Endogenous ureides are employed as a carbon source in Arabidopsis plants exposed to carbon starvation conditions.

Ureides, the degraded products of purine catabolism in Arabidopsis, have been shown to act as antioxidant and nitrogen sources. Herein we elucidate purine degraded metabolites as a carbon source using the Arabidopsis Atxdh1, Ataln, and Ataah knockout (KO) mutants vis-à-vis wild-type (WT) plants. Plants were grown under short-day conditions on agar plates containing half-strength MS medium with or without 1% sucrose. Notably, the absence of sucrose led to diminished biomass accumulation in both shoot and root tissues of the Atxdh1, Ataln, and Ataah mutants, while no such effect was observed in WT plants. Moreover, the application of sucrose resulted in a reduction of purine degradation metabolite levels, specifically xanthine and allantoin, predominantly within the roots of WT plants. Remarkably, an increase in proteins associated with the purine degradation pathway was observed in WT plants in the presence of sucrose. Lower glyoxylate levels in the roots but not in the shoot of the Atxdh1 mutant in comparison to WT, were observed under sucrose limitation, and improved by sucrose application in root, indicating that purine degradation provided glyoxylate in the root. Furthermore, the deficit of purine-degraded metabolites in the roots of mutants subjected to carbon starvation was partially mitigated through allantoin application. Collectively, these findings signify that under conditions of sucrose limitation and short-day growth, purines are primarily remobilized within the root system to augment the availability of ureides, serving as an additional carbon (as well as nitrogen) source to support plant growth.

Determination of ROS Generated by Arabidopsis Xanthine Dehydrogenase1 (AtXDH1) Using Nitroblue Tetrazolium (NBT) and 3,3'-Diaminobenzidine (DAP).

Plants generate reactive oxygen species (ROS) during different metabolic processes, which play an essential role in coordinating growth and response. ROS levels are sensitive to environmental stresses and are often used as a marker for stress in plants. While various methods can detect ROS changes, histochemical staining with nitroblue tetrazolium (NBT) and 3,3'-diaminobenzidine (DAB) is a popular method, though it has faced criticism. This staining method is advantageous as it enables both the quantification and localization of ROS and the identification of the enzymatic origin of ROS in plants, cellular compartments, or gels. In this protocol, we describe the use of NBT and DAP staining to detect ROS generation under different stresses such as nitrogen starvation, wounding, or UV-C. Additionally, we describe the use of NBT staining for detecting enzymatic generation of ROS in native and native SDS PAGE gels. Our protocol also outlines the separation and comparison of the origin of ROS generated by xanthine dehydrogenase1 (XDH1) using different substrates.

Desalination, Water Re-use, and Halophyte Cultivation in Salinized Regions: A Highly Productive Groundwater Treatment System.

Groundwater salinization is a problem affecting access to water in many world regions. Though desalination by conventional reverse osmosis (RO) can upgrade groundwater quality for drinking, its disadvantages include unmanaged brine discharge and accelerated groundwater depletion. Here, we propose a new approach combining RO, forward osmosis (FO), and halophyte cultivation, in which FO optimally adjusts the concentration of the RO reject brine for irrigation of Salicornia or Sarcocornia. The FO also re-uses wastewater, thus, reducing groundwater extraction and the wastewater effluent volume. To suit different groundwater salinities in the range 1-8 g/L, three practical designs are proposed and analyzed. Results include specific groundwater consumption (SGC), specific energy consumption (SEC), wastewater volume reduction, peak RO pressure, permeate water quality, efficiency of water resource utilization, and halophyte yield. Compared to conventional brackish water RO, the results show superior performance in almost all aspects. For example, SGC is reduced from 1.25 to 0.9 m3 per m3 of drinking water output and SEC is reduced from 0.79 to 0.70 kW h/m3 by a FO-RO-FO system treating groundwater of salinity 8 g/L. This system can produce 1.1 m3 of high-quality drinking water and up to 4.9 kg of edible halophyte per m3 of groundwater withdrawn.

Risk Factors and Multidimensional Assessment of Long Coronavirus Disease Fatigue: A Nested Case-Control Study.

BACKGROUND: Fatigue is the most prevalent and debilitating long-COVID (coronavirus disease) symptom; however, risk factors and pathophysiology of this condition remain unknown. We assessed risk factors for long-COVID fatigue and explored its possible pathophysiology. METHODS: This was a nested case-control study in a COVID recovery clinic. Individuals with (cases) and without (controls) significant fatigue were included. We performed a multidimensional assessment evaluating various parameters, including pulmonary function tests and cardiopulmonary exercise testing, and implemented multivariable logistic regression to assess risk factors for significant long-COVID fatigue. RESULTS: A total of 141 individuals were included. The mean age was 47 (SD: 13) years; 115 (82%) were recovering from mild coronavirus disease 2019 (COVID-19). Mean time for evaluation was 8 months following COVID-19. Sixty-six (47%) individuals were classified with significant long-COVID fatigue. They had a significantly higher number of children, lower proportion of hypothyroidism, higher proportion of sore throat during acute illness, higher proportions of long-COVID symptoms, and of physical limitation in daily activities. Individuals with long-COVID fatigue also had poorer sleep quality and higher degree of depression. They had significantly lower heart rate [153.52 (22.64) vs 163.52 (18.53); P = .038] and oxygen consumption per kilogram [27.69 (7.52) vs 30.71 (7.52); P = .036] at peak exercise. The 2 independent risk factors for fatigue identified in multivariable analysis were peak exercise heart rate (OR: .79 per 10 beats/minute; 95% CI: .65-.96; P = .019) and long-COVID memory impairment (OR: 3.76; 95% CI: 1.57-9.01; P = .003). CONCLUSIONS: Long-COVID fatigue may be related to autonomic dysfunction, impaired cognition, and decreased mood. This may suggest a limbic-vagal pathophysiology. CLINICAL TRIALS REGISTRATION: NCT04851561.

The Effect of Topo-Climate Variation on the Secondary Metabolism of Berries in White Grapevine Varieties (Vitis vinifera).

Exploiting consistent differences in radiation and average air temperature between two experimental vineyards (Ramat Negev, RN and Mitzpe Ramon, MR), we examined the impact of climate variations on total carotenoids, redox status, and phenylpropanoid metabolism in the berries of 10 white wine grapevine (Vitis vinifera) cultivars across three consecutive seasons (2017-2019). The differences in carotenoid and phenylpropanoid contents between sites were seasonal and varietal dependent. However, the warmer RN site was generally associated with higher H2O2 levels and carotenoid degradation, and lower flavonol contents than the cooler MR site. Enhanced carotenoid degradation was positively correlated with radiation and daily degree days, leading to a greater drop in content from véraison to harvest in Colombard, Sauvignon Blanc, and Semillon berries. Analyses of berry H2O2 and phenylpropanoids suggested differences between cultivars in the links between H2O2 and flavonol contents. Generally, however, grapes with higher H2O2 content seem to have lower flavonol contents. Correlative network analyses revealed that phenylpropanoids at the warmer RN site are tightly linked to the radiation and temperature regimes during fruit ripening, indicating potentially harmful effect of warmer climates on berry quality. Specifically, flavan-3-ols were negatively correlated with radiation at RN. Principal component analysis showed that Muscat Blanc, Riesling, Semillon, and Sauvignon Blanc were the most site sensitive cultivars. Our results suggest that grapevine biodiversity is likely the key to withstand global warming hazards.

Active O-acetylserine-(thiol) lyase A and B confer improved selenium resistance and degrade l-Cys and l-SeCys in Arabidopsis.

The roles of cytosolic O-acetylserine-(thiol)-lyase A (OASTLA), chloroplastic OASTLB, and mitochondrial OASTLC in plant selenate resistance were studied in Arabidopsis. Impairment in OASTLA and OASTLB resulted in reduced biomass, chlorophyll and soluble protein content compared with selenate-treated OASTLC-impaired and wild-type plants. The generally lower total selenium (Se), protein-Se, organic-sulfur and protein-sulfur (S) content in oastlA and oastlB compared with wild-type and oastlC leaves indicated that Se accumulation was not the main cause for the stress symptoms in these mutants. Notably, the application of selenate positively induced S-starvation markers and the OASTLs, followed by increased sulfite reductase, sulfite oxidase activities, and increased sulfite and sulfide concentrations. Taken together, our results indicate a futile anabolic S-starvation response that resulted in lower glutathione and increased oxidative stress symptoms in oastlA and oastlB mutants. In-gel assays of l-cysteine and l-seleno-cysteine, desulfhydrase activities revealed that two of the three OASTL activity bands in each of the oastl single mutants were enhanced in response to selenate, whereas the impaired proteins exhibited a missing activity band. The absence of differently migrated activity bands in each of the three oastl mutants indicates that these OASTLs are major components of desulfhydrase activity, degrading l-cysteine and l-seleno-cysteine in Arabidopsis.

Double-stranded RNA targeting fungal ergosterol biosynthesis pathway controls Botrytis cinerea and postharvest grey mould.

Pathogenic fungi cause major postharvest losses. During storage and ripening, fruit becomes highly susceptible to fungi that cause postharvest disease. Fungicides are effective treatments to limit disease. However, due to increased public concern for their possible side effects, there is a need to develop new strategies to control postharvest fungal pathogens. Botrytis cinerea, a common postharvest pathogen, was shown to uptake small double-stranded RNA (dsRNA) molecules from the host plant. Such dsRNA can regulate gene expression through the RNA interference system. This work aimed to develop a synthetic dsRNA simultaneously targeting three essential transcripts active in the fungal ergosterol biosynthesis pathway (dsRNA-ERG). Our results show initial uptake of dsRNA in the emergence zone of the germination tube that spreads throughout the fungus and results in down-regulation of all three targeted transcripts. Application of dsRNA-ERG decreased B. cinerea germination and growth in in vitro conditions and various fruits, leading to reduce grey-mould decay. The inhibition of growth or decay was reversed by the addition of ergosterol. While dual treatment with dsRNA-ERG and ergosterol-inhibitor fungicide reduced by 100-fold the required amount of fungicide to achieve the same protection rate. The application of dsRNA-ERG induced systemic protection as shown by decreased decay development at inoculation points distant from the treatment point in tomato and pepper fruits. Overall, this study suggests that dsRNA-ERG can effectively control B. cinerea growth and grey-mould development suggesting its efficacy as a future method for postharvest control of fungal pathogens.

Ureides are accumulated similarly in response to UV-C irradiation and wounding in Arabidopsis leaves but are remobilized differently during recovery.

Purine degradation products have been shown to play roles in plant response to stresses such as drought, salinity, extended dark, nitrogen deficiency, and pathogen infection. In this study, we used Arabidopsis wild-type (WT) and an Atxdh1-knockout mutant defective in xanthine dehydrogenase1 (XDH1) to examine the role of degraded purine metabolites in the responses to wounding or UV-C stress applied to the middle leaves of the plant. Wounding or UV-C stress in the mutant resulted in lower fresh-weight, increased senescence symptoms, and increased cell death compared to WT plants. In addition, WT plants exhibited lower levels of oxidative stress indicators, reactive oxygen species, and malondialdehyde in their leaves than the mutant. Notably, transcripts and proteins functioning in the purine degradation pathway were regulated in such a way that it led to enhanced ureide levels in WT leaves 24h after applying the UV-C or wound stress. However, different remobilization of the accumulated ureides was observed after 72h of stress. In plants treated with UV-C, the concentration of allantoin was highest in young leaves, whereas in wounded plants it was lowest in these leaves and instead accumulated mainly in the middle leaves that had been wounded. These results indicated that in WT plants treated with UV-C, ureides were remobilized from the lower older and damaged leaves to support young leaf growth during the recovery period from stress. After wounding, however, whilst some ureides were remobilized to the young leaves, more remained in the wounded middle leaves to function as antioxidants and/or healing agents.

A Review on Sarcocornia Species: Ethnopharmacology, Nutritional Properties, Phytochemistry, Biological Activities and Propagation.

Sarcocornia A. J. Scott is a halophytic edible succulent plant belonging to the Amaranthaceae family. To date, the genus includes 28 species distributed worldwide in saline environments, usually salt marshes. Sarcocornia (Scott) is similar to Salicornia (L.), which has a recognized commercial value in morphological and taxonomical traits. Species of both genera are commonly named samphire or glassworts in Europe, and their fleshy shoots are commercialized under their traditional names. Due to their nutritional, organoleptic and medicinal properties, Sarcocornia species have a high economic potential in various biotechnology sectors. Being highly tolerant to salt, they can be cultivated in saline conditions, and dissimilar to Salicornia, they are perennial, i.e., they can be harvested year-round. Therefore, Sarcocornia species are considered promising gourmet vegetables to be explored in the context of climate change, soil and water salinization and eco-sustainability. We hereby put together and reviewed the most relevant information on Sarcocornia taxonomy, morphology, nutritional and pharmacological properties, uses in ethnomedicine, potential applications in biotechnology, and propagation strategies.

Arabidopsis aldehyde oxidase 3, known to oxidize abscisic aldehyde to abscisic acid, protects leaves from aldehyde toxicity.

The Arabidopsis thaliana aldehyde oxidase 3 (AAO3) catalyzes the oxidation of abscisic aldehyde (ABal) to abscisic acid (ABA). Besides ABal, plants generate other aldehydes that can be toxic above a certain threshold. AAO3 knockout mutants (aao3) exhibited earlier senescence but equivalent relative water content compared with wild-type (WT) during normal growth or upon application of UV-C irradiation. Aldehyde profiling in leaves of 24-day-old plants revealed higher accumulation of acrolein, crotonaldehyde, 3Z-hexenal, hexanal and acetaldehyde in aao3 mutants compared with WT leaves. Similarly, higher levels of acrolein, benzaldehyde, crotonaldehyde, propionaldehyde, trans-2-hexenal and acetaldehyde were accumulated in aao3 mutants upon UV-C irradiation. Aldehydes application to plants hastened profuse senescence symptoms and higher accumulation of aldehydes, such as acrolein, benzaldehyde and 4-hydroxy-2-nonenal, in aao3 mutant leaves as compared with WT. The senescence symptoms included greater decrease in chlorophyll content and increase in transcript expression of the early senescence marker genes, Senescence-Related-Gene1, Stay-Green-Protein2 as well as NAC-LIKE, ACTIVATED-BY AP3/P1. Notably, although aao3 had lower ABA content than WT, members of the ABA-responding genes SnRKs were expressed at similar levels in aao3 and WT. Moreover, the other ABA-deficient mutants [aba2 and 9-cis-poxycarotenoid dioxygenase3-2 (nced3-2), that has functional AAO3] exhibited similar aldehydes accumulation and chlorophyll content like WT under normal growth conditions or UV-C irradiation. These results indicate that the absence of AAO3 oxidation activity and not the lower ABA and its associated function is responsible for the earlier senescence symptoms in aao3 mutant.

Level of Sulfite Oxidase Activity Affects Sulfur and Carbon Metabolism in Arabidopsis.

Molybdenum cofactor containing sulfite oxidase (SO) enzyme is an important player in protecting plants against exogenous toxic sulfite. It was also demonstrated that SO activity is essential to cope with rising dark-induced endogenous sulfite levels and maintain optimal carbon and sulfur metabolism in tomato plants exposed to extended dark stress. The response of SO and sulfite reductase to direct exposure of low and high levels of sulfate and carbon was rarely shown. By employing Arabidopsis wild-type, sulfite reductase, and SO-modulated plants supplied with excess or limited carbon or sulfur supply, the current study demonstrates the important role of SO in carbon and sulfur metabolism. Application of low and excess sucrose, or sulfate levels, led to lower biomass accumulation rates, followed by enhanced sulfite accumulation in SO impaired mutant compared with wild-type. SO-impairment resulted in the channeling of sulfite to the sulfate reduction pathway, resulting in an overflow of organic S accumulation. In addition, sulfite enhancement was followed by oxidative stress contributing as well to the lower biomass accumulation in SO-modulated plants. These results indicate that the role of SO is not limited to protection against elevated sulfite toxicity but to maintaining optimal carbon and sulfur metabolism in Arabidopsis plants.

Adenosine 5' phosphosulfate reductase and sulfite oxidase regulate sulfite-induced water loss in Arabidopsis.

Chloroplast-localized adenosine-5'-phosphosulphate reductase (APR) generates sulfite and plays a pivotal role in reduction of sulfate to cysteine. The peroxisome-localized sulfite oxidase (SO) oxidizes excess sulfite to sulfate. Arabidopsis wild type, SO RNA-interference (SO Ri) and SO overexpression (SO OE) transgenic lines infiltrated with sulfite showed increased water loss in SO Ri plants, and smaller stomatal apertures in SO OE plants compared with wild-type plants. Sulfite application also limited sulfate and abscisic acid-induced stomatal closure in wild type and SO Ri. The increases in APR activity in response to sulfite infiltration into wild type and SO Ri leaves resulted in an increase in endogenous sulfite, indicating that APR has an important role in sulfite-induced increases in stomatal aperture. Sulfite-induced H2O2 generation by NADPH oxidase led to enhanced APR expression and sulfite production. Suppression of APR by inhibiting NADPH oxidase and glutathione reductase2 (GR2), or mutation in APR2 or GR2, resulted in a decrease in sulfite production and stomatal apertures. The importance of APR and SO and the significance of sulfite concentrations in water loss were further demonstrated during rapid, harsh drought stress in root-detached wild-type, gr2 and SO transgenic plants. Our results demonstrate the role of SO in sulfite homeostasis in relation to water consumption in well-watered plants.

COVID-19 in a patient receiving adjuvant breast cancer chemotherapy with granulocyte olony-stimulating factor (G-CSF) support: A case report.

Patients receiving chemotherapy are at high risk for severe infections and complications such as acute respiratory syndrome. The most commonly used adjuvant chemotherapy protocols (docetaxel-cyclophosphamide every 3 weeks or the dose-dense regimen, doxorubicin-cyclophosphamide every 2 weeks followed by paclitaxel) incorporate granulocyte-colony stimulating factor (G-CSF). G-CSF is routinely administered to prevent chemotherapy-associated neutropenia but often results in significant neutrophilia. The present case describes a patient with breast cancer who was successfully treated for severe COVID-19 respiratory syndrome while under adjuvant chemotherapy (docetaxel-cyclophosphamide) treatment and long-term G-CSF support. In addition, the potential effect of G-CSF on the respiratory deterioration of the patient given its cardinal role in innate inflammation and, accordingly, the cytokine storm associated with COVID-19 was described. The case described in the present study indicated how solutions to the immunity challenges faced when treating a patient with chemotherapy may be the source of a larger problem within the coronavirus COVID-19 pandemic.

A possible increased risk of metamizole-associated neutropenia among COVID-19 patients.

Metamizole is commonly used as analgesic and antipyretic drug. The use of metamizole is prohibited in several countries due to its rare side effect of neutropenia and even agranulocytosis. Among the many symptoms of COVID-19, fever and diffuse pain predominant and therefore it can be assumed that metamizole may be widely used in the current epidemic period. So far, there have been no reports on the safety of metamizole in COVID-19 patients. We describe a series of 3 patients who developed severe neutropenia under metamizole treatment, raising a concern of a possible increased risk of this side effect among COVID-19 patients.

Combined network analysis and machine learning allows the prediction of metabolic pathways from tomato metabolomics data.

The identification and understanding of metabolic pathways is a key aspect in crop improvement and drug design. The common approach for their detection is based on gene annotation and ontology. Correlation-based network analysis, where metabolites are arranged into network formation, is used as a complentary tool. Here, we demonstrate the detection of metabolic pathways based on correlation-based network analysis combined with machine-learning techniques. Metabolites of known tomato pathways, non-tomato pathways, and random sets of metabolites were mapped as subgraphs onto metabolite correlation networks of the tomato pericarp. Network features were computed for each subgraph, generating a machine-learning model. The model predicted the presence of the ß-alanine-degradation-I, tryptophan-degradation-VII-via-indole-3-pyruvate (yet unknown to plants), the ß-alanine-biosynthesis-III, and the melibiose-degradation pathway, although melibiose was not part of the networks. In vivo assays validated the presence of the melibiose-degradation pathway. For the remaining pathways only some of the genes encoding regulatory enzymes were detected.

Early Senescence in Older Leaves of Low Nitrate-Grown Atxdh1 Uncovers a Role for Purine Catabolism in N Supply.

The nitrogen (N)-rich ureides allantoin and allantoate, which are products of purine catabolism, play a role in N delivery in Leguminosae. Here, we examined their role as an N source in nonlegume plants using Arabidopsis (Arabidopsis thaliana) plants mutated in XANTHINE DEHYDROGENASE1 (AtXDH1), a catalytic bottleneck in purine catabolism. Older leaves of the Atxdh1 mutant exhibited early senescence, lower soluble protein, and lower organic N levels as compared with wild-type older leaves when grown with 1 mm nitrate but were comparable to the wild type under 5 mm nitrate. Similar nitrate-dependent senescence phenotypes were evident in the older leaves of allantoinase (Ataln) and allantoate amidohydrolase (Ataah) mutants, which also are impaired in purine catabolism. Under low-nitrate conditions, xanthine accumulated in older leaves of Atxdh1, whereas allantoin accumulated in both older and younger leaves of Ataln but not in wild-type leaves, indicating the remobilization of xanthine-degraded products from older to younger leaves. Supporting this notion, ureide transporter expression was enhanced in older leaves of the wild type in low-nitrate as compared with high-nitrate conditions. Elevated transcripts and proteins of AtXDH and AtAAH were detected in low-nitrate-grown wild-type plants, indicating regulation at protein and transcript levels. The higher nitrate reductase activity in Atxdh1 leaves compared with wild-type leaves indicated a need for nitrate assimilation products. Together, these results indicate that the absence of remobilized purine-degraded N from older leaves of Atxdh1 caused senescence symptoms, a result of higher chloroplastic protein degradation in older leaves of low-nitrate-grown plants.

Differential influence of molybdenum and tungsten on the growth of barley seedlings and the activity of aldehyde oxidase under salinity.

The influence of molybdenum, tungsten on germination and growth of barley Hordeum vulgare L. was studied. Results of this study revealed the differential effect of heavy metals on seedlings growth. Exogenous molybdenum treatment stimulated the growth of seedlings. The addition of the metal significantly stimulated root elongation. Contrastingly, the addition of tungsten resulted in increased seed germination and inhibits the growth of seedlings. The negative effect of tungsten on the growth of barley was more profound for roots of plants. In addition, the influence of metals on the growth of plants was also tested in saline conditions. It is shown that under salinity stress plant growth drastically decreased in presence of tungsten. Results of this study showed that activity of molybdenum-containing aldehyde oxidase (AO; EC 1.2.3.1) was also significantly affected by metals. The activity of AO in leaves and roots enhanced with increasing concentrations of molybdate, while tungstate treatment inhibited the enzyme activity. Perhaps, the differential influence of molybdenum and tungsten on the growth of barley is a direct effect of metals on aldehyde oxidase activity in plants. Moreover, the intense negative effect of tungsten treatment on barley growth under salinity conditions emphasizes an important role of aldehyde oxidase in plant resistance to stress factors.

Zinc oxide nanoparticles phytotoxicity on halophyte from genus Salicornia.

This study deals with the effect of zinc oxide nanoparticles (ZnO NPs) on halophyte from the genus Salicornia. The presence of ZnO nanoparticles (100 and 1000 mg/L) in the solid culture medium resulted in the negative effects on plant growth in the concentration-dependent manner. The shoot length of plant cultivated with 1000 mg/L ZnO NPs decreased by more than 50% compared to non-treated plants. The phytotoxicity was associated with the release of free zinc(II) ions, which was determined by atomic absorption spectroscopy and fluorescence microscopy. Another mechanism involved in ZnO NPs phytotoxicity was closely connected with generation of reactive oxygen species (ROS), which was accompanied by changes in activities and amounts of antioxidant enzymes. Histochemical evaluation showed that ROS were present also in the shoot of plant, which was not in direct contact with NPs. The reduction of activity and amount of antioxidant enzymes such as gamma-ESC, GR, SOD, PER, APX and higher concentration of ROS lead to lipid peroxidation, the latter being almost 3 times higher for the plant treated with 1000 mg/L NPs compared to control. The misbalance in zinc homeostasis and creation of ROS with subsequent oxidative stress led to the initiation of processes of programmed cell death, which was demonstrated by the loss of mitochondrial potential and increase of intracellular calcium (II) ions. Despite halophytes exhibit higher stress resistance than glycophytes, they are prone to negative changes if incubated in the environment containing ZnO nanoparticles.

Higher Novel L-Cys Degradation Activity Results in Lower Organic-S and Biomass in Sarcocornia than the Related Saltwort, Salicornia.

Salicornia and Sarcocornia are almost identical halophytes whose edible succulent shoots hold promise for commercial production in saline water. Enhanced sulfur nutrition may be beneficial to crops naturally grown on high sulfate. However, little is known about sulfate nutrition in halophytes. Here we show that Salicornia europaea (ecotype RN) exhibits a significant increase in biomass and organic-S accumulation in response to supplemental sulfate, whereas Sarcocornia fruticosa (ecotype VM) does not, instead exhibiting increased sulfate accumulation. We investigated the role of two pathways on organic-S and biomass accumulation in Salicornia and Sarcoconia: the sulfate reductive pathway that generates Cys and l-Cys desulfhydrase that degrades Cys to H2S, NH3, and pyruvate. The major function of O-acetyl-Ser-(thiol) lyase (OAS-TL; EC 2.5.1.47) is the formation of l-Cys, but our study shows that the OAS-TL A and OAS-TL B of both halophytes are enzymes that also degrade l-Cys to H2S. This activity was significantly higher in Sarcocornia than in Salicornia, especially upon sulfate supplementation. The activity of the sulfate reductive pathway key enzyme, adenosine 5'-phosphosulfate reductase (APR, EC 1.8.99.2), was significantly higher in Salicornia than in Sarcocornia These results suggest that the low organic-S level in Sarcocornia is the result of high l-Cys degradation rate by OAS-TLs, whereas the greater organic-S and biomass accumulation in Salicornia is the result of higher APR activity and low l-Cys degradation rate, resulting in higher net Cys biosynthesis. These results present an initial road map for halophyte growers to attain better growth rates and nutritional value of Salicornia and Sarcocornia.

Determination of Enzymes Associated with Sulfite Toxicity in Plants: Kinetic Assays for SO, APR, SiR, and In-Gel SiR Activity.

The amino acid cysteine plays a major role in plant response to abiotic stress by being the donor of elemental sulfur for the sulfuration of the molybdenum cofactor, otherwise the last step of ABA biosynthesis, the oxidation of abscisic aldehyde, is inactivated. Additionally, cysteine serves as a precursor for the biosynthesis of glutathione, the reactive oxygen species scavenger essential for redox status homeostasis during stress. Cysteine is generated by the sulfate reductive pathway where sulfite oxidase (SO; EC 1.8.3.1) is an important enzyme in the homeostasis of sulfite levels (present either as a toxic intermediate in the pathway or as a toxic air pollutant that has penetrated the plant tissue via the stomata). SO is localized to the peroxisomes and detoxifies excess sulfite by catalyzing its oxidation to sulfate. Here we show a kinetic assay that relies on fuchsin colorimetric detection of sulfite, a substrate of SO activity. This SO assay is highly specific, technically simple, and readily performed in any laboratory.5'-adenylylsulfate (APS) reductase (APR, E.C. 1.8.4.9) enzyme regulates a crucial step of sulfate assimilation in plants, algae and some human pathogens. The enzyme is upregulated in response to oxidative stress induced by abiotic stresses, such as salinity and hydrogen peroxide, to generate sulfite an intermediate for cysteine generation essential for the biosynthesis of glutathione, the hydrogen peroxide scavenger. Here we present two robust, sensitive, and simple colorimetric methods of APR activity based on sulfite determination by fuchsin.Sulfite reductase (SiR) is one of the key enzymes in the primary sulfur assimilation pathway. It has been shown that SiR is an important plant enzyme for protection plant against sulfite toxicity and premature senescence. Here we describe two methods for SiR activity determination: a kinetic assay using desalted extract and an in-gel assay using crude extract.Due to the energetically favorable equilibrium, sulfurtransferase (ST) activity measured as sulfite generation or consumption. Sulfite-generating ST activity is determined by colorimetric detection of SCN- formation at 460 nm as the red Fe(SCN)3 complex from cyanide and thiosulfate using acidic iron reagent. Sulfite-consuming (MST) activity is detected as sulfite disappearance in the presence of thiocyanate (SCN-) or as SCN- disappearance. To abrogate interfering SO activity, total ST activities is detected by inhibiting SO activity with tungstate.