and the antisense line with the lowest sucrose synthase activity also developed the smallest plants. In most of the plant lines, the leaf-to-root dry weight ratios were not changed, suggesting that sucrose synthase in carrot is a major determinant of plant growth rather than of sucrose partitioning. In contrast to the acid invertases, which are critical for partitioning of assimilated carbon between source leaves and tap roots (Tang et al., Plant Cell 11: 177–189 (1999)), sucrose synthase appears to be the main sucrose-cleaving activity, feeding sucrose into metabolism.