Overexpression of the Vacuolar Sugar Carrier AtSWEET16 Modifies Germination,Growth, and Stress Tolerance in Arabidopsis

Here, we report that SUGARS WILL EVENTUALLY BE EXPORTED TRANSPORTER (SWEET16) from Arabidopsis (Arabidopsis thaliana) is a vacuole-located carrier, transporting glucose (Glc), fructose (Fru), and sucrose (Suc) after heterologous expression in Xenopus laevis oocytes. The SWEET16 gene, similar to the homologs gene SWEET17, is mainly expressed in vascular parenchyma cells. Application of Glc, Fru, or Suc, as well as cold, osmotic stress, or low nitrogen, provoke the down-regulation of SWEET16 messenger RNA accumulation. SWEET16 overexpressors (35S_Pro:SWEET16) showed a number of peculiarities related to differences in sugar accumulation, such as less Glc, Fru, and Suc at the end of the night. Under cold stress, 35S_Pro:SWEET16 plants are unable to accumulate Fru, while under nitrogen starvation, both Glc and Fru, but not Suc, were less abundant. These changes of individual sugars indicate that the consequences of an increased SWEET16 activity are dependent upon the type of external stimulus. Remarkably, 35S_Pro:SWEET16 lines showed improved germination and increased freezing tolerance. The latter observation, in combination with the modified sugar levels, points to a superior function of Glc and Suc for frost tolerance. 35S_Pro:SWEET16 plants exhibited increased growth efficiency when cultivated on soil and showed improved nitrogen use efficiency when nitrate was sufficiently available, while under conditions of limiting nitrogen, wild-type biomasses were higher than those of 35S_Pro:SWEET16 plants. Our results identify SWEET16 as a vacuolar sugar facilitator, demonstrate the substantial impact of SWEET16 overexpression on various critical plant traits, and imply that SWEET16 activity must be tightly regulated to allow optimal Arabidopsis development under nonfavorable conditions.Sugars are of enormous importance for plant properties and the agronomic values of most crop species (John, 1992). In plants, they serve as energy reserves, as building blocks for carbohydrate polymers like starch or cellulose, as precursors for amino and carboxylic acids, and as osmolytes required for the molecular antifreezing program initiated after exposure to cold temperatures (Nägele et al., 2010).Sugars in leaves are synthesized either during the day via photosynthesis or in the night as a product of starch degradation. The major sugar synthesized in most plants during the day is Suc, which, after the export of triose phosphates from the chloroplast, is synthesized in the cytosol. During nocturnal starch degradation, maltose leaves the chloroplast and serves as a substrate for the cytosolic synthesis of heteroglycans (Fettke et al., 2005). Subsequent to this, heteroglycans are degraded by phosphorylases (Fettke et al., 2005) and act as a carbon source to synthesize Suc, which can be hydrolyzed by cytosolic or vacuolar invertases to monosaccharides (Roitsch and González, 2004). These processes, in sum, enable leaf mesophyll cells to synthesize Glc and Fru, in addition to Suc, during the day and at night.Besides these metabolic processes, sugars are transported between different intracellular compartments and between different cells in order to serve as a long-distance carbon supply for sink organs. Due to their large size and hydrate shell, the movement of neutral sugars like Suc, Glc, or Fru across membranes requires the presence of membrane-bound carriers. For example, in the plant plasma membrane, a wide number of monosaccharide- and Suc-specific carriers were identified and have been analyzed with biochemical and molecular approaches. The Arabidopsis (Arabidopsis thaliana) genome harbors more than 50 isoforms of putative monosaccharide carriers, most of which belong to the sugar transport protein subfamily (Büttner and Sauer, 2000), while about 20 putative disaccharide carriers sucrose transporters (named SUT and SUC) are present in this plant species (Lalonde et al., 2004). Most of the sugar transport protein, SUT, or SUC carriers analyzed so far reside in the plasma membrane and import, as proton-coupled transporters, apoplastic sugars against a concentration gradient (Lalonde et al., 2004). This proton-driven sugar import allows a substantial accumulation of Suc in phloem sieve elements, building the driving force for interorgan long-distance sugar transport (Turgeon and Wolf, 2009). All monosaccharide and disaccharide carriers mentioned above exhibit 12 predicted transmembrane domains and group into the large major facilitator superfamily of carriers (Marger and Saier, 1993).In both photosynthetic active mesophyll cells as well as storage tissues, the large central vacuole represents the internal storage compartment for sugars (Martinoia et al., 2007, 2012), leading, in sugar beet (Beta vulgaris) or sugarcane (Saccharum officinarum), up to even 20% sugars per fresh biomass (John, 1992). Suc import into the vacuole occurs either via facilitated diffusion (Kaiser and Heber, 1984) or electrogenically via antiport against protons (Willenbrink and Doll, 1979). The latter process is driven by the significant proton-motive force across the vacuolar membrane (Schumacher and Krebs, 2010) and allows a substantial Suc accumulation in storage organs of high-sugar species (Getz, 1987; Getz and Klein, 1995). However, no Suc importer at the vacuolar membrane (tonoplast) has been identified on the molecular level yet, while tonoplast-located Suc exporters have been identified. This vacuolar Suc export is mediated by members of the SUT4-type clade of carriers, in cereals named SUT2 (Endler et al., 2006; Eom et al., 2011), procuring a proton-driven Suc export into the cytosol (Schulz et al., 2011). Loss of function of this type of carrier in Arabidopsis, poplar (Populus spp.), or rice (Oryza sativa) leads to an accumulation of Suc in leaves (Eom et al., 2011; Payyavula et al., 2011; Schneider et al., 2012), elegantly proving that this type of carrier fulfills an export function under in vivo conditions.In contrast to vacuolar Suc import, the import of monosaccharides into this compartment has been deciphered on the molecular level. In the Arabidopsis tonoplast, two different monosaccharide importers have been identified, namely the vacuolar Glc transporter protein and three isoforms of the tonoplast monosaccharide transporter (TMT; Wormit et al., 2006; Aluri and Büttner, 2007). While vacuolar Glc transporter loss-of-function plants do not show significant changes in monosaccharide levels (Aluri and Büttner, 2007), decreased TMT activity correlates with impaired vacuolar sugar import and low levels of both Glc and Fru in leaves (Wormit et al., 2006). This fact and the observations that (1) TMT1 is a sugar/proton antiporter (Schulz et al., 2011), (2) increased TMT activity provokes improved seed biomass (Wingenter et al., 2010), and (3) TMT activity is highly regulated via protein phosphorylation (Wingenter et al., 2011) clearly underline the superior function of TMT for monosaccharide loading into the plant vacuole.So far, two carriers, ESL1 and ERDL6, have been found to be responsible for Glc export from the plant vacuole (Yamada et al., 2010; Poschet et al., 2011). ESL1 (for early responsive to dehydration6-like1) represents a carrier majorly expressed in pericycle and xylem parenchyma cells and is known to be induced by drought stress (Yamada et al., 2010). Loss-of-function mutants of the ERDL6 (for early responsive to dehydration6-like6) carrier show increased leaf Glc levels and decreased seed weight, indicating that controlled Glc export via this carrier is critical for interorgan movement of sugars in Arabidopsis (Poschet et al., 2011). ESL1 seems to transport Glc in a facilitated diffusion, while in contrast to the plasma membrane-located sugar carriers and to TMT, the transport mode of ERDL6 has not been identified so far.In marked contrast to the carriers mentioned above, the recent identification of the so-called SWEET proteins opened our understanding of how cellular sugar export is achieved. SWEET proteins occur in plants as well as in animals and humans and consist of only seven predicted transmembrane domains (Chen et al., 2010). The observation that the expression of several plant SWEET proteins is strongly induced by various pathogens indicated that they serve as sugar exporters. That hypothesis has been proven for some SWEET isoforms by heterologous expression in Xenopus laevis oocytes (Chen et al., 2010), and detailed analysis revealed that Arabidopsis SWEET11 and SWEET12 catalyze Suc export from source leaves and are critical for interorgan sugar transport (Chen et al., 2012).In a recent quantitative trait locus analysis, we identified SWEET17 as a novel determinant of leaf Fru content, especially under cold conditions and conditions of low nitrogen supply (Chardon et al., 2013). In fact, a detailed molecular-physiological analysis revealed that SWEET17 is the first vacuole-located SWEET protein and that it serves as a Fru-specific exporter, connecting the vacuolar lumen to the cytosol. In contrast to SWEET17, the subcellular localization of its closest homolog, SWEET16, is elusive. Moreover, transport properties of SWEET16 are unknown, and the effect of increased SWEET16 activity (or any other SWEET proteins) on plant properties has not been determined. The latter aspect is of particular interest, since most genes coding for SWEET proteins are only comparably weakly expressed or are only expressed in certain cell types (Chen et al., 2010; Chardon et al., 2013).In this report, we analyzed the intracellular localization of SWEET16 and studied its transport properties in X. laevis oocytes. Moreover, we constructed constitutive SWEET16-overexpressing Arabidopsis lines and report the impact of this overexpression of a vacuolar SWEET protein on plant development and stress tolerance. Our results support the hypothesis that the activity of a SWEET facilitator has to be controlled in planta to cope with altering environmental and developmental conditions.     

LONG‐TERM CHANGES IN THE MACROALGAL VEGETATION OF THE INNER GULLMAR FJORD,SWEDISH SKAGERRAK COAST1

We examined long‐term changes in the macroalgal vegetation at Stora Bornö Island in the inner Gullmar Fjord on the Swedish Skagerrak coast. This was made possible by access to a 1941 diving investigation. The same sites were reinvestigated in 1998. Community composition and depth distributions of species were compared and changes were analyzed with focus on functional groups (size, thallus shape, and life‐history traits). We discovered a significant decrease in the depth extension of macroalgal species and a dramatic decline of species richness in the lower littoral (below 16 m of depth) compared with 57 years earlier. Ordination analysis revealed that there was a significant difference in the community composition between the two study periods. In general, small (<10 cm), thin, filamentous, and aseasonal ephemerals increased in relative abundance, whereas larger (>10 cm), coarsely branched, and perennial algae decreased. Calibrations of individual species to local sediment cover, using canonical correspondence analysis, indicated that part of the change in species composition was related to sediment load. Furthermore, large‐scale climate differences (NAO Winter Index) between the study periods indicated a higher impact of Baltic Sea and Kattegat water in the nutrient dynamics of the fjord in the 1998 study. We concluded that the observed long‐term changes in the macroalgal community at Stora Bornö Island were consistent with an increased nutrient availability.     

Fluid-structure interaction simulation of tissue degradation and its effects on intra-aneurysm hemodynamics

Biomechanics and Modeling in Mechanobiology - Tissue degradation plays a crucial role in vascular diseases such as atherosclerosis and aneurysms. Computational modeling of vascular hemodynamics...     

Dominance by a canopy forming seaweed modifies resource and consumer control of bloom-forming macroalgae

Degradation of ecological resources by large-scale disturbances highlights the need to demonstrate biological properties that increase resistance to change and promote the resilience of ecosystem regimes. Coastal eutrophication is a global-scale disturbance that drives ecosystem change by increasing primary production and favouring ephemeral and bloom-forming life-forms. Recent synthesis indicates that consumption processes increase the resistance of coastal communities to nutrient loading by controlling the responses of ephemeral macroalgae. Here we suggest a similar ecological function for canopy cover by demonstrating that the presence of a canopy species modifies both resource and consumer control of bloom-forming algae associated with nutrient enrichment. We tested effects of canopy presence on the interaction between consumer and resource control, by field-manipulations of a dominant canopy forming seaweed ( Fucus vesiculosus ), grazer presence (dominated by the gastropod Littorina littorea ) and nutrient enrichment (common agricultural NPK fertilizer). Canopy cover and grazers jointly controlled strong increases of ephemeral bloom-forming algae (dominated by Ulva spp) from nutrient enrichment; nutrients increased ephemeral recruitment almost 10-fold, but only in the absence of both grazers and canopy cover. Recruitment success of the canopy-forming seaweed itself decreased additively with 56.1, 71.3 and 50.5% from independent effects of canopy cover, grazers and nutrient enrichment, respectively. A meta-analysis of nine nutrient enrichment experiments including seaweed, seagrass and stream communities, showed that in the presence of canopies average nutrient effects were reduced by more than 90% compared to without canopies. This corroborates the generality of our finding that dominating canopy species are important for aquatic ecosystems by increasing community resistance to the propagation of nutrient effects.     

Directional changes of species spatial dispersion and realized environmental niches drive plant community assembly during early plant succession

Aims Probabilistic models of species co-occurrences predict aggregated intraspecific spatial distributions that might decrease the degree of joint species occurrences and increase community richness. Yet, little is known about the influence of intraspecific aggregation on the co-occurrence of species in natural, species-rich communities. Here, we focus on early plant succession and ask how changes in intraspecific aggregation of colonizing plant species influence the pattern of species co-existence, richness and turnover.     

The structure of Rpf2–Rrs1 explains its role in ribosome biogenesis

The assembly of eukaryotic ribosomes is a hierarchical process involving about 200 biogenesis factors and a series of remodeling steps. The 5S RNP consisting of the 5S rRNA, RpL5 and RpL11 is recruited at an early stage, but has to rearrange during maturation of the pre-60S ribosomal subunit. Rpf2 and Rrs1 have been implicated in 5S RNP biogenesis, but their precise role was unclear. Here, we present the crystal structure of the Rpf2–Rrs1 complex from Aspergillus nidulans at 1.5 Å resolution and describe it as Brix domain of Rpf2 completed by Rrs1 to form two anticodon-binding domains with functionally important tails. Fitting the X-ray structure into the cryo-EM density of a previously described pre-60S particle correlates with biochemical data. The heterodimer forms specific contacts with the 5S rRNA, RpL5 and the biogenesis factor Rsa4. The flexible protein tails of Rpf2–Rrs1 localize to the central protuberance. Two helices in the Rrs1 C-terminal tail occupy a strategic position to block the rotation of 25S rRNA and the 5S RNP. Our data provide a structural model for 5S RNP recruitment to the pre-60S particle and explain why removal of Rpf2–Rrs1 is necessary for rearrangements to drive 60S maturation.     

Biodiversity mediates top–down control in eelgrass ecosystems: a global comparative‐experimental approach

Nutrient pollution and reduced grazing each can stimulate algal blooms as shown by numerous experiments. But because experiments rarely incorporate natural variation in environmental factors and biodiversity, conditions determining the relative strength of bottom–up and top–down forcing remain unresolved. We factorially added nutrients and reduced grazing at 15 sites across the range of the marine foundation species eelgrass (Zostera marina) to quantify how top–down and bottom–up control interact with natural gradients in biodiversity and environmental forcing. Experiments confirmed modest top–down control of algae, whereas fertilisation had no general effect. Unexpectedly, grazer and algal biomass were better predicted by cross‐site variation in grazer and eelgrass diversity than by global environmental gradients. Moreover, these large‐scale patterns corresponded strikingly with prior small‐scale experiments. Our results link global and local evidence that biodiversity and top–down control strongly influence functioning of threatened seagrass ecosystems, and suggest that biodiversity is comparably important to global change stressors.