The Arabidopsis vacuolar sugar transporter SWEET2 limits carbon sequestration from roots and restricts Pythium infection

Plant roots secrete a significant portion of their assimilated carbon into the rhizosphere. The putative sugar transporter SWEET2 is highly expressed in Arabidopsis roots. Expression patterns of SWEET2–β‐glucuronidase fusions confirmed that SWEET2 accumulates highly in root cells and thus may contribute to sugar secretion, specifically from epidermal cells of the root apex. SWEET2–green fluorescent protein fusions localized to the tonoplast, which engulfs the major sugar storage compartment. Functional analysis of SWEET2 activity in yeast showed low uptake activity for the glucose analog 2‐deoxyglucose, consistent with a role in the transport of glucose across the tonoplast. Loss‐of‐function sweet2 mutants showed reduced tolerance to excess glucose, lower glucose accumulation in leaves, and 15–25% higher glucose‐derived carbon efflux from roots, suggesting that SWEET2 has a role in preventing the loss of sugar from root tissue. SWEET2 root expression was induced more than 10‐fold during Pythium infection. Importantly, sweet2 mutants were more susceptible to the oomycete, showing impaired growth after infection. We propose that root‐expressed vacuolar SWEET2 modulates sugar secretion, possibly by reducing the availability of glucose sequestered in the vacuole, thereby limiting carbon loss to the rhizosphere. Moreover, the reduced availability of sugar in the rhizosphere due to SWEET2 activity contributes to resistance to Pythium.     

SWEET17, a Facilitative Transporter,Mediates Fructose Transport across the Tonoplast of Arabidopsis Roots and Leaves

Fructose (Fru) is a major storage form of sugars found in vacuoles, yet the molecular regulation of vacuolar Fru transport is poorly studied. Although SWEET17 (for SUGARS WILL EVENTUALLY BE EXPORTED TRANSPORTERS17) has been characterized as a vacuolar Fru exporter in leaves, its expression in leaves is low. Here, RNA analysis and SWEET17-β-glucuronidase/-GREEN FLUORESCENT PROTEIN fusions expressed in Arabidopsis (Arabidopsis thaliana) reveal that SWEET17 is highly expressed in the cortex of roots and localizes to the tonoplast of root cells. Expression of SWEET17 in roots was inducible by Fru and darkness, treatments that activate accumulation and release of vacuolar Fru, respectively. Mutation and ectopic expression of SWEET17 led to increased and decreased root growth in the presence of Fru, respectively. Overexpression of SWEET17 specifically reduced the Fru content in leaves by 80% during cold stress. These results intimate that SWEET17 functions as a Fru-specific uniporter on the root tonoplast. Vacuoles overexpressing SWEET17 showed increased [¹⁴C]Fru uptake compared with the wild type. SWEET17-mediated Fru uptake was insensitive to ATP or treatment with NH₄Cl or carbonyl cyanide m-chlorophenyl hydrazone, indicating that SWEET17 functions as an energy-independent facilitative carrier. The Arabidopsis genome contains a close paralog of SWEET17 in clade IV, SWEET16. The predominant expression of SWEET16 in root vacuoles and reduced root growth of mutants under Fru excess indicate that SWEET16 also functions as a vacuolar transporter in roots. We propose that in addition to a role in leaves, SWEET17 plays a key role in facilitating bidirectional Fru transport across the tonoplast of roots in response to metabolic demand to maintain cytosolic Fru homeostasis.Sugars are main energy sources for generating ATP, major precursors to various storage carbohydrates as well as key signaling molecules important for normal growth in higher plants (Rolland et al., 2006). Depending on the metabolic demand, sugars are translocated over long distances or stored locally. SWEET (for SUGARS WILL EVENTUALLY BE EXPORTED TRANSPORTERS) and SUC/SUT (for Sucrose transporter/Sugar transporter)-type transporters are responsible for transfer of Suc from the phloem parenchyma into the sieve element companion cell complex for long-distance translocation (Riesmeier et al., 1992; Sauer, 2007; Kühn and Grof, 2010; Chen et al., 2012). Suc or hexoses derived from Suc hydrolysis in the cell wall are then taken up into sink cells by SUT (Braun and Slewinski, 2009) or monosaccharide transporters, such as sugar transporter1 (Sauer et al., 1990; Pego and Smeekens, 2000; Sherson et al., 2003). Alternatively, sugars are thought to move between cells via plasmodesmata (Voitsekhovskaja et al., 2006; Ayre, 2011). Major sugar storage pools within plant cells are soluble sugars stored in the vacuole, starch in plastids, and lipids in oil bodies.Vacuoles, which can account for approximately 90% of the cell volume (Winter et al., 1993), play central roles in temporary and long-term storage of soluble sugars (Martinoia et al., 2007; Etxeberria et al., 2012). Some agriculturally important crops such as sugar beet (Beta vulgaris; Leigh, 1984; Getz and Klein, 1995), citrus (Citrus spp.; Echeverria and Valich, 1988), sugarcane (Saccharum officinarum; Thom et al., 1982), and carrot (Daucus carota; Keller, 1988) can store considerable amounts (>10% of plant dry weight) of Suc, Glc, or Fru in vacuoles of the storage parenchyma. Due to a high capacity of vacuoles for storing sugars, vacuolar sugars can serve as an important carbohydrate source during energy starvation, e.g. after starch has been exhausted (Echeverria and Valich, 1988), as well as for the production of other compounds (e.g. osmoprotectants). Sugars are known to regulate photosynthesis; therefore, the release of sugars from vacuoles could be important for modulating photosynthesis (Kaiser and Heber, 1984). Moreover, vacuole-derived sugars are commercially used to produce biofuels, such as ethanol, from sugarcane. Knowledge of the key transporters involved in sugar exchange between the vacuole and cytoplasm is thus relevant in the context of bioenergy (Grennan and Gragg, 2009).To facilitate the exchange of sugars across the tonoplast, plant vacuoles are equipped with a multitude of transporters (Neuhaus, 2007; Etxeberria et al., 2012; Martinoia et al., 2012) comprising both facilitated diffusion and active transport systems of vacuolar sugars (Martinoia et al., 2000). Typically, Suc is actively imported into vacuoles by tonoplast monosaccharide transporter (AtTMT1/AtTMT2; Schulz et al., 2011) and exported by the SUT4 family (AtSUC4, OsSUT2; Eom et al., 2011; Payyavula et al., 2011; Schulz et al., 2011). Two H⁺-dependent sugar antiporters, the vacuolar Glc transporter (AtVGT1; Aluri and Büttner, 2007) and AtTMT1 (Wormit et al., 2006), mediate Glc uptake across the tonoplast to promote carbohydrate accumulation in Arabidopsis (Arabidopsis thaliana). The Early Responsive to Dehydration-Like6 protein has been shown to export vacuolar Glc into the cytosol (Poschet et al., 2011), likely via an energy-independent diffusion mechanism (Yamada et al., 2010). Defects in these vacuolar sugar transporters alter carbohydrate partitioning and allocation and inhibit plant growth and seed yield (Aluri and Büttner, 2007; Wingenter et al., 2010; Eom et al., 2011; Poschet et al., 2011).In contrast to numerous studies on vacuolar transport of Suc and Glc, limited efforts have been devoted to the molecular mechanism of vacuolar Fru transport even though Fru is predominantly located in vacuoles (Martinoia et al., 1987; Voitsekhovskaja et al., 2006; Tohge et al., 2011). Vacuolar Fru is important for the regulation of turgor pressure (Pontis, 1989), antioxidative defense (Bogdanović et al., 2008), and signal transduction during early seedling development (Cho and Yoo, 2011; Li et al., 2011). Thus, control of Fru transport across the tonoplast is thought to be important for plant growth and development. One vacuolar Glc transporter from the Arabidopsis monosaccharide transporter family, VGT1, has been reported to mediate low-affinity Fru uptake when expressed in yeast (Saccharomyces cerevisiae) vacuoles (Aluri and Büttner, 2007). Yet, the high vacuolar uptake activity to Fru intimates the existence of additional high-capacity Fru-specific vacuolar transporters (Thom et al., 1982). Recently, quantitative mapping of a quantitative trait locus for Fru content of leaves led to the identification of the Fru-specific vacuolar transporter SWEET17 (Chardon et al., 2013).SWEET17 belongs to the recently identified SWEET (PFAM:PF03083) super family, which contains 17 members in Arabidopsis and 21 in rice (Oryza sativa; Chen et al., 2010; Frommer et al., 2013; Xuan et al., 2013). Based on homology with 27% to 80% amino acid identity, plant SWEET proteins were grouped into four subclades (Chen et al., 2010). Analysis of GFP fusions indicated that most SWEET transporters are plasma membrane localized. Transport assays using radiotracers in Xenopus laevis oocytes and sugar nanosensors in mammalian cells showed that they function as largely pH-independent low-affinity uniporters with both uptake and efflux activity (Chen et al., 2010, 2012). In particular, clade I and II SWEETs transport monosaccharides and clade III SWEETs transport disaccharides, mainly Suc (Chen et al., 2010, 2012). Mutant phenotypes and developmental expression of several SWEET transporters support important roles in sugar translocation between organs. The clade III SWEETs, in particular SWEET11 and 12, mediate the key step of Suc efflux from phloem parenchyma cells for phloem translocation (Chen et al., 2012). Moreover, SWEETs are coopted by pathogens, likely to provide energy resources and carbon at the site of infection (Chen et al., 2010). Mutations of SWEET8/Ruptured pollen grain1 in Arabidopsis, and RNA inhibition of OsSWEET11 (also called Os8N3 or Xa13) in rice, and petunia (Petunia hybrida) NEC1 resulted in male sterility (Ge et al., 2001; Yang et al., 2006; Guan et al., 2008), possibly caused by inhibiting the Glc supply to developing pollen (Guan et al., 2008). Interestingly, two members, SWEET16 and SWEET17, of the family localize to the tonoplast (Chardon et al., 2013; Klemens et al., 2013). Allelic variation or mutations that affect SWEET17 expression caused Fru accumulation in Arabidopsis leaves, indicating that it plays a key role in exporting Fru from leaf vacuoles (Chardon et al., 2013). A more recent study demonstrated that SWEET16 also functions as a vacuolar sugar transporter (Klemens et al., 2013). Surprisingly, however, SWEET17 expression in mature leaves was comparatively low (Chardon et al., 2013), which leads us to ask whether SWEET17 could mainly function in other tissues under specific developmental or environmental conditions. Although Arabidopsis SWEET17 has been shown to transport Fru in a heterologous system where it accumulated in part at the plasma membrane (Chardon et al., 2013), the biochemical properties of SWEET17 were still elusive. SWEET16 and SWEET17 from Arabidopsis belong to the clade IV SWEETs. Whether clade IV proteins both transport vacuolar sugars in planta deserves further studies.Here, we used GUS/GFP fusions to reveal the root-dominant expression and vacuolar localization of the SWEET17 protein in vivo and its regulation by Fru levels. Phenotypes of mutants and overexpressors were consistent with a role of SWEET17 in bidirectional Fru transport across root vacuoles. The uniport feature of SWEET17 transport was further confirmed using isolated mesophyll vacuoles. Similarly, SWEET16 is also shown to function in vacuolar sugar transport in roots. Our work, performed in parallel to the two other studies (Chardon et al., 2013; Klemens et al., 2013), provides direct evidence for Fru uniport by SWEET17 and presents functional analyses to uncover important roles of these vacuolar transporters in maintaining intracellular Fru homeostasis in roots.     

Chloroplast protein PLGG1 is involved in abscisic acid-regulated lateral root development and stomatal movement in Arabidopsis

The plant hormone abscisic acid (ABA) plays a crucial role in root architecture; however, the molecular mechanism of ABA-regulated lateral root (LR) growth is not well known. We screened an Arabidopsis thaliana mutant with LR growth that was sensitive to ABA from a T-DNA insertion mutant library, which was an allelic mutant of plgg1-1, termed plgg1-2. PLGG1 encodes a chloroplast protein that transports plastidic glycolate and glycerate. The length and number of LRs at the root-hypocotyl junction of plgg1-1 and plgg1-2 were significantly impaired under exogenous ABA treatment, and the transgenic plant complementary lines of plgg1-2 restored LR growth in response to ABA. In addition, we found that PLGG1 is involved in other major ABA responses, including ABA-inhibited seed germination, ABA-mediated stomatal movement, and drought tolerance. These findings open new perspectives on elucidating the mechanism of ABA response, and provide clues for analysing the functions of chloroplast proteins in regulating root growth.     

Identification of drought tolerance determinants by genetic analysis of root response to drought stress and abscisic Acid

Drought stress is a common adverse environmental condition that seriously affects crop productivity worldwide. Due to the complexity of drought as a stress signal, deciphering drought tolerance mechanisms has remained a major challenge to plant biologists. To develop new approaches to study plant drought tolerance, we searched for phenotypes conferred by drought stress and identified the inhibition of lateral root development by drought stress as an adaptive response to the stress. This drought response is partly mediated by the phytohormone abscisic acid. Genetic screens using Arabidopsis (Arabidopsis thaliana) were devised, and drought inhibition of lateral root growth (dig) mutants with altered responses to drought or abscisic acid in lateral root development were isolated. Characterization of these dig mutants revealed that they also exhibit altered drought stress tolerance, indicating that this root response to drought stress is intimately linked to drought adaptation of the entire plant and can be used as a trait to access the elusive drought tolerance machinery. Our study also revealed that multiple mechanisms coexist and together contribute to whole-plant drought tolerance.     

Expression of an Arabidopsis vacuolar H+‐pyrophosphatase gene (AVP1) in cotton improves drought‐ and salt tolerance and increases fibre yield in the field conditions

The Arabidopsis gene AVP1 encodes a vacuolar pyrophosphatase that functions as a proton pump on the vacuolar membrane. Overexpression of AVP1 in Arabidopsis, tomato and rice enhances plant performance under salt and drought stress conditions, because up‐regulation of the type I H⁺‐PPase from Arabidopsis may result in a higher proton electrochemical gradient, which facilitates enhanced sequestering of ions and sugars into the vacuole, reducing water potential and resulting in increased drought‐ and salt tolerance when compared to wild‐type plants. Furthermore, overexpression of AVP1 stimulates auxin transport in the root system and leads to larger root systems, which helps transgenic plants absorb water more efficiently under drought conditions. Using the same approach, AVP1‐expressing cotton plants were created and tested for their performance under high‐salt and reduced irrigation conditions. The AVP1‐expressing cotton plants showed more vigorous growth than wild‐type plants in the presence of 200 mm NaCl under hydroponic growth conditions. The soil‐grown AVP1‐expressing cotton plants also displayed significantly improved tolerance to both drought and salt stresses in greenhouse conditions. Furthermore, the fibre yield of AVP1‐expressing cotton plants is at least 20% higher than that of wild‐type plants under dry‐land conditions in the field. This research indicates that AVP1 has the potential to be used for improving crop’s drought‐ and salt tolerance in areas where water and salinity are limiting factors for agricultural productivity.     

A Cascade of Sequentially Expressed Sucrose Transporters in the Seed Coat and Endosperm Provides Nutrition for the Arabidopsis Embryo

Developing plant embryos depend on nutrition from maternal tissues via the seed coat and endosperm, but the mechanisms that supply nutrients to plant embryos have remained elusive. Sucrose, the major transport form of carbohydrate in plants, is delivered via the phloem to the maternal seed coat and then secreted from the seed coat to feed the embryo. Here, we show that seed filling in Arabidopsis thaliana requires the three sucrose transporters SWEET11, 12, and 15. SWEET11, 12, and 15 exhibit specific spatiotemporal expression patterns in developing seeds, but only a sweet11;12;15 triple mutant showed severe seed defects, which include retarded embryo development, reduced seed weight, and reduced starch and lipid content, causing a “wrinkled” seed phenotype. In sweet11;12;15 triple mutants, starch accumulated in the seed coat but not the embryo, implicating SWEET-mediated sucrose efflux in the transfer of sugars from seed coat to embryo. This cascade of sequentially expressed SWEETs provides the feeding pathway for the plant embryo, an important feature for yield potential.     

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.     

Abscisic acid promotes pre-emergence stages of lateral root development in Medicago truncatula

The plant root system is important for plant anchorage and nutrition. Among the different characteristics of the root system, root branching is a major factor of plasticity and adaptation to changing environments. Indeed, many biotic and abiotic stresses, such as drought or symbiotic interactions, influence root branching. Many studies concerning root development and root branching were performed on the model plant Arabidopsis thaliana, but this model plant has a very simplified root structure and is not able to establish any symbiotic interactions. We have recently described 7 stages for lateral root development in the model legume Medicago truncatula and found significant differences in the tissular contribution of root cell layers to the formation of new lateral roots (LR). We have also described 2 transgenic lines expressing the DR5:GUS and DR5:VENUS-N7 reporter genes that are useful to follow LR formation at early developmental stages. Here, we describe the use of these transgenic lines to monitor LR developmental responses of M. truncatula to the phytohormone abscisic acid (ABA) which is a major actor of stress and symbiotic interactions. We show that ABA promotes the formation of new lateral root primordia and their development, mostly at the late, pre-emergence stage.     

Aluminum induces low phosphate adaptive responses and modulates primary and lateral root growth by differentially affecting auxin signaling in Arabidopsis seedlings

Aims

The aims of this work were to investigate the aluminum (Al) and phosphate (P) interactions in the regulation of root system architecture of Arabidopsis thaliana seedlings and the contribution of auxin signaling in primary and lateral root growth in response to Al toxicity.

Methods

Detailed analyses of root system architecture and cell division were performed in Arabidopsis WT seedlings and in low phosphorus insensitive mutants lpi1-3 and lpr1-1 lpr2-1 in response to Al. Expression studies of P-deficiency regulated phosphate transporter AtPT2 were also conducted. The role of auxin as a mediator of root morphogenetic changes by Al was evaluated by using the auxin-signaling mutants tir1, tir1 afb2 afb3, and arf7 arf19.

Results

Al inhibited primary root growth by affecting cell cycle progression and causing differentiation of cells in the root meristem. These effects were reduced in low phosphorus insensitive lpi1-3 and low phosphate resistant lpr1-1 lpr2-1 Arabidopsis mutants. Al also activated the expression of the low phosphate-induced P transporter AtPT2 in roots. Lateral root formation by Al decreased in tir1 afb2 afb3 while arf7 arf19 mutants were highly resistant to Al in both primary root inhibition and lateral root induction.

Conclusions

Our results suggest that lateral root formation in response to Al toxicity and P deficiency may involve common signaling mechanisms, while a pathway involving ARF7 and ARF19 is important for primary root growth inhibition by Al.     

AtCYT-INV1 in Arabidopsis Sugar Signaling

Sugar acts as a signal molecule and plays a pivotal role in plant development and stress response. Neutral/alkaline invertases found only in photosynthetic bacteria and plants is sucrose-specific enzymes cleave sucrose into glucose and fructose. We have identified a gene for neutral/alkaline invertase in Arabidopsis designated as AtCYT-INV1 which is involved in sugar/ABA signaling and plays multiple roles in plant development and osmotic stress-induced inhibition on lateral root growth.Key Words: Arabidopsis thaliana, AtCTY-INV1, sugar signaling     

Pharmacological and genetical evidence supporting nitric oxide requirement for 2,4-epibrassinolide regulation of root architecture in Arabidopsis thaliana

Brassinosteroids (BRs) regulate various physiological processes, such as tolerance to stresses and root growth. Recently, a connection was reported between BRs and nitric oxide (NO) in plant responses to abiotic stress. Here we present evidence supporting NO functions in BR signaling during root growth process. Arabidopsis seedlings treated with BR 24-epibrassinolide (BL) show increased lateral roots (LR) density, inhibition of primary root (PR) elongation and NO accumulation. Similar effects were observed adding the NO donor GSNO to BR-receptor mutant bri1-1. Furthermore, BL-induced responses in the root were abolished by the specific NO scavenger c-PTIO. The activities of nitrate reductase (NR) and nitric oxide synthase (NOS)-like, two NO generating enzymes were involved in BR signaling. These results demonstrate that BR increases the NO concentration in root cells, which is required for BR-induced changes in root architecture.     

The involvement of auxin in root architecture plasticity in Arabidopsis induced by heterogeneous phosphorus availability

Homogeneous low phosphorus availability was reported to regulate root architecture in Arabidopsis via auxin, but the roles of auxin in root architecture plasticity to heterogeneous P availability remain unclear. In this study, we employed auxin biosynthesis-, transport- and signalling-related mutants. Firstly, we found that in contrast to low P (LP) content in the whole medium, primary root (PR) growth of Arabidopsis was partially rescued in the medium divided into two parts: upper with LP and lower with high P (HP) content or in the reverse arrangement. The down part LP was more effective to arrest PR growth as well as to decrease density of lateral roots (DLR) than the upper LP, and effects were dependent on polar auxin transport. Secondly, we verified that auxin receptor TIR1 was involved in the responses of PR growth and lateral root (LR) development to P supply and loss of function of TIR1 inhibited LR development. Thirdly, effects of heterogeneous P on LRD in the upper part of PR was dependent on PIN2 and PIN4, and in the down part on PIN3 and PIN4, whereas density of total LRs was dependent on auxin transporters PIN2 and PIN7. Finally, heterogeneous P availability altered the accumulation of auxin in PR tip and the expression of auxin biosynthesisrelated genes TAA1, YUC1, YUC2, and YUC4. Taken together, we provided evidences for the involvement of auxin in root architecture plasticity in response to heterogeneous phosphorus availability in Arabidopsis.     

A glucuronokinase gene in<Emphasis Type="Italic"> Arabidopsis</Emphasis>, <Emphasis Type="Italic">AtGlcAK</Emphasis>, is involved in drought tolerance by modulating sugar metabolism

Arabidopsis glucuronokinase (AtGlcAK), as a member of the GHMP kinases family, is implicated in the de novo synthesis of UDP-glucuronic acid (UDP-GlcA) by the myo-inositol oxygenation pathway. In this study, two T-DNA insertion homozygous mutants of AtGlcAK, atglcak-1 and atglcak-2, were identified. AtGlcAK was highly expressed in roots and flowers. There was reduced primary root elongation and lateral root formation in atglcak mutants under osmotic stress. The atglcak mutants displayed enhanced stomatal opening in response to abscisic acid (ABA), elevated water loss and impaired drought tolerance. Under water stress, the accumulation of reducing and soluble sugars was reduced in atglcak mutants, and the metabolism of glucose and sucrose was affected by the synthetic pathway of UDP-GlcA. Furthermore, a reduced level of starch in atglcak mutants was observed under normal conditions. The phylogenetic analysis suggested that GlcAK was conserved in numerous dicots and monocots plants. In short, AtGlcAK mutants displayed hypersensitivity to ABA and reduced root development under water stress, rendering the plants more susceptible to drought stress.     

Drought and salt stress tolerance of an Arabidopsis glutathione S-transferase U17 knockout mutant are attributed to the combined effect of glutathione and abscisic acid

Although glutathione S-transferases (GSTs) are thought to play major roles in oxidative stress metabolism, little is known about the regulatory functions of GSTs. We have reported that Arabidopsis (Arabidopsis thaliana) GLUTATHIONE S-TRANSFERASE U17 (AtGSTU17; At1g10370) participates in light signaling and might modulate various aspects of development by affecting glutathione (GSH) pools via a coordinated regulation with phytochrome A. Here, we provide further evidence to support a negative role of AtGSTU17 in drought and salt stress tolerance. When AtGSTU17 was mutated, plants were more tolerant to drought and salt stresses compared with wild-type plants. In addition, atgstu17 accumulated higher levels of GSH and abscisic acid (ABA) and exhibited hyposensitivity to ABA during seed germination, smaller stomatal apertures, a lower transpiration rate, better development of primary and lateral root systems, and longer vegetative growth. To explore how atgstu17 accumulated higher ABA content, we grew wild-type plants in the solution containing GSH and found that they accumulated ABA to a higher extent than plants grown in the absence of GSH, and they also exhibited the atgstu17 phenotypes. Wild-type plants treated with GSH also demonstrated more tolerance to drought and salt stresses. Furthermore, the effect of GSH on root patterning and drought tolerance was confirmed by growing the atgstu17 in solution containing l-buthionine-(S,R)-sulfoximine, a specific inhibitor of GSH biosynthesis. In conclusion, the atgstu17 phenotype can be explained by the combined effect of GSH and ABA. We propose a role of AtGSTU17 in adaptive responses to drought and salt stresses by functioning as a negative component of stress-mediated signal transduction pathways.