RESUMO
Dedicated water channels are involved in the facilitated diffusion of water molecules across the cell membrane in plants. Transporter proteins are also known to transport water molecules along with substrates, however the molecular mechanism of water permeation is not well understood in plant transporters. Here, we show plant sugar transporters from the SWEET (Sugar Will Eventually be Exported Transporter) family act as water-conducting carrier proteins via a variety of passive and active mechanisms that allow diffusion of water molecules from one side of the membrane to the other. This study provides a molecular perspective on how plant membrane transporters act as water carrier proteins, a topic that has not been extensively explored in literature. Water permeation in membrane transporters could occur via four distinct mechanisms which form our hypothesis for water transport in SWEETs. These hypothesis are tested using molecular dynamics simulations of the outward-facing, occluded, and inward-facing state of AtSWEET1 to identify the water permeation pathways and the flux associated with them. The hydrophobic gates at the center of the transport tunnel act as a barrier that restricts water permeation. We have performed in silico single and double mutations of the hydrophobic gate residues to examine the changes in the water conductivity. Surprisingly, the double mutant allows the water permeation to the intracellular half of the membrane and forms a continuous water channel. These computational results are validated by experimentally examining the transport of hydrogen peroxide molecules by the AtSWEET family of transporters. We have also shown that the transport of hydrogen peroxide follows the similar mechanism as water transport in AtSWEET1. Finally, we conclude that similar water-conduction states are also present in other SWEET transporters due to the high sequence and structure conservation exhibited by this transporter family.
RESUMO
Sweet sorghum has emerged as a promising source of bioenergy mainly due to its high biomass and high soluble sugar yield in stems. Studies have shown that loss-of-function Dry locus alleles have been selected during sweet sorghum domestication, and decapitation can further boost sugar accumulation in sweet sorghum, indicating that the potential for improving sugar yields is yet to be fully realized. To maximize sugar accumulation, it is essential to gain a better understanding of the mechanism underlying the massive accumulation of soluble sugars in sweet sorghum stems in addition to the Dry locus. We performed a transcriptomic analysis upon decapitation of near-isogenic lines for mutant (d, juicy stems, and green leaf midrib) and functional (D, dry stems and white leaf midrib) alleles at the Dry locus. Our analysis revealed that decapitation suppressed photosynthesis in leaves, but accelerated starch metabolic processes in stems. SbbHLH093 negatively correlates with sugar levels supported by genotypes (DD vs. dd), treatments (control vs. decapitation), and developmental stages post anthesis (3d vs.10d). D locus gene SbNAC074A and other programmed cell death-related genes were downregulated by decapitation, while sugar transporter-encoding gene SbSWEET1A was induced. Both SbSWEET1A and Invertase 5 were detected in phloem companion cells by RNA in situ assay. Loss of the SbbHLH093 homolog, AtbHLH093, in Arabidopsis led to a sugar accumulation increase. This study provides new insights into sugar accumulation enhancement in bioenergy crops, which can be potentially achieved by reducing reproductive sink strength and enhancing phloem unloading.
RESUMO
Galactose is an abundant and essential sugar used for the biosynthesis of many macromolecules in different organisms, including plants. Galactose metabolism is tightly and finely controlled, since excess galactose and its derivatives are inhibitory to plant growth. In Arabidopsis (Arabidopsis thaliana), root growth and pollen germination are strongly inhibited by excess galactose. However, the mechanism of galactose-induced inhibition during pollen germination remains obscure. In this study, we characterized a plasma membrane-localized transporter, Arabidopsis Sugars Will Eventually be Exported Transporter 5, that transports glucose and galactose. SWEET5 protein levels started to accumulate at the tricellular stage of pollen development and peaked in mature pollen, before rapidly declining after pollen germinated. SWEET5 levels are responsible for the dosage-dependent sensitivity to galactose, and galactokinase is essential for these inhibitory effects during pollen germination. However, sugar measurement results indicate that galactose flux dynamics and sugar metabolism, rather than the steady-state galactose level, may explain phenotypic differences between sweet5 and Col-0 in galactose inhibition of pollen germination.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Galactoquinase/metabolismo , Galactoquinase/farmacologia , Galactose/metabolismo , Galactose/farmacologia , Germinação , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Transporte de Monossacarídeos/genética , Proteínas de Transporte de Monossacarídeos/metabolismo , PólenRESUMO
Abiotic stresses affect plant growth and development by causing cellular damage and/or restricting resources. Plants often respond to stresses through abscisic acid (ABA) signaling. Exogenous ABA application can therefore be used to mimic stress responses, which can be overridden by glucose (Glc) addition during seed germination. It remains unclear whether ABA-mediated germination inhibition is due to regional or global suppression of Glc availability in germinating Arabidopsis seeds. We used a genetically engineered Förster resonance energy transfer (FRET) sensor to ascertain whether ABA affects the spatiotemporal distribution of Glc, 14 C-Glc uptake assays to track potential effects of ABA on sugar import, and transcriptome and mutant analyses to identify genes associated with Glc availability that are involved in ABA-inhibited seed germination. Abscisic acid limits Glc in the hypocotyl largely by suppressing sugar allocation as well as altering sugar metabolism. Mutant plants carrying loss-of-function ABA-inducible sucrose-phosphate synthase (SPS) genes accumulated more Glc, leading to ABA-insensitive germination. We reveal that Glc antagonizes ABA by globally counteracting the ABA influence at the transcript level, including expansin (EXP) family genes suppressed by ABA. This study presents a new perspective on how ABA affects Glc distribution, which likely reflects what occurs when seeds are subjected to abiotic stresses such as drought and salt stress.
Assuntos
Ácido Abscísico , Proteínas de Arabidopsis , Ácido Abscísico/farmacologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , Germinação , Glucose , Hipocótilo/metabolismo , Sementes/metabolismoRESUMO
Soybean accounts for more than half of the global production of oilseed and more than a quarter of the protein used globally for human food and animal feed. Soybean domestication involved parallel increases in seed size and oil content, and a concomitant decrease in protein content. However, science has not yet discovered whether these effects were due to selective pressure on a single gene or multiple genes. Here, re-sequencing data from >800 genotypes revealed a strong selection during soybean domestication on GmSWEET10a. The selection of GmSWEET10a conferred simultaneous increases in soybean-seed size and oil content as well as a reduction in the protein content. The result was validated using both near-isogenic lines carrying substitution of haplotype chromosomal segments and transgenic soybeans. Moreover, GmSWEET10b was found to be functionally redundant with its homologue GmSWEET10a and to be undergoing selection in current breeding, leading the the elite allele GmSWEET10b, a potential target for present-day soybean breeding. Both GmSWEET10a and GmSWEET10b were shown to transport sucrose and hexose, contributing to sugar allocation from seed coat to embryo, which consequently determines oil and protein contents and seed size in soybean. We conclude that past selection of optimal GmSWEET10a alleles drove the initial domestication of multiple soybean-seed traits and that targeted selection of the elite allele GmSWEET10b may further improve the yield and seed quality of modern soybean cultivars.
RESUMO
The SWEET family belongs to a class of transporters in plants that undergoes large conformational changes to facilitate transport of sugar molecules across the cell membrane (SWEET, Sugars Will Eventually Be Exported Transporter). However, the structures of their functionally relevant conformational states in the transport cycle have not been reported. In this study, we have characterized the conformational dynamics and complete transport cycle of glucose in the OsSWEET2b transporter using extensive molecular dynamics simulations. Using Markov state models, we estimated the free energy barrier associated with different states as well as for the glucose transport mechanism. SWEETs undergo a structural transition to outward-facing (OF), occluded (OC), and inward-facing (IF) and strongly support an alternate access transport mechanism. The glucose diffuses freely from outside to inside the cell without causing major conformational changes which means that the conformations of glucose unbound and bound snapshots are exactly the same for OF, OC, and IF states. We identified a network of hydrophobic core residues at the center of the transporter that restricts the glucose entry to the cytoplasmic side and acts as an intracellular hydrophobic gate. The mechanistic predictions from molecular dynamics simulations are validated using site-directed mutagenesis experiments. Our simulation also revealed hourglass-like intermediate states making the pore radius narrower at the center. This work provides new fundamental insights into how substrate-transporter interactions actively change the free energy landscape of the transport cycle to facilitate enhanced transport activity.
RESUMO
It has long been recognized that stomatal movement modulates CO2 availability and as a consequence the photosynthetic rate of plants, and that this process is feedback-regulated by photoassimilates. However, the genetic components and mechanisms underlying this regulatory loop remain poorly understood, especially in monocot crop species. Here, we report the cloning and functional characterization of a maize (Zea mays) mutant named closed stomata1 (cst1). Map-based cloning of cst1 followed by confirmation with the clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR associated protein 9 system identified the causal mutation in a Clade I Sugars Will Eventually be Exported Transporters (SWEET) family gene, which leads to the E81K mutation in the CST1 protein. CST1 encodes a functional glucose transporter expressed in subsidiary cells, and the E81K mutation strongly impairs the oligomerization and glucose transporter activity of CST1. Mutation of CST1 results in reduced stomatal opening, carbon starvation, and early senescence in leaves, suggesting that CST1 functions as a positive regulator of stomatal opening. Moreover, CST1 expression is induced by carbon starvation and suppressed by photoassimilate accumulation. Our study thus defines CST1 as a missing link in the feedback-regulation of stomatal movement and photosynthesis by photoassimilates in maize.
Assuntos
Proteínas Facilitadoras de Transporte de Glucose/metabolismo , Fotossíntese/fisiologia , Proteínas Facilitadoras de Transporte de Glucose/genética , Fotossíntese/genética , Folhas de Planta/metabolismo , Estômatos de Plantas/metabolismo , Zea mays/metabolismoRESUMO
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-ß-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.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Proteínas de Transporte de Monossacarídeos/metabolismo , Doenças das Plantas/microbiologia , Raízes de Plantas/metabolismo , Pythium/patogenicidade , Arabidopsis/microbiologia , Proteínas de Arabidopsis/genética , Sequestro de Carbono , Regulação da Expressão Gênica de Plantas , Glucose/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Interações Hospedeiro-Patógeno , Proteínas de Transporte de Monossacarídeos/genética , Folhas de Planta/metabolismo , Raízes de Plantas/microbiologia , Plantas Geneticamente Modificadas , Vacúolos/metabolismoRESUMO
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-ß-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.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Frutose/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Folhas de Planta/metabolismo , Raízes de Plantas/metabolismo , Vacúolos/metabolismo , Adaptação Fisiológica/efeitos dos fármacos , Adaptação Fisiológica/genética , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Transporte Biológico/genética , Temperatura Baixa , Frutose/farmacologia , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Genes de Plantas/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Membrana Transportadoras/genética , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/genética , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Vacúolos/efeitos dos fármacosRESUMO
Destruxins are fungal toxins used as insecticides. Recent reports demonstrated the potential anti-cancer activities of destruxin B (DB). This study is to discover the effects of DB in lymphoma. Flow cytometry and Western blotting were used to analyze apoptosis and protein expression, respectively, in Toledo human non-Hodgkin lymphoma cells in response to DB. Administration of DB, induced apoptosis via death receptor pathway activating Fas associated death domain (FADD), caspase 8 and caspase 3, and suppressed the cell growth. In addition, DB alterated mitochondrial membrane potential by increasing the expressions of tBid and Bax, but decreasing the levels of Bcl-2, resulting in the release of apoptosis-inducing factor (AIF). In conclusion, apoptosis of human non-Hodgkin lymphoma cells in response to DB is induced through the death receptor pathway and involves an alteration of the mitochondrial membrane potential. These findings may aid the development of novel treatment of non-Hodgkin lymphoma.