The calcium-dependent protein kinase CPK16 regulates hypoxia-induced ROS production by phosphorylating the NADPH oxidase RBOHD in Arabidopsis.

Reactive oxygen species (ROS) production is a key event in modulating plant responses to hypoxia and post-hypoxia reoxygenation. However, the molecular mechanism by which hypoxia-associated ROS homeostasis is controlled remains largely unknown. Here, we showed that the calcium-dependent protein kinase CPK16 regulates plant hypoxia tolerance by phosphorylating the plasma membrane-anchored NADPH oxidase respiratory burst oxidase homolog D (RBOHD) to regulate ROS production in Arabidopsis (Arabidopsis thaliana). In response to hypoxia or reoxygenation, CPK16 was activated through phosphorylation of its Ser274 residue. The cpk16 knockout mutant displayed enhanced hypoxia tolerance, whereas CPK16-overexpressing (CPK16-OE) lines showed increased sensitivity to hypoxic stress. In agreement with these observations, hypoxia and reoxygenation both induced ROS accumulation in the rosettes of CPK16-OEs more strongly than in the rosettes of the cpk16-1 mutant or the wild type. Moreover, CPK16 interacted with and phosphorylated the N-terminus of RBOHD at 4 serine residues (Ser133, Ser148, Ser163, and Ser347) that were necessary for hypoxia- and reoxygenation-induced ROS accumulation. Furthermore, the hypoxia-tolerant phenotype of cpk16-1 was fully abolished in the cpk16 rbohd double mutant. Thus, we have uncovered a regulatory mechanism by which the CPK16-RBOHD module shapes the ROS production during hypoxia and reoxygenation in Arabidopsis.

Real-time volumetric reconstruction of biological dynamics with light-field microscopy and deep learning.

Light-field microscopy has emerged as a technique of choice for high-speed volumetric imaging of fast biological processes. However, artifacts, nonuniform resolution and a slow reconstruction speed have limited its full capabilities for in toto extraction of dynamic spatiotemporal patterns in samples. Here, we combined a view-channel-depth (VCD) neural network with light-field microscopy to mitigate these limitations, yielding artifact-free three-dimensional image sequences with uniform spatial resolution and high-video-rate reconstruction throughput. We imaged neuronal activities across moving Caenorhabditis elegans and blood flow in a beating zebrafish heart at single-cell resolution with volumetric imaging rates up to 200 Hz.

Polyunsaturated linolenoyl-CoA modulates ERF-VII-mediated hypoxia signaling in Arabidopsis.

In plants, submergence from flooding causes hypoxia, which impairs energy production and affects plant growth, productivity, and survival. In Arabidopsis, hypoxia induces nuclear localization of the group VII ethylene-responsive transcription factor RELATED TO AP2.12 (RAP2.12), following its dissociation from the plasma membrane-anchored ACYL-COA BINDING PROTEIN1 (ACBP1) and ACBP2. Here, we show that polyunsaturated linolenoyl-CoA (18:3-CoA) regulates RAP2.12 release from the plasma membrane. Submergence caused a significant increase in 18:3-CoA, but a significant decrease in 18:0-, 18:1-, and 18:2-CoA. Application of 18:3-CoA promoted nuclear accumulation of the green fluorescent protein (GFP) fusions RAP2.12-GFP, HYPOXIA-RESPONSIVE ERF1-GFP, and RAP2.3-GFP, and enhanced transcript levels of hypoxia-responsive genes. Plants with decreased ACBP1 and ACBP2 (acbp1 ACBP2-RNAi, produced by ACBP2 RNA interference in the acbp1 mutant) had reduced tolerance to hypoxia and impaired 18:3-CoA-induced expression of hypoxia-related genes. In knockout mutants and overexpression lines of LONG-CHAIN ACYL-COA SYNTHASE2 (LACS2) and FATTY ACID DESATURASE 3 (FAD3), the acyl-CoA pool size and 18:3-CoA levels were closely related to ERF-VII-mediated signaling and hypoxia tolerance. These findings demonstrate that polyunsaturation of long-chain acyl-CoAs functions as important mechanism in the regulation of plant hypoxia signaling, by modulating ACBP-ERF-VII dynamics.

DIACYLGLYCEROL ACYLTRANSFERASE and DIACYLGLYCEROL KINASE Modulate Triacylglycerol and Phosphatidic Acid Production in the Plant Response to Freezing Stress.

Plants accumulate the lipids phosphatidic acid (PA), diacylglycerol (DAG), and triacylglycerol (TAG) during cold stress, but how plants balance the levels of these lipids to mediate cold responses remains unknown. The enzymes ACYL-COENZYME A:DIACYLGLYCEROL ACYLTRANSFERASE (DGAT) and DIACYLGLYCEROL KINASE (DGK) catalyze the conversion of DAG to TAG and PA, respectively. Here, we show that DGAT1, DGK2, DGK3, and DGK5 contribute to the response to cold in Arabidopsis (Arabidopsis thaliana). With or without cold acclimation, the dgat1 mutants exhibited higher sensitivity upon freezing exposure compared with the wild type. Under cold conditions, the dgat1 mutants showed reduced expression of C-REPEAT/DRE BINDING FACTOR2 and its regulons, which are essential for the acquisition of cold tolerance. Lipid profiling revealed that freezing significantly increased the levels of PA and DAG while decreasing TAG in the rosettes of dgat1 mutant plants. During freezing stress, the accumulation of PA in dgat1 plants stimulated NADPH oxidase activity and enhanced RbohD-dependent hydrogen peroxide production compared with the wild type. Moreover, the cold-inducible transcripts of DGK2, DGK3, and DGK5 were significantly more up-regulated in the dgat1 mutants than in the wild type during cold stress. Consistent with this observation, dgk2, dgk3, and dgk5 knockout mutants showed improved tolerance and attenuated PA production in response to freezing temperatures. Our findings demonstrate that the conversion of DAG to TAG by DGAT1 is critical for plant freezing tolerance, acting by balancing TAG and PA production in Arabidopsis.

Jasmonate Regulates Plant Responses to Postsubmergence Reoxygenation through Transcriptional Activation of Antioxidant Synthesis.

Submergence induces hypoxia in plants; exposure to oxygen following submergence, termed reoxygenation, produces a burst of reactive oxygen species. The mechanisms of hypoxia sensing and signaling in plants have been well studied, but how plants respond to reoxygenation remains unclear. Here, we show that reoxygenation in Arabidopsis (Arabidopsis thaliana) involves rapid accumulation of jasmonates (JAs) and increased transcript levels of JA biosynthesis genes. Application of exogenous methyl jasmonate improved tolerance to reoxygenation in wild-type Arabidopsis; also, mutants deficient in JA biosynthesis and signaling were very sensitive to reoxygenation. Moreover, overexpression of the transcription factor gene MYC2 enhanced tolerance to posthypoxic stress, and myc2 knockout mutants showed increased sensitivity to reoxygenation, indicating that MYC2 functions as a key regulator in the JA-mediated reoxygenation response. MYC2 transcriptionally activates members of the VITAMIN C DEFECTIVE (VTC) and GLUTATHIONE SYNTHETASE (GSH) gene families, which encode rate-limiting enzymes in the ascorbate and glutathione synthesis pathways. Overexpression of VTC1 and GSH1 in the myc2-2 mutant suppressed the posthypoxic hypersensitive phenotype. The JA-inducible accumulation of antioxidants may alleviate oxidative damage caused by reoxygenation, improving plant survival after submergence. Taken together, our findings demonstrate that JA signaling interacts with the antioxidant pathway to regulate reoxygenation responses in Arabidopsis.

Preparation of a silica-based high-performance hydrophobic interaction chromatography stationary phase for protein separation and renaturation.

In this work, based on the structural characteristics of bio-membrane molecules, a novel type of high-performance hydrophobic interaction chromatography stationary phase was prepared using cholesterol as a ligand. Investigating the separation performance of this stationary phase, the effect of pH and salt concentration of the mobile phase on the retention time, the absorption capacity, and the hydrophobic ability revealed that this stationary phase had a high loading capacity and moderate hydrophobic interactions compared with four different hydrophobic interaction chromatography stationary phase ligands. Five types of standard proteins could be baseline separated with a great selection for protein separation. When 3.0 M urea was added to the mobile phase, it could be refolded with simultaneous purification of denatured lysozyme by one-step chromatography. The mass recovery of lysozyme reached 89.5%, and the active recovery was 96.8%. Compared with traditional hydrophobic interaction chromatography, this new stationary phase has a good hydrophobic ability and a significant refolding efficiency.

Refolding and simultaneous purification of recombinant human proinsulin from inclusion bodies on protein-folding liquid-chromatography columns.

Protein-folding liquid chromatography (PFLC) is an effective and scalable method for protein renaturation with simultaneous purification. However, it has been a challenge to fully refold inclusion bodies in a PFLC column. In this work, refolding with simultaneous purification of recombinant human proinsulin (rhPI) from inclusion bodies from Escherichia coli were investigated using the surface of stationary phases in immobilized metal ion affinity chromatography (IMAC) and high-performance size-exclusion chromatography (HPSEC). The results indicated that both the ligand structure on the surface of the stationary phase and the composition of the mobile phase (elution buffer) influenced refolding of rhPI. Under optimized chromatographic conditions, the mass recoveries of IMAC column and HPSEC column were 77.8 and 56.8% with purifies of 97.6 and 93.7%, respectively. These results also indicated that the IMAC column fails to refold rhPI, and the HPSEC column enables efficient refolding of rhPI with a low-urea gradient-elution method. The refolded rhPI was characterized by circular dichroism spectroscopy. The molecular weight of the converted human insulin was further confirmed with SDS-18% PAGE, Matrix-Assisted Laser Desorption/ Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS) and the biological activity assay by HP-RPLC.

Study on the mechanical properties of flax fiber-reinforced silty clay contaminated by zinc-ion solution.

Mechanical properties of fiber-reinforced soil after soaking in heavy metal ion solution have great influences on safety and stability of the reinforcement, herein the mechanical properties of optimum moisture content of different concentrations of zinc ions contaminated soil were studied through shear test, compression test and triaxial test. The compressive modulus, compression coefficient and porosity ratio of different concentrations of the zinc-ion contaminated soil under different pressure were studied, and the variation characteristics of internal friction angle and cohesion were also investigated, thereafter, the causes of the change of cohesion and internal friction angle were analyzed from the microscopic perspective. The results show that the shear strength of contaminated reinforced soil increases with the increase of confining pressure at the same zinc ions concentration. And at the same confining pressure, with the increase of zinc ions concentration, the shear strength of contaminated reinforced soil first increases and then decreases. With the increase of zinc ions concentration, the internal friction angle and compression coefficient increase, the cohesion and the modulus of compression decrease. With the increase of normal stress, the compression coefficient decreases firstly and then increases, and the compression modulus increases and then decreases. With the concentration of zinc ions increases at lower normal stress, the amount of shrinkage increases and the compression coefficient decreases. While at higher normal stress, the compressive modulus decreases and the compression coefficient increases.

Long-Chain acyl-CoA Synthetase LACS2 Contributes to Submergence Tolerance by Modulating Cuticle Permeability in Arabidopsis.

In Arabidopsis thaliana, LONG-CHAIN ACYL-COA SYNTHETASEs (LACSs) catalyze the synthesis of long-chain acyl-CoAs and function in diverse biological processes. We have recently revealed that LACS2 is primarily involved in the production of polyunsaturated linolenoyl-CoA, essential for the activation of ethylene response transcription factors-mediated hypoxia signaling. Here, we further reported the dual role of LACS2 in the regulation of submergence tolerance by modulating cuticle permeability in Arabidopsis cells. LACS2-overexpressors (LACS2-OEs) showed improved tolerance to submergence, with higher accumulation of cuticular wax and cutin in their rosettes. In contrast, knockout of LACS2 in the lacs2-3 mutant resulted in hypersensitivity to submergence with reduced wax crystals and thinner cutin layer. By analyses of plant surface permeability, we observed that the hypoxic sensitivities in the LACS2-OEs and lacs2-3 mutant were physiologically correlated with chlorophyll leaching, water loss rates, ionic leakage, and gas exchange. Thus, our findings suggest the role of LACS2 in plant response to submergence by modulating cuticle permeability in plant cells.

Efficient and cost-effective 3D cellular imaging by sub-voxel-resolving light-sheet add-on microscopy.

Light-sheet fluorescence microscopy (LSFM) allows volumetric live imaging at high-speed and with low photo-toxicity. Various LSFM modalities are commercially available, but their size and cost limit their access by the research community. A new method, termed sub-voxel-resolving (SVR) light-sheet add-on microscopy (SLAM), is presented to enable fast, resolution-enhanced light-sheet fluorescence imaging from a conventional wide-field microscope. This method contains two components: a miniature add-on device to regular wide-field microscopes, which contains a horizontal laser light-sheet illumination path to confine fluorophore excitation at the vicinity of the focal plane for optical sectioning; an off-axis scanning strategy and a SVR algorithm that utilizes sub-voxel spatial shifts to reconstruct the image volume that results in a twofold increase in resolution. SLAM method has been applied to observe the muscle activity change of crawling C. elegans, the heartbeat of developing zebrafish embryo, and the neural anatomy of cleared mouse brains, at high spatiotemporal resolution. It provides an efficient and cost-effective solution to convert the vast number of in-service microscopes for fast 3D live imaging with voxel-super-resolved capability.

High-throughput, high-resolution deep learning microscopy based on registration-free generative adversarial network.

We combine a generative adversarial network (GAN) with light microscopy to achieve deep learning super-resolution under a large field of view (FOV). By appropriately adopting prior microscopy data in an adversarial training, the neural network can recover a high-resolution, accurate image of new specimen from its single low-resolution measurement. Its capacity has been broadly demonstrated via imaging various types of samples, such as USAF resolution target, human pathological slides, fluorescence-labelled fibroblast cells, and deep tissues in transgenic mouse brain, by both wide-field and light-sheet microscopes. The gigapixel, multi-color reconstruction of these samples verifies a successful GAN-based single image super-resolution procedure. We also propose an image degrading model to generate low resolution images for training, making our approach free from the complex image registration during training data set preparation. After a well-trained network has been created, this deep learning-based imaging approach is capable of recovering a large FOV (~95 mm2) enhanced resolution of ~1.7 µm at high speed (within 1 second), while not necessarily introducing any changes to the setup of existing microscopes.

A poly(alkyl methacrylate-divinylbenzene-vinylbenzyl trimethylammonium chloride) monolithic column for solid-phase microextraction.

In this study, an organic polymer monolithic columns, which were prepared via in situ polymerization of alkyl methacrylate-ester (AMA), divinylbenzene (DVB) and vinylbenzyl trimethylammonium chloride (VBTA, charged monomer), were developed as adsorbent for solid-phase microextraction (SPME). Different parameters affecting the extraction efficiency for nine (9) non-steroidal anti-inflammatory drugs (NSAIDs) such as the ratio of the stearyl methacrylate (SMA) to DVB monomer, column length, sample pH, extraction flow rate and desorption solvent were investigated to obtain the optimal SPME condition. Also, the permeability for each poly(AMA-DVB-VBTA) monolithic column was investigated by adding porogenic solvent (poly(ethylene glycol), PEG). Using the optimized condition, a series of AMA-based poly(AMA-DVB-VBTA) monolith columns were developed to determine the effect the extraction efficiency of NSAIDs by varying the alkyl chain length of the methacrylate ester (methyl-, butyl-, octyl-, or lauryl-methacrylate; (MMA, BMA, OMA, LMA)). Results showed that decreasing the AMA chain length increases the extraction efficiency of some NSAIDs (i.e. sulindac (sul), naproxen (nap), ketoprofen (ket) and indomethacin (idm)). Among the poly(AMA-DVB-VBTA) monolithic columns, poly(BMA-DVB-VBTA) showed a highly repeatable extraction efficiency for NSAIDs with recoveries ranging from 85.0 to 100.2% with relative standard deviation (RSD) less than 6.8% (n=3). The poly(BMA-DVB-VBTA) can also be reused for at least 50 times without any significant effect in extraction efficiency for NSAIDs. Finally, using the established conditions, the poly(BMA-DVB-VBTA) was used to extract trace-level NSAIDs (100µgL(-1)) in river water with good recoveries ranging from 75.8 to 90.8% (RSD<14.9%).