We investigated the effects of salt-sensitive signaling molecules on ionic fluxes and gene expression related to K+/Na+ homeostasis in a perennial herb, Glycyrrhiza uralensis, during short-term NaCl stress (100 mM, 24 h). Salt treatment caused more pronounced Na+ accumulation in root cells than in leaf cells. Na+ ions were mostly compartmentalized in vacuoles. Roots exposed to NaCl showed increased levels of extracellular ATP (eATP), cytosolic Ca2+, H2O2, and NO. Steady-state flux recordings revealed that these salt-sensitive signaling molecules enhanced NaCl-responsive Na+ efflux, due to the activated Na+/H+ antiport system in the plasma membrane (PM). Moreover, salt-elicited K+ efflux, which was mediated by depolarization-activated cation channels, was reduced with the addition of Ca2+, H2O2, NO, and eATP. The salt-adaptive effects of these molecules (Na+ extrusion and K+ maintenance) were reduced by pharmacological agents, including LaCl3 (a PM Ca2+ channel inhibitor), DMTU (a reactive oxygen species scavenger), cPTIO (an NO scavenger), or PPADS (an antagonist of animal PM purine P2 receptors). RT-qPCR data showed that the activation of the PM Na+/H+ antiport system in salinized roots most likely resulted from the upregulation of two genes, GuSOS1 and GuAHA, which encoded the PM Na+/H+ antiporter, salt overly sensitive 1 (SOS1) and H+-ATPase, respectively. Clear interactions occurred between these salt-sensitive agonists to accelerate transcription of salt-responsive signaling pathway genes in G. uralensis roots. For example, Ca2+, H2O2, NO, and eATP promoted transcription of GuSOS3 (salt overly sensitive 3) and/or GuCIPK (CBL-interacting protein kinase) to activate the predominant Ca2+-SOS signaling pathway in salinized liquorice roots. eATP, a novel player in the salt response of G. uralensis, increased the transcription of GuSOS3, GuCIPK, GuRbohD (respiratory burst oxidase homolog protein D), GuNIR (nitrate reductase), GuMAPK3, and GuMAPK6 (the mitogen-activated protein kinases 3 and 6). Moreover, GuMAPK3 and GuMAPK6 expression levels were enhanced by H2O2 in NaCl-stressed G. uralensis roots. Our results indicated that eATP triggered downstream components and interacted with Ca2+, H2O2, and NO signaling to maintain K+/Na+ homeostasis. We propose that a multiple signaling network regulated K+/Na+ homeostasis in NaCl-stressed G. uralensis roots.
In order to improve the accuracy of building structure identification using remote sensing images, a building structure classification method based on multi-feature fusion of UAV remote sensing image is proposed in this paper. Three identification approaches of remote sensing images are integrated in this method: object-oriented, texture feature, and digital elevation based on DSM and DEM. So RGB threshold classification method is used to classify the identification results. The accuracy of building structure classification based on each feature and the multi-feature fusion are compared and analyzed. The results show that the building structure classification method is feasible and can accurately identify the structures in large-area remote sensing images.
Apyrase and extracellular ATP play crucial roles in mediating plant growth and defense responses. In the cold-tolerant poplar, Populus euphratica, low temperatures up-regulate APYRASE2 (PeAPY2) expression in callus cells. We investigated the biochemical characteristics of PeAPY2 and its role in cold tolerance. We found that PeAPY2 predominantly localized to the plasma membrane, but punctate signals also appeared in the endoplasmic reticulum and Golgi apparatus. PeAPY2 exhibited broad substrate specificity, but it most efficiently hydrolyzed purine nucleotides, particularly ATP. PeAPY2 preferred Mg2+ as a cofactor, and it was insensitive to various, specific ATPase inhibitors. When PeAPY2 was ectopically expressed in Arabidopsis (Arabidopsis thaliana), cold tolerance was enhanced, based on root growth measurements and survival rates. Moreover, under cold stress, PeAPY2-transgenic plants maintained plasma membrane integrity and showed reduced cold-elicited electrolyte leakage compared with wild-type plants. These responses probably resulted from efficient plasma membrane repair via vesicular trafficking. Indeed, transgenic plants showed accelerated endocytosis and exocytosis during cold stress and recovery. We found that low doses of extracellular ATP accelerated vesicular trafficking, but high extracellular ATP inhibited trafficking and reduced cell viability. Cold stress caused significant increases in root medium extracellular ATP. However, under these conditions, PeAPY2-transgenic lines showed greater control of extracellular ATP levels than wild-type plants. We conclude that Arabidopsis plants that overexpressed PeAPY2 could increase membrane repair by accelerating vesicular trafficking and hydrolyzing extracellular ATP to avoid excessive, cold-elicited ATP accumulation in the root medium and, thus, reduced ATP-induced inhibition of vesicular trafficking.
Abstract To investigate physiological and transcriptomic regulation mechanisms underlying the distinct net fluxes of NH4+ and NO3− in different root segments of Populus species under low nitrogen (N) conditions, we used saplings of Populus × canescens supplied with either 500 (normal N) or 50 (low N) μM NH4NO3. The net fluxes of NH4+ and NO3−, the concentrations of NH4+, amino acids and organic acids and the enzymatic activities of nitrite reductase (NiR) and glutamine synthetase (GS) in root segment II (SII, 35–70 mm to the apex) were lower than those in root segment I (SI, 0–35 mm to the apex). The net NH4+ influxes and the concentrations of organic acids were elevated, whereas the concentrations of NH4+ and NO3− and the activities of NiR and GS were reduced in SI and SII in response to low N. A number of genes were significantly differentially expressed in SII vs SI and in both segments grown under low vs normal N conditions, and these genes were mainly involved in the transport of NH4+ and NO3−, N metabolism and adenosine triphosphate synthesis. Moreover, the hub gene coexpression networks were dissected and correlated with N physiological processes in SI and SII under normal and low N conditions. These results suggest that the hub gene coexpression networks play pivotal roles in regulating N uptake and assimilation, amino acid metabolism and the levels of organic acids from the tricarboxylic acid cycle in the two root segments of poplars in acclimation to low N availability.
Sodium chloride (NaCl) induced expression of a jacalin-related mannose-binding lectin (JRL) gene in leaves, roots, and callus cultures of Populus euphratica (salt-resistant poplar). To explore the mechanism of the PeJRL in salinity tolerance, the full length of PeJRL was cloned from P. euphratica and was transformed into Arabidopsis. PeJRL was localized to the cytoplasm in mesophyll cells. Overexpression of PeJRL in Arabidopsis significantly improved the salt tolerance of transgenic plants, in terms of seed germination, root growth, and electrolyte leakage during seedling establishment. Under NaCl stress, transgenic plants retained K+ and limited the accumulation of Na+. PeJRL-transgenic lines increased Na+ extrusion, which was associated with the upward regulation of SOS1, AHA1, and AHA2 genes encoding plasma membrane Na+/proton (H+) antiporter and H+-pumps. The activated H+-ATPases in PeJRL-overexpressed plants restricted the channel-mediated loss of K+ that was activated by NaCl-induced depolarization. Under salt stress, PeJRL–transgenic Arabidopsis maintained reactive oxygen species (ROS) homeostasis by activating the antioxidant enzymes and reducing the production of O2− through downregulation of NADPH oxidases. Of note, the PeJRL-transgenic Arabidopsis repressed abscisic acid (ABA) biosynthesis, thus reducing the ABA-elicited ROS production and the oxidative damage during the period of salt stress. A schematic model was proposed to show the mediation of PeJRL on ABA response, and ionic and ROS homeostasis under NaCl stress.
This work aimed at investigating the interactive effects of salt-signaling molecules, i.e., ethylene, extracellular ATP (eATP), H2O2, and cytosolic Ca2+ ([Ca2+]cyt), on the regulation of K+/Na+ homeostasis in Arabidopsisthaliana. The presence of eATP shortened Col-0 hypocotyl length under no-salt conditions. Moreover, eATP decreased relative electrolyte leakage and lengthened root length significantly in salt-treated Col-0 plants but had no obvious effects on the ethylene-insensitive mutants etr1-1 and ein3-1eil1-1. Steady-state ionic flux kinetics showed that exogenous 1-aminocyclopropane-1-carboxylic acid (ACC, an ethylene precursor) and eATP-Na2 (an eATP donor) significantly increased Na+ extrusion and suppressed K+ loss during short-term NaCl treatment. Moreover, ACC remarkably raised the fluorescence intensity of salt-elicited H2O2 and cytosolic Ca2+. Our qPCR data revealed that during 12 h of NaCl stress, application of ACC increased the expression of AtSOS1 and AtAHA1, which encode the plasma membrane (PM) Na+/H+ antiporters (SOS1) and H+-ATPase (H+ pumps), respectively. In addition, eATP markedly increased the transcription of AtEIN3, AtEIL1, and AtETR1, and ACC treatment of Col-0 roots under NaCl stress conditions caused upregulation of AtRbohF and AtSOS2/3, which directly contribute to the H2O2 and Ca2+ signaling pathways, respectively. Briefly, ethylene was triggered by eATP, a novel upstream signaling component, which then activated and strengthened the H2O2 and Ca2+ signaling pathways to maintain K+/Na+ homeostasis under salinity.
We elucidated the extracellular ATP (eATP) signalling cascade active in programmed cell death (PCD) using cell cultures of Populus euphratica. Millimolar amounts of eATP induced a dose- and time-dependent reduction in viability, and the agonist-treated cells displayed hallmark features of PCD. eATP caused an elevation of cytosolic Ca(2+) levels, resulting in Ca(2+) uptake by the mitochondria and subsequent H(2) O(2) accumulation. P. euphratica exhibited an increased mitochondrial transmembrane potential, and cytochrome c was released without opening of the permeability transition pore over the period of ATP stimulation. Moreover, the eATP-induced increase of intracellular ATP, essential for the activation of caspase-like proteases and subsequent PCD, was found to be related to increased mitochondrial transmembrane potential. NO is implicated as a downstream component of the cytosolic Ca(2+) concentration but plays a negligible role in eATP-stimulated cell death. We speculate that ATP binds purinoceptors in the plasma membrane, leading to the induction of downstream intermediate signals, as the proposed sequence of events in PCD signalling was terminated by the animal P2 receptor antagonist suramin.
Abstract Different root zones have distinct capacities for nitrate (NO3−) uptake in Populus species, but the underlying physiological and microRNA (miRNA) regulatory mechanisms remain largely unknown. To address this question, two root zones of Populus × canescens (Ait.) Smith. with contrasting capacities for NO3− uptake were investigated. The region of 0–40 mm (root zone I) to the root apex displayed net influxes, whereas the region of 40–80 mm (root zone II) exhibited net effluxes. Concentrations of NO3− and ammonium (NH4+) as well as nitrate reductase activity were lower in zone II than in zone I. Forty one upregulated and twenty three downregulated miRNAs, and 576 targets of these miRNAs were identified in zone II in comparison with zone I. Particularly, growth-regulating factor 4 (GRF4), a target of upregulated ptc-miR396g-5p and ptc-miR396f_L + 1R-1, was downregulated in zone II in comparison with zone I, probably contributing to lower NO3− uptake rates and assimilation in zone II. Furthermore, several miRNAs and their targets, members of C2H2 zinc finger family and APETALA2/ethylene-responsive element binding protein family, were found in root zones, which probably play important roles in regulating NO3− uptake. These results indicate that differentially expressed miRNA–target pairs play key roles in regulation of distinct NO3− uptake rates and assimilation in different root zones of poplars.
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