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.

Receptor-like Kinases (LRR-RLKs) in Response of Plants to Biotic and Abiotic Stresses.

Plants live under different biotic and abiotic stress conditions, and, to cope with the adversity and severity, plants have well-developed resistance mechanisms. The mechanism starts with perception of the stimuli followed by molecular, biochemical, and physiological adaptive measures. The family of LRR-RLKs (leucine-rich repeat receptor-like kinases) is one such group that perceives biotic and abiotic stimuli and also plays important roles in different biological processes of development. This has been mostly studied in the model plant, Arabidopsis thaliana, and to some extent in other plants, such as Solanum lycopersicum, Nicotiana benthamiana, Brassica napus, Oryza sativa, Triticum aestivum, Hordeum vulgare, Brachypodium distachyon, Medicago truncatula, Gossypium barbadense, Phaseolus vulgaris, Solanum tuberosum, and Malus robusta. Most LRR-RLKs tend to form different combinations of LRR-RLKs-complexes (dimer, trimer, and tetramers), and some of them were observed as important receptors in immune responses, cell death, and plant development processes. However, less is known about the function(s) of LRR-RLKs in response to abiotic and biotic stresses. Here, we give recent updates about LRR-RLK receptors, specifically focusing on their involvement in biotic and abiotic stresses in the model plant, A. thaliana. Furthermore, the recent studies on LRR-RLKs that are homologous in other plants is also reviewed in relation to their role in triggering stress response processes against biotic and abiotic stimuli and/or in exploring their additional function(s). Furthermore, we present the interactions and combinations among LRR-RLK receptors that have been confirmed through experiments. Moreover, based on GENEINVESTIGATOR microarray database analysis, we predict some potential LRR-RLK genes involved in certain biotic and abiotic stresses whose function and mechanism may be explored.

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.

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.

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.

Morphological, Physiological and Molecular Markers for Salt-Stressed Plants.

Plant growth and development is adversely affected by different kind of stresses. One of the major abiotic stresses, salinity, causes complex changes in plants by influencing the interactions of genes. The modulated genetic regulation perturbs metabolic balance, which may alter plant's physiology and eventually causing yield losses. To improve agricultural output, researchers have concentrated on identification, characterization and selection of salt tolerant varieties and genotypes, although, most of these varieties are less adopted for commercial production. Nowadays, phenotyping plants through Machine learning (deep learning) approaches that analyze the images of plant leaves to predict biotic and abiotic damage on plant leaves have increased. Here, we review salinity stress related markers on molecular, physiological and morphological levels for crops such as maize, rice, ryegrass, tomato, salicornia, wheat and model plant, Arabidopsis. The combined analysis of data from stress markers on different levels together with image data are important for understanding the impact of salt stress on plants.

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.

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.

Aldehyde Oxidase 4 Plays a Critical Role in Delaying Silique Senescence by Catalyzing Aldehyde Detoxification.

The Arabidopsis (Arabidopsis thaliana) aldehyde oxidases are a multigene family of four oxidases (AAO1-AAO4) that oxidize a variety of aldehydes, among them abscisic aldehyde, which is oxidized to the phytohormone abscisic acid. Toxic aldehydes are generated in plants both under normal conditions and in response to stress. The detoxification of such aldehydes by oxidation is attributed to aldehyde dehydrogenases but never to aldehyde oxidases. The feasibility of the detoxification of aldehydes in siliques via oxidation by AAO4 was demonstrated, first, by its ability to efficiently oxidize an array of aromatic and aliphatic aldehydes, including the reactive carbonyl species (RCS) acrolein, hydroxyl-2-nonenal, and malondialdehyde. Next, exogenous application of several aldehydes to siliques in AAO4 knockout (KO) Arabidopsis plants induced severe tissue damage and enhanced malondialdehyde levels and senescence symptoms, but not in wild-type siliques. Furthermore, abiotic stresses such as dark and ultraviolet C irradiation caused an increase in endogenous RCS and higher expression levels of senescence marker genes, leading to premature senescence of KO siliques, whereas RCS and senescence marker levels in wild-type siliques were hardly affected. Finally, in naturally senesced KO siliques, higher endogenous RCS levels were associated with enhanced senescence molecular markers, chlorophyll degradation, and earlier seed shattering compared with the wild type. The aldehyde-dependent differential generation of superoxide and hydrogen peroxide by AAO4 and the induction of AAO4 expression by hydrogen peroxide shown here suggest a self-amplification mechanism for detoxifying additional reactive aldehydes produced during stress. Taken together, our results indicate that AAO4 plays a critical role in delaying senescence in siliques by catalyzing aldehyde detoxification.