Nondestructive detection of saline-alkali stress in wheat (Triticum aestivum L.) seedlings via fusion technology.

BACKGROUND: Wheat (Triticum aestivum L.) is an important grain crops in the world, and its growth and development in different stages is seriously affected by saline-alkali stress, especially in seedling stage. Therefore, nondestructive detection of wheat seedlings under saline-alkali stress can provide more comprehensive technical support for wheat breeding, cultivation and management. RESULTS: This research focused on moisture signal prediction and classification of saline-alkali stress in wheat seedlings using fusion techniques. After collecting and analyzing transverse relaxation time and Multispectral imaging (MSI) information of wheat seedlings, four regression models were used to predict the moisture signal. K-Nearest Neighbor (KNN) and Gaussian-Naïve Bayes (GNB) models were combined with fivefold cross validation to classify the prediction of wheat seedling stress. The results showed that wheat seedlings would increase the bound water content through a certain mechanism to enhance their saline-alkali stress. Under the same Na concentration, the effect of alkali stress on moisture, growth and spectrum of wheat seedlings is stronger than salt stress. The Gradient Boosting Decision Regression Tree model performs the best in predicting wheat moisture signals, with a coefficient of determination (R2P) of 0.98 and a root mean square error of 109.60. It also had a short training time (1.48 s) and an efficient prediction speed (1300 obs/s). The KNN and GNB demonstrated significantly enhanced predictive performance when classifying the fused dataset, compared to using single datasets individually. In particular, the GNB model performing best on the fused dataset, with Precision, Recall, Accuracy, and F1-score of 90.30, 88.89%, 88.90%, and 0.90, respectively. CONCLUSIONS: Under the same Na concentration, the effects of alkali stress on water content, spectrum, and growth of wheat were stronger than that of salt stress, which was more unfavorable to the growth of wheat. The fusion of low-field nuclear magnetic resonance and MSI technology can improve the classification of wheat stress, and provide an effective technical method for rapid and accurate monitoring of wheat seedlings under saline-alkali stress.

Flexible and wearable sensor for in situ monitoring of gallic acid in plant leaves.

Gallic acid (GA) is one of the main phenolic components naturally occurring in many plants and foods and has been a subject of increasing interest owing to its antioxidant and anti-mutagenic properties. This study introduces a novel flexible sensor designed for in situ detecting GA in plant leaves. The sensor employs a laser-induced graphene (LIG) flexible electrode, enhanced with MXene and molybdenum disulfide (MoS2) nanosheets. The MXene/MoS2/LIG flexible sensor not only demonstrates exceptional mechanical properties, covering a wide detection range of 1-1000 µM for GA, but also exhibits remarkable selectivity and stability. The as-prepared sensor was successfully applied to in situ determination of GA content in strawberry leaves under salt stress. This innovative sensor opens an attractive avenue for in situ measurement of metabolites in plant bodies with flexible electronics.

Genome-wide analysis of MYB transcription factor family and AsMYB1R subfamily contribution to ROS homeostasis regulation in Avena sativa under PEG-induced drought stress.

BACKGROUND: The myeloblastosis (MYB) transcription factor (TF) family is one of the largest and most important TF families in plants, playing an important role in a life cycle and abiotic stress. RESULTS: In this study, 268 Avena sativa MYB (AsMYB) TFs from Avena sativa were identified and named according to their order of location on the chromosomes, respectively. Phylogenetic analysis of the AsMYB and Arabidopsis MYB proteins were performed to determine their homology, the AsMYB1R proteins were classified into 5 subgroups, and the AsMYB2R proteins were classified into 34 subgroups. The conserved domains and gene structure were highly conserved among the subgroups. Eight differentially expressed AsMYB genes were screened in the transcriptome of transcriptional data and validated through RT-qPCR. Three genes in AsMYB2R subgroup, which are related to the shortened growth period, stomatal closure, and nutrient and water transport by PEG-induced drought stress, were investigated in more details. The AsMYB1R subgroup genes LHY and REV 1, together with GST, regulate ROS homeostasis to ensure ROS signal transduction and scavenge excess ROS to avoid oxidative damage. CONCLUSION: The results of this study confirmed that the AsMYB TFs family is involved in the homeostatic regulation of ROS under drought stress. This lays the foundation for further investigating the involvement of the AsMYB TFs family in regulating A. sativa drought response mechanisms.

Development of surface molecular-imprinted electrochemical sensor for palmitic acid with machine learning assistance.

Palmitic acid (PA) is a kind of saturated high fatty acid, which is involved in physiological safety and food quality. A surface molecularly imprinted polymer (MIP) electrochemical sensor was prepared on MXene surface using dopamine (DA) as functional monomer. The electrode was modified with gold nanoparticles (AuNPs), ferrocene-graphene oxide-multiwalled carbon nanotubes (Fc-GO-MWCNT) composite to enhance the electroactive area and conductivity. The sensor was characterized by scanning electron microscope (SEM), energy-dispersive X-ray spectroscopy (EDS), electrochemical impedance spectroscopy (EIS) and Differential pulse voltammetry (DPV), respectively. The parameters concerning this assay and various regeneration conditions have been carefully studied. The sensor can detect PA in the range of 1 nM-1 mM (R2 = 0.995), the limit of detection (LOD) is 0.48 nM (S/N = 3), and the limit of quantification (LOQ) is 1.61 nM. The artificial neural network (ANN) model in machine learning is further used to analyze the data collected by the sensor. The results show that the back propagation (BP) neural network in ANN is more suitable for the intelligent analysis of PA. The practicality of the sensor was confirmed by detecting PA in pork samples. This is the first MIP-based electrochemical sensor for PA, and it has great potential in practical applications.

An ultrasensitive probe-free electrochemical immunosensor for gibberellins employing polydopamine-antibody nanoparticles modified electrode.

Gibberellins (GA3) is an ubiquitous plant hormone, which plays a regulatory role in different growth stages of plants, so it is of great significance to develop a sensitive quantitative analysis method for GA3. In this study, carboxylated graphene oxide- carboxylated multi-walled carbon nanotubes-Fc (GO-MWNT-Fc) composite material and PDANPs-antibody (PDANPs-Ab) were sequentially modified to screen-printed electrodes (SPEs), and an ultrasensitive probe-free immunosensor for GA3 was developed. Fc was applied to generate electrochemical signals. GO-COOH and MWNT-COOH can increase the catalytic ability of the sensor and bind the PDANPs-Ab nanoparticles. PDANPs nanomaterial were synthetized by a facile self-polymerization and used to bind with antibody, so as to increase the antibody loading of the sensor. The as-prepared immunosensor has the widest detection range (100 aM-1 mM) and lowest detection limit (17.4 aM) for GA3 up to date. To our knowledge, it is the first electrochemical immunosensor for GA3. By changing the GA3 antibody to ABA antibody, a sensitive and selective immunosensor for ABA was also fabricated. This immunosensor platform is simple, sensitive, and low cost. It opens broad prospect in on-site applications for biosensors in detecting of various biomolecules in precision agriculture.

A novel COOH-GO-COOH-MWNT/pDA/AuNPs based electrochemical aptasensor for detection of AFB1.

Aflatoxin B1 (AFB1), one of the most common mycotoxins in food matrixes, has been identified as the most toxic contaminant with mutagenic, teratogenic, immunosuppressive, and carcinogenic effects. In this study, an electrochemical aptamer sensor was developed for the on-site detection of AFB1. Carboxylated graphene oxide (COOH-GO) and carboxylated multi-walled carbon nanotubes (COOH-MWNT) nanocomposites, dopamine polymers (pDA) and gold nanoparticles (AuNPs) were used to enhance the electrochemical activity and the biocompatibility of the screen-printed electrodes (SPE). Once AFB1 was captured by the aptamer immobilized on the electrode surface, the redox current of [Fe(CN)6]3-/4- decreased. Therefore, the binding of aptamer (Apt) and AFB1 can be reflected by the change of the peak current. The as-prepared sensor showed a wide detection range of 0.1 fg ml-1-100 pg ml-1 and a low detection limit of 15.16 ag ml-1. It is also simple and low-cost, which shows great potential in practical application.

In Vivo Detection of Glutamate in Tomatoes by an Enzyme-Based Electrochemical Biosensor.

The in vivo and on-site detection of key physiology parameters in plants will be of great relevance for precision agriculture and food technology. In this work, a sensitive enzymatic glutamate sensor was successfully developed. To enhance the conductivity and catalytic ability and to fix the glutamate oxidase, Au-Pt nanoparticles were first deposited on screen-printed electrodes, and then carboxylated graphene oxide and carboxylated multiwalled carbon nanotubes were fabricated for the synthesis of the electrode. The detection range of the glutamate sensor is widest (2 µM to 16 mM) up to date, and its detection limit is relatively low (0.14 µM). A number of standard curves were built in the pH range of 3.5-7.5, which can be applied in various plants and fruits. Using this sensor, the glutamate level in tomatoes was determined in vivo. This glutamate sensor has important practical value in precision agriculture. Our strategy also provides a way to establish the detection modes for other biomolecules in plants.

A probe-free electrochemical immunosensor for methyl jasmonate based on a Cu-MOF-carboxylated graphene oxide platform.

Methyl jasmonate (MeJA) is an important phytohormone which can regulate plant growth and stress tolerance. It is very necessary to develop sensitive and accurate detection methods for MeJA. In this work, a probe-free electrochemical immunosensor for MeJA detection was developed based on a Cu-MOF-carboxylated graphene oxide (COOH-GO) platform. The Cu2+ in the Cu-MOFs was used to provide redox signals, which avoids the application of an external redox probe in the electrolyte solutions as conventional immunosensors. COOH-GO was used to improve the structural stability and provide more sites for binding MeJA antibodies. The linear range of the MeJA immunosensor is from 10 pM to 100 µM, which can cover the whole concentration range of MeJA in most plants. And its detection limit is very low (0.35 pM), and it can detect very low concentrations of MeJA. This immunosensor is simple, low cost, and does not need redox probe solutions for measurements. It shows remarkable potential for on-site application in precision agriculture.

Enzyme-Free Electrochemical Sensors for in situ Quantification of Reducing Sugars Based on Carboxylated Graphene-Carboxylated Multiwalled Carbon Nanotubes-Gold Nanoparticle-Modified Electrode.

The reducing sugars of plants, including glucose, fructose, arabinose, galactose, xylose, and mannose, are not only the energy source of plants, but also have the messenger function of hormones in signal transduction. Moreover, they also determine the quality and flavor of agricultural products. Therefore, the in situ quantification of reducing sugars in plants or agriculture products is very important in precision agriculture. However, the upper detection limit of the currently developed sugar sensor is not high enough for in situ detection. In this study, an enzyme-free electrochemical sensor for in situ detection of reducing sugars was developed. Three-dimensional composite materials based on carboxylated graphene-carboxylated multi-walled carbon nanotubes attaching with gold nanoparticles (COOH-GR-COOH-MWNT-AuNPs) were formed and applied for the non-enzymatic determination of glucose, fructose, arabinose, mannose, xylose, and galactose. It was demonstrated that the COOH-GR-COOH-MWNT-AuNP-modified electrode exhibited a good catalysis behavior to these reducing sugars due to the synergistic effect of the COOH-GR, COOH-MWNT, and AuNPs. The detection range of the sensor for glucose, fructose, arabinose, mannose, xylose, and galactose is 5-80, 2-20, 2-50, 5-60, 2-40, and 5-40 mM, respectively. To our knowledge, the upper detection limit of our enzyme-free sugar sensor is the highest compared to previous studies, which is more suitable for in situ detection of sugars in agricultural products and plants. In addition, this sensor is simple and portable, with good reproducibility and accuracy; it will have broad practical application value in precision agriculture.

A probe-free electrochemical immunosensor for methyl jasmonate based on ferrocene functionalized-carboxylated graphene-multi-walled carbon nanotube nanocomposites.

Methyl jasmonate (MeJA) is an endogenous plant hormone, which plays an important role in agriculture production. A novel probe-free electrochemical immunosensor was fabricated for detecting of MeJA. Fc, carboxylated graphene (COOH-GR) and carboxylated multi-walled carbon nanotubes (COOH-MWNT) composite was formed and used to fabricate screen-printed electrode (SPE). Fc was used as the electronic medium. COOH-GR and COOH-MWNT were used to improve the conductivity and catalytic activity of the sensor and to immobilize the MeJA antibody. Thus, the immunosensor can be used to detect MeJA without external redox probe solution. The designed sensor can detect MeJA in a wide range of 100 fM-100 µM, and its detection limit is as low as 31.26 fM (S/N = 3). The as-prepared probe-free immunosensor is simple, low cost, and does not need redox probe solutions for measurements, which shows great promise for future application in precision agriculture.

Antioxidant Enzymatic Activity and Osmotic Adjustment as Components of the Drought Tolerance Mechanism in Carex duriuscula.

Drought stress is a major environmental constraint for plant growth. Climate-change-driven increases in ambient temperatures resulted in reduced or unevenly distributed rainfalls, leading to increased soil drought. Carex duriuscula C. A. Mey is a typical drought-tolerant sedge, but few reports have examined the mechanisms conferring its tolerant traits. In the present study, the drought responses of C. duriuscula were assessed by quantifying activity of antioxidant enzymes in its leaf and root tissues and evaluating the relative contribution of organic and inorganic osmolyte in plant osmotic adjustment, linking it with the patterns of the ion acquisition by roots. Two levels of stress-mild (MD) and severe (SD) drought treatments-were used, followed by re-watering. Drought stress caused reduction in a relative water content and chlorophyll content of leaves; this was accompanied by an increase in the hydrogen peroxide (H2O2) and superoxide (O2-) contents in leaves and roots. Under MD stress, the activities of catalase (CAT), peroxidase (POD), and glutathione peroxidase (GPX) increased in leaves, whereas, in roots, only CAT and POD activities increased. SD stress led to an increase in the activities of CAT, POD, superoxide dismutase (SOD), and GPX in both tissues. The levels of proline, soluble sugars, and soluble proteins in the leaves also increased. Under both MD and SD stress conditions, C. duriuscula increased K+, Na+, and Cl- uptake by plant roots, which resulted in an increased K+, Na+, and Cl- concentrations in leaves and roots. This reliance on inorganic osmolytes enables a cost-efficient osmotic adjustment in C. duriuscula. Overall, this study revealed that C. duriuscula was able to survive arid environments due to an efficient operation of its ROS-scavenging systems and osmotic adjustment mechanisms.

Kandelia candel Thioredoxin f Confers Osmotic Stress Tolerance in Transgenic Tobacco.

Water deficit caused by osmotic stress and drought limits crop yield and tree growth worldwide. Screening and identifying candidate genes from stress-resistant species are a genetic engineering strategy to increase drought resistance. In this study, an increased concentration of mannitol resulted in elevated expression of thioredoxin f (KcTrxf) in the nonsecretor mangrove species Kandelia candel. By means of amino acid sequence and phylogenetic analysis, the mangrove Trx was classified as an f-type thioredoxin. Subcellular localization showed that KcTrxf localizes to chloroplasts. Enzymatic activity characterization revealed that KcTrxf recombinant protein possesses the disulfide reductase function. KcTrxf overexpression contributes to osmotic and drought tolerance in tobacco in terms of fresh weight, root length, malondialdehyde (MDA) content, and hydrogen peroxide (H2O2) production. KcTrxf was shown to reduce the stomatal aperture by enhancing K+ efflux in guard cells, which increased the water-retaining capacity in leaves under drought conditions. Notably, the abscisic acid (ABA) sensitivity was increased in KcTrxf-transgenic tobacco, which benefits plants exposed to drought by reducing water loss by promoting stomatal closure. KcTrxf-transgenic plants limited drought-induced H2O2 in leaves, which could reduce lipid peroxidation and retain the membrane integrity. Additionally, glutathione (GSH) contributing to reactive oxygen species (ROS) scavenging and transgenic plants are more efficient at regenerating GSH from oxidized glutathione (GSSG) under conditions of drought stress. Notably, KcTrxf-transgenic plants had increased glucose and fructose contents under drought stress conditions, presumably resulting from KcTrxf-promoted starch degradation under water stress. We conclude that KcTrxf contributes to drought tolerance by increasing the water status, by enhancing osmotic adjustment, and by maintaining ROS homeostasis in transgene plants.

A multifunctional ratiometric electrochemical sensor for combined determination of indole-3-acetic acid and salicylic acid.

Indole-3-acetic acid (IAA) and salicylic acid (SA) are two important phytohormones. In this work, for the first time, a ratiometric electrochemical sensor was developed for quantifying IAA and SA simultaneously. A composite of multi wall carbon nanotubes (MWNT) and carbon black (CB) was used to enhance the sensitivity of electrochemical detection. Ferrocene (Fc) was used as the reference molecule to offer a built-in correction to improve the accuracy. A good linearity was constructed between the I IAA/I Fc and the concentration of IAA from 25 µM to 1000 µM. The linear equation was y = 0.00159x + 0.124 (R 2 = 0.9887). The LOD for IAA was 1.99 µM. Meanwhile, the I SA/I Fc gradually increased with increasing concentration of SA. The linear regression equation for SA was y = 0.00107x + 0.34465 (R 2 = 0.9488) with the LOD of 3.30 µM. Thus, the as-prepared multifunctional ratiometric electrochemical sensor was successfully applied to detect IAA and SA at the same time. This sensor was also successfully used to detect IAA and SA in the homogenates of soybean seedlings under salt stress, confirming the practical applicability of the sensor. And the obtained results agreed well with those obtained by the ultra performance liquid chromatography-mass spectrometry (UPLC-MS) method.

Illumination/Darkness-Induced Changes in Leaf Surface Potential Linked With Kinetics of Ion Fluxes.

A highly reproducible plant electrical signal-light-induced bioelectrogenesis (LIB) was obtained by means of periodic illumination/darkness stimulation of broad bean (Vicia faba L.) leaves. By stimulating the same position of the same leaf with different concentrations of NaCl, we observed that the amplitude and waveform of the LIB was correlated with the intensity of stimulation. This method allowed us to link dynamic ion fluxes induced by periodic illumination/darkness to salt stress. The self-referencing ion electrode technique was used to explore the ionic mechanisms of the LIB. Fluxes of H+, Ca2+, K+, and Cl- showed periodic changes under periodic illumination/darkness before and after 50 mM NaCl stimulation. Gray relational analysis was used to analyze correlations between each of these ions and LIB. The results showed that different ions are involved in surface potential changes at different stages under periodic illumination/darkness. The gray relational grade reflected the contribution of each ion to the change in surface potential at a certain time period. The ion fluxes data obtained under periodic illumination/darkness stimulation will contribute to the future development of a dynamic model for interpretation of electrophysiological events in plant cells.

Disposable stainless steel-based electrochemical microsensor for in vivo determination of indole-3-acetic acid in soybean seedlings.

In vivo detecting of plants signal molecules is of great importance for the precision farming, crop management and plant phenotyping. In this work, for in vivo detecting indole-3-acetic acid (IAA), one of phytohormones, fine stainless steel (SS) wire was used as electrode material. Highly ordered nanopores, popcorn-like Au nanostructures, Pt nanoparticles and reduced graphene oxide (ERGO) nanocomposite films, and polymerized ST film (PST) were fabricated on the SS microelectrode in turn for improving the detection effect. Using the as-prepared SS microelectrode as working electrode, two untreated SS wires as reference electrode and counter electrode respectively, a disposable electrochemical microsensor for IAA were developed. The microsensor exhibited excellent selectivity and high sensitivity with low detection limit (LOD) of 43 pg mL-1. The limit of quantity (LOQ) is 143 pg mL-1. The RSD was 7% for 12 different PST/Pt-ERGO/Au/a-SS microsensors in presence of 100 µg mL-1 IAA. Using this microsensor, IAA of the stem of soybean seedlings was detected in vivo under salt stress. Our result was also confirmed by ultra-performance liquid chromatography-mass spectrum (UPLC-MS). This is the first report for the in vivo detection of IAA in plants using SS-based electrochemical microsensor. Our sensor provides an excellent sensing platform for detecting IAA in plants in vivo.

In vivo detection of salicylic acid in sunflower seedlings under salt stress.

Salicylic acid (SA) is an important phytohormone. It plays an essential role in regulating many physiological processes of plants. Most of the conventional methods for SA detection are based on in vitro processes. More attention should be paid to develop in vivo methods for SA detection. In this work, Pt nanoflowers and GO were simultaneously electrodeposited and reduced on a Pt wire microelectrode in one step. The Pt nanoflowers/ERGO modified Pt microsensor demonstrated high sensitivity and selectivity for SA. SA could be detected from 100 pM to 1 µM with a detection limit of 48.11 pM. Then this microsensor was used to detect SA in the stem of sunflower seedlings under different salt stresses in vivo. The result showed that with the increasing concentration of salt, SA levels decreased. Our result was also confirmed by UPLC-MS and gene expression analysis. To the best of our knowledge, this is the first report of in vivo detection of SA in plants using the Pt nanoflowers/ERGO modified Pt microelectrode. It is foreseeable that our strategy could pave the way for the in vivo detection of phytohormones in plants.

Populus euphratica APYRASE2 Enhances Cold Tolerance by Modulating Vesicular Trafficking and Extracellular ATP in Arabidopsis Plants.

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 Mg(2+) 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.

Populus euphratica HSF binds the promoter of WRKY1 to enhance salt tolerance.

Poplar species increase expressions of transcription factors to deal with salt environments. We assessed the salt-induced transcriptional responses of heat-shock transcription factor (HSF) and WRKY1 in Populus euphratica, and their roles in salt tolerance. High NaCl (200mM) induced PeHSF and PeWRKY1 expressions in P. euphratica, with a rapid rise in roots than in leaves. Moreover, the salt-elicited PeHSF reached its peak level 6h earlier than PeWRKY1 in leaves. PeWRKY1 was down-regulated in salinized P. euphratica when PeHSF was silenced by tobacco rattle virus-based gene silencing. Subcellular assays in onion epidermal cells and Arabidopsis protoplasts revealed that PeHSF and PeWRKY1 were restricted to the nucleus. Transgenic tobacco plants overexpressing PeWRKY1 showed improved salt tolerance in terms of survival rate, root growth, photosynthesis, and ion fluxes. We further isolated an 1182-bp promoter fragment upstream of the translational start of PeWRKY1 from P. euphratica. Promoter sequence analysis revealed that PeWRKY1 harbours four tandem repeats of heat shock element (HSE) in the upstream regulatory region. Yeast one-hybrid assay showed that PeHSF directly binds the cis-acting HSE. To determine whether the HSE cluster was important for salt-induced PeWRKY1 expression, the promoter-reporter construct PeWRKY1-pro::GUS was transferred to tobacco plants. ß-glucuronidase activities increased in root, leaf, and stem tissues under salt stress. Therefore, we conclude that salinity increased PeHSF transcription in P. euphratica, and that PeHSF binds the cis-acting HSE of the PeWRKY1 promoter, thus activating PeWRKY1 expression.

Overexpression of copper/zinc superoxide dismutase from mangrove Kandelia candel in tobacco enhances salinity tolerance by the reduction of reactive oxygen species in chloroplast.

Na(+) uptake and transport in Kandelia candel and antioxidative defense were investigated under rising NaCl stress from 100 to 300 mM. Salinized K. candel roots had a net Na(+) efflux with a declined flux rate during an extended NaCl exposure. Na(+) buildup in leaves enhanced H2O2 levels, superoxide dismutase (SOD) activity, and increased transcription of CSD gene encoding a Cu/Zn SOD. Sequence and subcellular localization analyses have revealed that KcCSD is a typical Cu/Zn SOD in chloroplast. The transgenic tobacco experimental system was used as a functional genetics model to test the effect of KcCSD on salinity tolerance. KcCSD-transgenic lines were more Na(+) tolerant than wild-type (WT) tobacco in terms of lipid peroxidation, root growth, and survival rate. In the latter, 100 mM NaCl led to a remarkable reduction in chlorophyll content and a/b ratio, decreased maximal chlorophyll a fluorescence, and photochemical efficiency of photosystem II. NaCl stress in WT resulted from H2O2 burst in chloroplast. Na(+) injury to chloroplast was less pronounced in KcCSD-transgenic plants due to upregulated antioxidant defense. KcCSD-transgenic tobacco enhanced SOD activity by an increment in SOD isoenzymes under 100 mM NaCl stress from 24 h to 7 day. Catalase activity rose in KcCSD overexpressing tobacco plants. KcCSD-transgenic plants better scavenged NaCl-elicited reactive oxygen species (ROS) compared to WT ones. In conclusion, K. candel effectively excluded Na(+) in roots during a short exposure; and increased CSD expression to reduce ROS in chloroplast in a long-term and high saline environment.

Overexpression of PeHA1 enhances hydrogen peroxide signaling in salt-stressed Arabidopsis.

The plant plasma membrane (PM) H(+)-ATPase plays a crucial role in controlling K(+)/Na(+) homeostasis under salt stress. Our previous microarray analysis indicated that Populus euphratica retained a higher abundance of PM H(+)-ATPase transcript versus a salt-sensitive poplar. To clarify the roles of the PM H(+)-ATPase in salt sensing and adaptation, we isolated the PM H(+)-ATPase gene PeHA1 from P. euphratica and introduced it into Arabidopsis thaliana. Compared to wild-type, PeHA1-transgenic Arabidopsis had a greater germination rate, root length, and biomass under NaCl stress (50-150 mM). Ectopic expression of PeHA1 remarkably enhanced the capacity to control the homeostasis of ions and reactive oxygen species in salinized Arabidopsis. Flux data from salinized roots showed that transgenic plants exhibited a more pronounced Na(+)/H(+) antiport and less reduction of K(+) influx versus wild-type. Enhanced PM ATP hydrolytic activity, proton pumping, and Na(+)/H(+) antiport in PeHA1-transgenic plants, were consistent to those observed in vivo, i.e., H(+) extrusion, external acidification, and Na(+) efflux. Activities of the antioxidant enzymes ascorbate peroxidase and catalase were typically higher in transgenic seedlings irrespective of salt concentration. In transgenic Arabidopsis roots, H2O2 production was higher under control conditions and increased more rapidly than wild-type when plants were subjected to NaCl treatment. Interestingly, transgenic plants were unable to control K(+)/Na(+) homeostasis when salt-induced H2O2 production was inhibited by diphenylene iodonium, an inhibitor of NADPH oxidase. These observations suggest that PeHA1 accelerates salt tolerance partially through rapid H2O2 production upon salt treatment, which triggers adjustments in K(+)/Na(+) homeostasis and antioxidant defense in Arabidopsis.