Normal growth of Arabidopsis requires cytosolic invertase but not sucrose synthase

The entry of carbon from sucrose into cellular metabolism in plants can potentially be catalyzed by either sucrose synthase (SUS) or invertase (INV). These 2 routes have different implications for cellular metabolism in general and for the production of key metabolites, including the cell-wall precursor UDPglucose. To examine the importance of these 2 routes of sucrose catabolism in Arabidopsis thaliana (L.), we generated mutant plants that lack 4 of the 6 isoforms of SUS. These mutants (sus1/sus2/sus3/sus4 mutants) lack SUS activity in all cell types except the phloem. Surprisingly, the mutant plants are normal with respect to starch and sugar content, seed weight and lipid content, cellulose content, and cell-wall structure. Plants lacking the remaining 2 isoforms of SUS (sus5/sus6 mutants), which are expressed specifically in the phloem, have reduced amounts of callose in the sieve plates of the sieve elements. To discover whether sucrose catabolism in Arabidopsis requires INVs rather than SUSs, we further generated plants deficient in 2 closely related isoforms of neutral INV predicted to be the main cytosolic forms in the root (cinv1/cinv2 mutants). The mutant plants have severely reduced growth rates. We discuss the implications of these findings for our understanding of carbon supply to the nonphotosynthetic cells of plants.Most plant cells receive essentially all of their carbon as sucrose. Sucrose catabolism in plants is one of the largest metabolic fluxes on the planet, second only to fluxes in primary carbon assimilation. Only 2 enzymes can catalyze sucrose catabolism under physiological conditions: sucrose synthase (SUS) and invertase (INV); thus, most plant biomass is derived via 1 of these 2 routes. However, despite their central role in carbon partitioning and biomass accumulation, the precise roles and relative importance of these enzymes remain largely unknown.SUS and INV both occur as multiple, distinct isoforms. INV catalyzes the effectively irreversible hydrolysis of sucrose to glucose and fructose. Isoforms in the cell wall and vacuole (acid INV) differ in structure from those predicted to be in the cytosol, mitochondria and plastids (neutral/alkaline INV). SUS catalyzes the reversible conversion of sucrose to fructose and UDPglucose; SUS isoforms are believed to be cytosolic.Several lines of evidence indicate a predominant role for SUS in the entry of carbon into metabolism in nonphotosynthetic cells. Individual isoforms are needed for normal development in some plant organs, including potato tuber, pea and maize seed, tomato fruit, and cotton fibers (1–5). SUS is held to be important in determining sink strength, and in phloem loading (1, 6, 7). It is also proposed to have specific roles in cellulose synthesis, and in starch synthesis in leaves. In the widely cited model for cellulose synthesis, the substrate UDPglucose is channeled to the cellulose synthase complex in the plasma membrane via a SUS associated with the inner face of the complex (8, 9). Consistent with this idea, some SUS activity is associated with the plasma membrane (10–12). Leaf starch synthesis is generally believed to occur via a pathway in which the substrate ADPglucose is generated inside the chloroplast, without involvement of SUS. However, a recent alternative proposal is that ADPglucose is generated via SUS from sucrose in the cytosol, then imported into the chloroplast (13). Evidence for this pathway includes parallel alterations in starch levels in leaves of transgenic potato plants in which SUS activity has been altered (14). If correct, this proposal gives SUS a central role in photosynthetic carbon assimilation and partitioning.Most of the roles proposed for INVs are specific to particular developmental stages. Vacuolar INV is involved in mobilization of vacuolar sucrose in sucrose-storing organs (15, 16). It is required for normal root elongation in Arabidopsis, probably through its impact on vacuolar osmotic potential and, thus, on water uptake (17). Cell-wall INV activity is high after wounding and pathogen attack (18, 19), and in early seed development (20), and is required for normal kernel development in maize (21) and pollen tube extension (22). The functions of neutral INV are not known, but loss of 1 of the 6 isoforms in rice (OsCYT-INV1), or 1 of the 7 in Lotus japonicus (LjINV1), strongly affects plant growth and development (23, 41). Loss of 1 of the 9 isoforms in Arabidopsis (CINV1 or CYT-INV1) has much less pronounced effects. It reduces primary root extension by ≈30% and can reduce leaf and silique expansion (24, 25).The route of sucrose catabolism has important implications for energy conservation and carbon allocation in nonphotosynthetic cells. Conversion of sucrose to hexose phosphates via SUS uses only half the ATP needed for conversion via INV. The reversibility of the reactions of the SUS route means flux via this route is sensitive to hexose phosphate levels and, thus, to demand for glycolytic intermediates (26, 27). The requirement for PP_i (as a substrate for UDPglucose pyrophosphorylase) links sucrose catabolism via SUS to other PPi-requiring processes, including flux over the reversible glycolytic enzyme PPi-dependent fructose 6-phosphate phosphotransferase. In contrast, the INV-catalyzed reaction is effectively irreversible, and INV isoforms have no reported properties that would allow coordination of sucrose catabolism with carbon demand in nonphotosynthetic cells.Despite the accepted importance of SUS, we recently showed that none of the 6 isoforms in Arabidopsis is individually required for normal growth and reproduction, neither are any of the 3 pairs of most closely related isoforms (SUS1/SUS4, SUS2/SUS3, and SUS5/SUS6) (28). Thus, there must either be a high level of redundancy within the SUS family in Arabidopsis, or INV isoforms must be able to compensate for loss of SUS in this species. To explore the implications of this finding, we have generated a quadruple mutant (the sus1/sus2/sus3/sus4 mutant) that has no detectable soluble or membrane-bound SUS activity. Remarkably, the mutant is normal with respect to growth and development, metabolite levels, seed composition, and the composition of cell walls. In marked contrast, loss of 2 of the 9 isoforms of neutral INV (the cinv1/cinv2 mutant) results in severe inhibition of growth. We discuss the implications of these results for understanding of sucrose catabolism in the nonphotosynthetic cells of plants.     

Tobacco control and the inequitable socio-economic distribution of smoking: smokers’ discourses and implications for tobacco control

Warning: this article contains strong language.

This paper focuses on the ways in which social context structures smokers’ views of, and reactions to, tobacco control. This exploratory study examined the interactions between tobacco control and smokers’ social contexts and how this may be contributing to inequalities in smoking. We found in our sample that higher socio-economic status (SES) smokers are more likely to positively respond and adapt to tobacco control messages and policies, viewing them for their future health betterment. Lower SES smokers in our study, on the other hand, are in conflict with tobacco control and feel intransigent with regard to the effects that tobacco control is having on their smoking. A better understanding of how social context structures people's perceptions of tobacco control may help us to understand why social inequalities in smoking are deepening, and potentially what can be done better in tobacco control to decrease them.     

The cysteine-histidine-rich region of the movement protein of Cucumber mosaic virus contributes to plasmodesmal targeting, zinc binding and pathogenesis

Viral movement proteins (MPs) are central to the establishment of viral pathogenesis, and yet relatively little is understood about the structural and functional aspects of MPs or about the host factors on which they depend. Through chemical mutagenesis of transgenic Arabidopsis expressing Cucumber mosaic virus (CMV) MP fused with the green fluorescent protein, we have studied the function of a central region of the MP, defined by a number of conserved cysteine and histidine residues (Cys-His-rich region), which potentially functions as a zinc-binding domain. Transient expression of mutant MPs identified through an in planta screen for altered MP function or constructed with altered putative zinc ligands through site-directed mutagenesis showed that mutations in the Cys-His-rich region affected localization to and trafficking through plasmodesmata. In vitro zinc-binding analysis revealed that wild type (wt) CMV MP had the ability to bind zinc and that movement-defective mutants bound zinc with less affinity than wt MP. Furthermore, a correlation between the association of the MP with plasmodesmata and virus pathogenesis was shown. We discuss roles of the Cys-His region in biochemical and biological functions of the MP during virus movement.     

The pattern of accumulation of cauliflower mosaic virus-specific products in infected turnips

The concentrations of cauliflower mosaic virus (CaMV) DNA and protein products in the developing leaves of a host, turnip, have been measured and the results have been correlated with symptom production. Virus-specific products were limited to the symptomatic leaves. CaMV DNA was detected in the youngest foliar tissues showing full systemic symptoms and continued to accumulate as the leaf expanded, indicating that virus multiplication was not restricted to meristematic tissues of the host plant and that virus concentration was not a primary determinant for symptom production. Using specific antisera for Western blot analysis, the distribution of CaMV-specific proteins (P1-P6) in a range of subcellular fractions of infected tissue was determined. The protein products (P2-P6) of genes II-VI were all detected in fractions enriched for virus inclusion bodies, although P5 was present only at low levels. A high-speed pellet fraction enriched for virus replication complexes revealed P5 in higher concentrations, and also contained P4 and small amounts of P6 in proportions which indicated that replication complexes had been released from inclusion bodies. In the different leaves of the host, P2, 3, 4, 5, and 6 all increased in concentration in parallel with viral DNA, although there appeared to be a bias toward protein rather than DNA synthesis in the very young leaves. P1 showed a different pattern of accumulation; it was most concentrated in the very young and the oldest infected tissues, and showed a different spectrum of products between leaves. The experiments described provide a more complete picture of the relationship between CaMV multiplication and expression, and leaf development, and an increased understanding of how the disease syndrome is established.     

Immunocytochemical demonstration of 5-hydroxytryptamine (serotonin) in the nervous system of the liver fluke,Fasciola hepatica (Trematoda,Digenea)

The localisation and distribution of 5-hydroxytryptamine (5-HT or serotonin) in the nervous system ofFasciola hepatica has been determined by an indirect immunofluorescence technique. Cell bodies and nerve fibres immunoreactive to 5-HT are present in the anterior ganglia, and the longitudinal nerve cords and their commissures in the central nervous system. In the peripheral nervous system, similar immunoreactivity occurs in the nerve plexuses supplying the sub-tegumental muscle layers and the muscular lining of various reproductive ducts, including the ootype, uterus and cirrus pouch. The significance of these results in the light of previous studies on the role of 5-HT inF. hepatica is discussed.     

Serotonin and neuropeptide immunoreactivities in the intramolluscan stages of three marine trematode parasites

Using an indirect immunofluorescence technique interfaced with confocal scanning laser microscopy, whole-mount preparations of three general of marine trematode larvae,Cryptocotyle lingua, Cercaria emasculans andHimasthla leptosoma, were screened for 5-hydroxytryptamine (5-HT) and selected neuropeptide immunoreactivities (IRs). IRs for pancreatic polypeptide (PP), peptide YY (PYY) and FMRFamide were found in the central nervous systems of the three species of cercariae, immunostaining the paired ganglia and central commissure and the longitudinal nerve cords, with slight differences in both distribution and intensity of IRs being observed for the different antisera used. PP, PYY and FMRFamide IRs were evident in both central and peripheral components of the nervous system in the rediae ofC. lingua. 5-HT IR was confined to the peripheral nervous systems of the cercariae ofC. emasculans and the rediae ofC. lingua, appearing in the form of a network of immunoreactive fibres and associated large cell bodies. A moderate substance P IR was observed in the nervous system of the cercariae ofC. lingua. The patterns of immunostaining described were compared with those obtained using antiserum directed to the C-terminal decapeptide amide of neuropeptide F (NPF), a native parasitic peptide from the cestodeMoniezia expansa. Results demonstrated that serotoninergic and peptidergic components were present in the nervous systems of all of the trematode larvae studied and that some, if not all, of the IR for PP, PYY and FMRFamide was due to the presence of a trematode NPF homologue.     

Occurrence of 5-hydroxytryptamine (serotonin) in the nervous system of a monogenean,Diclidophora merlangi

Both whole-mount preparations and cryostat sections of the monogenean, Diclidophora merlangi, were screened for the presence of 5-hydroxytryptamine-like immunoreactivity, using an indirect immunofluorescence technique. Immunostaining was evident in cell bodies and fibres comprising the longitudinal cords of the central nervous system and in the peripheral plexuses that innervate the musculature of the adhesive organs, feeding apparatus and accessory ducts of the reproductive system. The subsurface nerve-net was reactive, and a distinctive collar of staining marked a plexus of nerves and somata that supply the subtegumental muscles of the extensile forebody.