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- Ackerschmalwand (1)
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Institute
- Julius-von-Sachs-Institut für Biowissenschaften (16) (remove)
The control of energy homeostasis is of pivotal importance for all living organisms. In the last years emerged the idea that many stress responses that are apparently unrelated, are actually united by a common increase of the cellular energy demand. Therefore, the so called energy signaling is activated by many kind of stresses and is responsible for the activation of the general stress response. In Arabidopsis thaliana the protein family SnF1- related protein kinases (SnRK1) is involved in the regulation of many physiological processes but is more known for its involvement in the regulation of the energy homeostasis in response to various stresses. To the SnRK1 protein family belong SnRK1.1 (also known as KIN10), SnRK1.2 (KIN11), and SnRK1.3 (KIN12). SnRK1 exerts its function regulating directly the activity of metabolic enzymes or those of key transcription factors (TFs). The only TFs regulated by SnRK1 identified so far is the basic leucine zipper (bZIP) 63. bZIP63 belongs to the C group of bZIPs (C-bZIPs) protein family together with bZIP9, bZIP10, and bZIP25. SnRK1.1 phosphorylates bZIP63 on three amino acids residues, serine (S) 29, S294, and S300. The phosphorylation of tbZIP63 is strongly related to the energy status of the plant, shifting from almost absent during the normal growth to strongly phosphorylated when the plant is exposed to extended dark. bZIPs normally bind the DNA as dimer in order to regulate the expression of their target genes. C-bZIPs preferentially form dimers with S1-bZIPs, constituting the so called C/S1- bZIPs network. The SnRk1 dependent phosphorylation of bZIP63 regulates its activation potential and its dimerization properties. In particular bZIP63 shift its dimerization preferences according to its phosphorylation status. The non-phosphorylated form of bZIP63 dimerize bZIP1, the phosphorylates ones, instead, forms dimer with bZIP1, bZIP11, and bZIP63 its self. Together with bZIP63, S1-bZIPs are important mediator of part of the huge transcriptional reprogramming induced by SnRK1 in response to extended dark. S1-bZIPs regulate, indeed, the expression of 4'000 of the 10'000 SnRK1-regulated genes in response to energy deprivation. In particular S1-bZIPs are very important for the regulation of many genes encoding for enzymes involved in the amino acid metabolism and for their use as alternative energy source. After the exposition for some hours to extended dark, indeed, the plant make use of every energy substrate and amino acids are considered an important energy source together with lipids and proteins. Interestingly, S1- bZIPs regulate the expression of ETFQO. ETFQO is a unique protein that convoglia the electrons provenienti from the branch chain amino acids catabolism into the mitochondrial electron transport chain. The dimer formed between bZIP63 and bZIP2 recruits SnRK1.1 directly on the chromatin of ETFQO promoter. The recruitment of SnRK1 on ETFQO promoter is associated with its acetylation on the lysine 14 of the histone protein 3 (K14H3). This chromatin modification is normally asociated with an euchromatic status of the DNA and therefore with its transcriptional activation. Beside the particular case of the regulation of ETFQO gene, S1-bZIPs are involved in the regulation of many other genes activated in response of different stresses. bZIP1 is for example an important mediator of the salt stress response. In particular bZIP1 regulates the primary C- and N-metabolism. The expression of bZIP1, in response of both salt ans energy stress seems to be regulated by SnRK1, as it is the expression of bZIP53 and bZIP63.
Beside its involvement in the regulation of the energy stress response and salt response, SnRK1 is the primary activators of the lipids metabolism during see germination. SnRK1, indeed, controls the expression of CALEOSINs and OLEOSINs. Those proteins are very important for lipids remobilization from oil droplets. Without their expression seed germination and subsequent establishment do not take place because of the absence of fuel to sustain these highly energy costly processes, which entirely depend on the catabolism of seed storages.
Background:
In previous studies, the gram-positive firmicute genus Paenibacillus was found with significant abundances in nests of wild solitary bees. Paenibacillus larvae is well-known for beekeepers as a severe pathogen causing the fatal honey bee disease American foulbrood, and other members of the genus are either secondary invaders of European foulbrood or considered a threat to honey bees. We thus investigated whether Paenibacillus is a common bacterium associated with various wild bees and hence poses a latent threat to honey bees visiting the same flowers.
Results:
We collected 202 samples from 82 individuals or nests of 13 bee species at the same location and screened each for Paenibacillus using high-throughput sequencing-based 16S metabarcoding. We then isolated the identified strain Paenibacillus MBD-MB06 from a solitary bee nest and sequenced its genome. We did find conserved toxin genes and such encoding for chitin-binding proteins, yet none specifically related to foulbrood virulence or chitinases. Phylogenomic analysis revealed a closer relationship to strains of root-associated Paenibacillus rather than strains causing foulbrood or other accompanying diseases. We found anti-microbial evidence within the genome, confirmed by experimental bioassays with strong growth inhibition of selected fungi as well as gram-positive and gram-negative bacteria.
Conclusions:
The isolated wild bee associate Paenibacillus MBD-MB06 is a common, but irregularly occurring part of wild bee microbiomes, present on adult body surfaces and guts and within nests especially in megachilids. It was phylogenetically and functionally distinct from harmful members causing honey bee colony diseases, although it shared few conserved proteins putatively toxic to insects that might indicate ancestral predisposition for the evolution of insect pathogens within the group. By contrast, our strain showed anti-microbial capabilities and the genome further indicates abilities for chitin-binding and biofilm-forming, suggesting it is likely a useful associate to avoid fungal penetration of the bee cuticula and a beneficial inhabitant of nests to repress fungal threats in humid and nutrient-rich environments of wild bee nests.
Two-component cyclase opsins of green algae are ATP-dependent and light-inhibited guanylyl cyclases
(2018)
Background:
The green algae Chlamydomonas reinhardtii and Volvox carteri are important models for studying light perception and response, expressing many different photoreceptors. More than 10 opsins were reported in C. reinhardtii, yet only two—the channelrhodopsins—were functionally characterized. Characterization of new opsins would help to understand the green algae photobiology and to develop new tools for optogenetics.
Results:
Here we report the characterization of a novel opsin family from these green algae: light-inhibited guanylyl cyclases regulated through a two-component-like phosphoryl transfer, called “two-component cyclase opsins” (2c-Cyclops). We prove the existence of such opsins in C. reinhardtii and V. carteri and show that they have cytosolic N- and C-termini, implying an eight-transmembrane helix structure. We also demonstrate that cGMP production is both light-inhibited and ATP-dependent. The cyclase activity of Cr2c-Cyclop1 is kept functional by the ongoing phosphorylation and phosphoryl transfer from the histidine kinase to the response regulator in the dark, proven by mutagenesis. Absorption of a photon inhibits the cyclase activity, most likely by inhibiting the phosphoryl transfer. Overexpression of Vc2c-Cyclop1 protein in V. carteri leads to significantly increased cGMP levels, demonstrating guanylyl cyclase activity of Vc2c-Cyclop1 in vivo. Live cell imaging of YFP-tagged Vc2c-Cyclop1 in V. carteri revealed a development-dependent, layer-like structure at the immediate periphery of the nucleus and intense spots in the cell periphery.
Conclusions:
Cr2c-Cyclop1 and Vc2c-Cyclop1 are light-inhibited and ATP-dependent guanylyl cyclases with an unusual eight-transmembrane helix structure of the type I opsin domain which we propose to classify as type Ib, in contrast to the 7 TM type Ia opsins. Overexpression of Vc2c-Cyclop1 protein in V. carteri led to a significant increase of cGMP, demonstrating enzyme functionality in the organism of origin. Fluorescent live cell imaging revealed that Vc2c-Cyclop1 is located in the periphery of the nucleus and in confined areas at the cell periphery.
Rhodopsin-cyclases for photocontrol of cGMP/cAMP and 2.3 Å structure of the adenylyl cyclase domain
(2018)
The cyclic nucleotides cAMP and cGMP are important second messengers that orchestrate fundamental cellular responses. Here, we present the characterization of the rhodopsinguanylyl cyclase from Catenaria anguillulae (CaRhGC), which produces cGMP in response to green light with a light to dark activity ratio > 1000. After light excitation the putative signaling state forms with tau = 31 ms and decays with tau = 570 ms. Mutations (up to 6) within the nucleotide binding site generate rhodopsin-adenylyl cyclases (CaRhACs) of which the double mutated YFP-CaRhAC (E497K/C566D) is the most suitable for rapid cAMP production in neurons. Furthermore, the crystal structure of the ligand-bound AC domain (2.25 angstrom) reveals detailed information about the nucleotide binding mode within this recently discovered class of enzyme rhodopsin. Both YFP-CaRhGC and YFP-CaRhAC are favorable optogenetic tools for non-invasive, cell-selective, and spatio-temporally precise modulation of cAMP/cGMP with light.
Sodium-glucose transporters (SGLT) belong to the solute carrier 5 family, which is characterized by sodium dependent transport of sugars and other solutes. In contrast, the human SGLT3 (hSGLT3) isoform, encoded by SLC5A4, acts as a glucose sensor that does not transport sugar but induces membrane depolarization by Na\(^{+}\) currents upon ligand binding. Whole-exome sequencing (WES) of several extended pedigrees with high density of attention-deficit/hyperactivity disorder (ADHD) identified a triplet ATG deletion in SLC5A4 leading to a single amino acid loss (ΔM500) in the hSGLT3 protein imperfectly co-segregating with the clinical phenotype of ADHD. Since mutations in homologous domains of hSGLT1 and hSGLT2 were found to affect intestinal and renal function, respectively, we analyzed the functional properties of hSGLT3[wt] and [ΔM500] by voltage clamp and current clamp recordings from cRNA-injected Xenopus laevis oocytes.
The cation conductance of hSGLT3[wt] was activated by application of glucose or the specific agonist 1-desoxynojirimycin (DNJ) as revealed by inward currents in the voltage clamp configuration and cell depolarization in the current clamp mode. Almost no currents and changes in membrane potential were observed when glucose or DNJ were applied to hSGLT3[ΔM500]-injected oocytes, demonstrating a loss of function by this amino acid deletion in hSGLT3. To monitor membrane targeting of wt and mutant hSGLT3, fusion constructs with YFP were generated, heterologously expressed in Xenopus laevis oocytes and analyzed for membrane fluorescence by confocal microscopy. In comparison to hSGLT3[wt] the fluorescent signal of mutant [ΔM500] was reduced by 43% indicating that the mutant phenotype might mainly result from inaccurate membrane targeting. As revealed by homology modeling, residue M500 is located in TM11 suggesting that in addition to the core structure (TM1-TM10) of the transporter, the surrounding TMs are equally crucial for transport/sensor function.
In conclusion, our findings indicate that the deletion [ΔM500] in hSGLT3 inhibits membrane targeting and thus largely disrupts glucose-induced sodium conductance, which may, in interaction with other ADHD risk-related gene variants, influence the risk for ADHD in deletion carriers.
Optogenetic manipulation of cells or living organisms became widely used in neuroscience following the introduction of the light-gated ion channel channelrhodopsin-2 (ChR2). ChR2 is a non-selective cation channel, ideally suited to depolarize and evoke action potentials in neurons. However, its calcium (Ca2\(^{2+}\)) permeability and single channel conductance are low and for some applications longer-lasting increases in intracellular Ca\(^{2+}\) might be desirable. Moreover, there is need for an efficient light-gated potassium (K\(^{+}\)) channel that can rapidly inhibit spiking in targeted neurons. Considering the importance of Ca\(^{2+}\) and K\(^{+}\) in cell physiology, light-activated Ca\(^{2+}\)-permeant and K\(^{+}\)-specific channels would be welcome additions to the optogenetic toolbox. Here we describe the engineering of novel light-gated Ca\(^{2+}\)-permeant and K\(^{+}\)-specific channels by fusing a bacterial photoactivated adenylyl cyclase to cyclic nucleotide-gated channels with high permeability for Ca\(^{2+}\) or for K\(^{+}\), respectively. Optimized fusion constructs showed strong light-gated conductance in Xenopus laevis oocytes and in rat hippocampal neurons. These constructs could also be used to control the motility of Drosophila melanogaster larvae, when expressed in motoneurons. Illumination led to body contraction when motoneurons expressed the light-sensitive Ca\(^{2+}\)-permeant channel, and to body extension when expressing the light-sensitive K\(^{+}\) channel, both effectively and reversibly paralyzing the larvae. Further optimization of these constructs will be required for application in adult flies since both constructs led to eclosion failure when expressed in motoneurons.
The number of plants pollinated by ants is surprisingly low given the abundance of ants and the fact that they are common visitors of angiosperms. Generally ants are considered as nectar robbers that do not provide pollination service. We studied the pollination system of the endangered dry grassland forb Euphorbia seguieriana and found two ant species to be the most frequent visitors of its flowers. Workers of Formica cunicularia carried five times more pollen than smaller Tapinoma erraticum individuals, but significantly more viable pollen was recovered from the latter. Overall, the viability of pollen on ant cuticles was significantly lower (p < 0.001)-presumably an antibiotic effect of the metapleural gland secretion. A marking experiment suggested that ants were unlikely to facilitate outcrossing as workers repeatedly returned to the same individual plant. In open pollinated plants and when access was given exclusively to flying insects, fruit set was nearly 100%. In plants visited by ants only, roughly one third of flowers set fruit, and almost none set fruit when all insects were excluded. The germination rate of seeds from flowers pollinated by flying insects was 31 +/- 7% in contrast to 1 +/- 1% resulting from ant pollination. We conclude that inbreeding depression may be responsible for the very low germination rate in ant pollinated flowers and that ants, although the most frequent visitors, play a negligible or even deleterious role in the reproduction of E. seguieriana. Our study reiterates the need to investigate plant fitness effects beyond seed set in order to confirm ant-plant mutualisms.
Arbuscular Mycorrhiza and Root Nodule Symbiosis are symbiotic interactions with a high benefit for plant growth and crop production. Thus, it is of great interest to understand the developmental process of these symbioses in detail. We analysed very early symbiotic responses of Medicago truncatula root hair cells, by stimulation with lipochitinoligosaccharides specific for the induction of nodules (Nod-LCOs), or the interaction with mycorrhiza (Myc-LCOs). Intracellular micro electrodes were used, in combination with Ca\(^{2+}\) sensitive reporter dyes, to study the relations between cytosolic Ca\(^{2+}\) signals and membrane potential changes. We found that sulfated Myc- as well as Nod-LCOs initiate a membrane depolarization, which depends on the chemical composition of these signaling molecules, as well as the genotype of the plants that were studied. A successive application of sulfated Myc-LCOs and Nod-LCOs resulted only in a single transient depolarization, indicating that Myc-LCOs can repress plasma membrane responses to Nod-LCOs. In contrast to current models, the Nod-LCO-induced depolarization precedes changes in the cytosolic Ca\(^{2+}\) level of root hair cells. The Nod-LCO induced membrane depolarization thus is most likely independent of cytosolic Ca\(^{2+}\) signals and nuclear Ca\(^{2+}\) spiking.
Auxin is a key regulator of plant growth and development, but the causal relationship between hormone transport and root responses remains unresolved. Here we describe auxin uptake, together with early steps in signaling, in Arabidopsis root hairs. Using intracellular microelectrodes we show membrane depolarization, in response to IAA in a concentration- and pH-dependent manner. This depolarization is strongly impaired in aux1 mutants, indicating that AUX1 is the major transporter for auxin uptake in root hairs. Local intracellular auxin application triggers Ca2+ signals that propagate as long-distance waves between root cells and modulate their auxin responses. AUX1-mediated IAA transport, as well as IAA- triggered calcium signals, are blocked by treatment with the SCFTIR1/AFB - inhibitor auxinole. Further, they are strongly reduced in the tir1afb2afb3 and the cngc14 mutant. Our study reveals that the AUX1 transporter, the SCFTIR1/AFB receptor and the CNGC14 Ca2+ channel, mediate fast auxin signaling in roots.
The plant cuticle is a continuous extracellular protective layer covering the outermost surfaces of higher plants that are in contact with the surrounding atmosphere. The primary function of the cuticular lipid membrane, which is mainly composed of biopolymer cutin and cuticular waxes, is to protect the plant organs against uncontrolled water loss. The chemical composition and the biophysical properties of cuticular waxes affect the rate of water diffusion across the cuticle. Fruit transpiration plays an important role in the development and the maintenance of fruit quality. The fruit has been suggested to present better dehydration stress tolerance than the leaf. However, the differences in transpiration and the chemical composition of cuticular waxes between fruit and leaf have yet to be comprehensively investigated.
The present study aims to investigate the water permeability and cuticular wax composition of fruit and leaf cuticles of a wide range of plant species and to elucidate the different roles of the cuticular wax components in the transpiration barrier. To address these objectives, fruit and leaf samples from 17 species were investigated. The cuticular transpiration of intact fruits and astomatous adaxial leaf surfaces and the minimum leaf conductance obtained by leaf drying curves for intact leaves were gravimetrically determined for a variety of plant species. The chemical composition of cuticular waxes of fruits and leaves was thoroughly analysed by gas chromatography with flame ionization and mass spectrometry.
The water permeability of fruits ranged from 3.7 x 10-5 m s-1 (Prunus domestica subsp. syriaca) to 37.4 x 10-5 m s-1 (Coffea arabica), whereas permeability for leaves varied between 1.6 x 10-5 m s-1 (Cornus officinalis) and 4.5 x 10-5 m s-1 (Prunus domestica subsp. syriaca (L.)). The interspecies range of water permeability of fruits was significantly higher than that of leaves. Chemical analyses of the cuticular waxes demonstrated that fatty acids, primary alcohols, n-alkanes, aldehydes and alkyl esters were the predominant very-long-chain aliphatic compound classes of fruit and leaf surfaces. Sterols, such as β-sitosterol and campesterol, and triterpenoids, such as oleanolic acid, ursolic acid, α-amyrin and ß-amyrin, were the major cyclic compound classes in the cuticular wax membrane.
The amount and composition of cuticular waxes of both fruits and leaves varied at an intraspecific level. There were no significant correlations between the total cuticular wax load or the individual cuticular wax composition and the water permeability of fruits or leaves independently or together. After combining the fruit and leaf data set, a significant correlation between the average chain length of very-long-chain aliphatic compounds and permeabilities was detected, i.e. the longer the average chain length, the lower the water permeability.
Interestingly, n-Nonacosane (C29) was abundantly detected in fruit waxes of Rosaceae species. These fruits exhibited a relatively low transpiration level, which was very close to their leaf cuticular permeability. The present study suggests that the lower cuticular permeability of leaves, in comparison to that of fruits, may be attributed to the longer average chain length of aliphatic compounds. The accumulation of total wax, triterpenoids and aliphatic compounds may not contribute to the transpiration barrier directly. The present results are highly consistent with the previous model assumptions for the cuticular structure and transport barrier. Furthermore, this comparative study on leaf and fruit cuticles provides further insights linking the cuticular wax chemistry to the physiological properties of the plant cuticle.
Malvaviscus arboreus Cav. is a medicinal plant belonging to family Malvaceae with both ethnomedical and culinary value; however, its phytochemical and biological profiles have been scarcely studied. Accordingly, this work was designed to explore the chemical composition and the hepatoprotective potential of M. arboreus against carbon tetrachloride (CCl\(_4\))-induced hepatotoxicity. The total extract of the aerial parts and its derived fractions (petroleum ether, dichloromethane, ethyl acetate, and aqueous) were orally administered to rats for six consecutive days, followed by injection of CCl\(_4\) (1:1 v/v, in olive oil, 1.5 ml/kg, i.p.) on the next day. Results showed that the ethyl acetate and dichloromethane fractions significantly alleviated liver injury in rats as indicated by the reduced levels of alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), total bilirubin (TB), and malondialdehyde (MDA), along with enhancement of the total antioxidant capacities of their livers, with the maximum effects were recorded by the ethyl acetate fraction. Moreover, the protective actions of both fractions were comparable to those of silymarin (100 mg/kg), and have been also substantiated by histopathological evaluations. On the other hand, liquid chromatography-high resolution electrospray ionization mass spectrometry (LC‒HR‒ESI‒MS) metabolomic profiling of the crude extract of M. arboreus aerial parts showed the presence of a variety of phytochemicals, mostly phenolics, whereas the detailed chemical analysis of the most active fraction (i.e. ethyl acetate) resulted in the isolation and identification of six compounds for the first time in the genus, comprising four phenolic acids; β-resorcylic, caffeic, protocatechuic, and 4-hydroxyphenylacetic acids, in addition to two flavonoids; trifolin and astragalin. Such phenolic principles, together with their probable synergistic antioxidant and liver-protecting properties, seem to contribute to the observed hepatoprotective potential of M. arboreus.
Objectives
Glycemic control by medical treatment represents one therapeutic strategy for diabetic patients. The Na+-d-glucose cotransporter 1 (SGLT1) is currently of high interest in this context. SGLT1 is known to mediate glucose absorption and incretin secretion in the small intestine. Recently, inhibition of SGLT1 function was shown to improve postprandial hyperglycemia. In view of the lately demonstrated SGLT1 expression in pancreatic islets, we investigated if loss of SGLT1 affects islet morphology and function.
Methods
Effects associated with the loss of SGLT1 on pancreatic islet (cyto) morphology and function were investigated by analyzing islets of a SGLT1 knockout mouse model, that were fed a glucose-deficient, fat-enriched diet (SGLT1−/−-GDFE) to circumvent the glucose-galactose malabsorption syndrome. To distinguish diet- and Sglt1−/−-dependent effects, wildtype mice on either standard chow (WT-SC) or the glucose-free, fat-enriched diet (WT-GDFE) were used as controls. Feeding a glucose-deficient, fat-enriched diet further required the analysis of intestinal SGLT1 expression and function under diet-conditions.
Results
Consistent with literature, our data provide evidence that small intestinal SGLT1 mRNA expression and function is regulated by nutrition. In contrast, pancreatic SGLT1 mRNA levels were not affected by the applied diet, suggesting different regulatory mechanisms for SGLT1 in diverse tissues. Morphological changes such as increased islet sizes and cell numbers associated with changes in proliferation and apoptosis and alterations of the β- and α-cell population are specifically observed for pancreatic islets of SGLT1−/−-GDFE mice. Glucose stimulation revealed no insulin response in SGLT1−/−-GDFE mice while WT-GDFE mice displayed only a minor increase of blood insulin. Irregular glucagon responses were observed for both, SGLT1−/−-GDFE and WT-GDFE mice. Further, both animal groups showed a sustained release of GLP-1 compared to WT-SC controls.
Conclusion
Loss or impairment of SGLT1 results in abnormal pancreatic islet (cyto)morphology and disturbed islet function regarding the insulin or glucagon release capacity from β- or α-cells, respectively. Consequently, our findings propose a new, additional role for SGLT1 maintaining proper islet structure and function.
Plants and animals in endosomes operate TPC1/SV-type cation channels. All plants harbor at least one TPC1 gene. Although the encoded SV channel was firstly discovered in the plant vacuole membrane two decades ago, its biological function has remained enigmatic. Recently, the structure of a plant TPC1/SV channel protein was determined. Insights into the 3D topology has now guided site-directed mutation approaches, enabling structure–function analyses of TPC1/SV channels to shed new light on earlier findings. Fou2 plants carrying a hyperactive mutant form of TPC1 develop wounding stress phenotypes. Recent studies with fou2 and mutants that lack functional TPC1 have revealed atypical features in local and long-distance stress signaling, providing new access to the previously mysterious biology of this vacuolar cation channel type in planta.
Agrobacterium tumefaciens is the causal agent of crown gall disease in a wide range of plants via a unique interkingdom DNA transfer from bacterial cells into the plant genome. Agrobacterium tumefaciens is capable of transferring its T-DNA into different plant parts at different developmental stages for transient and stable transformation. However, the plant genes and mechanisms involved in these transformation processes are not well understood. We used Arabidopsis thaliana Col-0 seedlings to reveal the gene expression profiles at early time points during Agrobacterium infection. Common and differentially expressed genes were found in shoots and roots. A gene ontology analysis showed that the glucosinolate (GS) biosynthesis pathway was an enriched common response. Strikingly, several genes involved in indole glucosinolate (iGS) modification and the camalexin biosynthesis pathway were up-regulated, whereas genes in aliphatic glucosinolate (aGS) biosynthesis were generally down-regulated, on Agrobacterium infection. Thus, we evaluated the impacts of GSs and camalexin during different stages of Agrobacterium-mediated transformation combining Arabidopsis mutant studies, metabolite profiling and exogenous applications of various GS hydrolysis products or camalexin. The results suggest that the iGS hydrolysis pathway plays an inhibitory role on transformation efficiency in Arabidopsis seedlings at the early infection stage. Later in the Agrobacterium infection process, the accumulation of camalexin is a key factor inhibiting tumour development on Arabidopsis inflorescence stalks. In conclusion, this study reveals the differential roles of GSs and camalexin at different stages of Agrobacterium-mediated transformation and provides new insights into crown gall disease control and improvement of plant transformation.
Recent studies have revealed that some responses of fern stomata to environmental signals differ from those of their relatives in seed plants. However, it is unknown whether the biophysical properties of guard cells differ fundamentally between species of both clades.
Intracellular micro-electrodes and the fluorescent Ca2+ reporter FURA2 were used to study voltage-dependent cation channels and Ca2+ signals in guard cells of the ferns Polypodium vulgare and Asplenium scolopendrium.
Voltage clamp experiments with fern guard cells revealed similar properties of voltage-dependent K+ channels as found in seed plants. However, fluorescent dyes moved within the fern stomata, from one guard cell to the other, which does not occur in most seed plants. Despite the presence of plasmodesmata, which interconnect fern guard cells, Ca2+ signals could be elicited in each of the cells individually.
Based on the common properties of voltage-dependent channels in ferns and seed plants, it is likely that these key transport proteins are conserved in vascular plants. However, the symplastic connections between fern guard cells in mature stomata indicate that the biophysical mechanisms that control stomatal movements differ between ferns and seed plants.
The membrane-bound proton-pumping pyrophosphatase (V-PPase), together with the V-type H+-ATPase, generates the proton motive force that drives vacuolar membrane solute transport. Transgenic plants constitutively overexpressing V-PPases were shown to have improved salinity tolerance, but the relative impact of increasing PPi hydrolysis and proton-pumping functions has yet to be dissected.
For a better understanding of the molecular processes underlying V-PPase-dependent salt tolerance, we transiently overexpressed the pyrophosphate-driven proton pump (NbVHP) in Nicotiana benthamiana leaves and studied its functional properties in relation to salt treatment by primarily using patch-clamp, impalement electrodes and pH imaging.
NbVHP overexpression led to higher vacuolar proton currents and vacuolar acidification. After 3 d in salt-untreated conditions, V-PPase-overexpressing leaves showed a drop in photosynthetic capacity, plasma membrane depolarization and eventual leaf necrosis. Salt, however, rescued NbVHP-hyperactive cells from cell death. Furthermore, a salt-induced rise in V-PPase but not of V-ATPase pump currents was detected in nontransformed plants.
The results indicate that under normal growth conditions, plants need to regulate the V-PPase pump activity to avoid hyperactivity and its negative feedback on cell viability. Nonetheless, V-PPase proton pump function becomes increasingly important under salt stress for generating the pH gradient necessary for vacuolar proton-coupled Na+ sequestration.