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.

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Salinity negatively influences crop growth, but several salt-tolerant plant species (halophytes) are viable crops. Sarcocornia fruticosa (ecotypes EL and VM) is currently cultivated, but there is demand for new crop candidates and higher biomass production. Salicornia brachiata Roxb. and Arthrocneum macrostachyum L. are considered novel crops, and to realize their potential, their response to salinity and nitrogen nutrition was compared to S. fruticosa ecotypes. Experiments revealed that higher N supplemented with lower NaCl significantly increased fresh and dry shoot biomass. Lower biomass was obtained at lower nitrogen supplemented with elevated NaCl, whereas total soluble solids content positively correlated with NaCl fertigation in both Sarcocornia ecotypes. Protein content increased with a lower nitrogen supply. Anthocyanins and oxygen radical absorbance capacity were highest in S. fruticosa EL and A. macrostachyum at higher NaCl supply. The results show that halophytes have a variety of strategies to cope with high NaCl, even between ecotypes of the same species. Notably, repetitive harvesting of S. brachiata delayed flowering enabling year-round biomass production. Additionally, S. brachiata accumulated higher biomass than Sarcocornia VM when grown in a greenhouse at higher radiation than in a growth room and strongly supports its inclusion as a cash-crop halophyte.

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.

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.

The roles of cytosolic O-acetylserine-(thiol)-lyase A (OASTLA), chloroplast-localized OASTLB and mitochondrion OASTLC in plant selenate resistance were studied in Arabidopsis. Impairment in OASTLA and OASTLB resulted in reduced biomass, chlorophyll and soluble protein levels compared with selenate treated impaired OASTLC and Wild-Type plants. The general lower total selenium (Se), protein-Se, organic-sulfur and protein-sulfur (S) in oastlA and oastlB levels compared to Wild-Type and oastlC leaves indicate that Se accumulation levels are not the main cause for the stress symptoms in these mutants. Notably, the application of selenate positively induced the S-starvation transcript markers: low-sulfur-induced1 (LSU1), LSU2 and ChaC like protein. Additionally, the activity of S-starvation enzymes markers, the adenosine-5'-phosphosulfate reductase and the OASTLs was increased followed by increased sulfite reductase and sulfite oxidase activities, as well as increased sulfite and sulfide levels. Taken together, the results indicate a futile anabolic S-starvation response that resulted in lower glutathione and increased oxidative stress symptoms in leaves of oastlA and oastlB mutants. Employing in-gel assays of L-cysteine and L-seleno-cysteine desulfhydrase activities revealed that two of the three OASTLs activity bands in each of the oastls single mutants were enhanced in response to selenate, whereas the impaired proteins exhibited a missing activity band in stressed and unstressed plants. The absence of differently migrated activity band in each of the three oastls indicates that these OASTLs are major components in Arabidopsis desulfhydrase activity, degrading not only L-cysteine but also L-Seleno-cysteine.

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.

Chloroplast-localized adenosine-5'-phosphosulphate reductase (APR) generates sulfite and plays a pivotal role in sulfate reduction to cysteine. The peroxisome-localized sulfite oxidase (SO), oxidizes excess sulfite to sulfate. Wild-type (WT), SO RNA-interference (SO Ri) and SO overexpression (SO OE) Arabidopsis mutants were infiltrated with sulfite. In SO Ri plants, water loss was increased due to enhancement of stomatal aperture compared to WT leaves, whereas in SO OE plants, stomatal aperture was smaller than that of the WT plants, and hence water loss was lower. Sulfite application also limited sulfate and ABA-induced stomatal closure in WT and SO Ri. The increases in APR activity in response to sulfite infiltration into WT and SO Ri leaves resulted in an increase in sulfite beyond the level of the applied sulfite, indicating that APR has an important role in sulfite-induced increases in stomatal aperture. Notably, 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) by diphenyleneiodonium, or mutation in APR2 or GR2, resulted in decrease in sulfite production and stomatal aperture size, further supporting the role of APR in stomatal aperture size. The importance of APR and SO in the set-up of sulfite level in leaves, and the significance of sulfite level in water loss were further demonstrated during fast and harsh drought stress in root-detached WT, gr2 and SO modified plants. The role of SO in sulfite homeostasis in relation to water consumption was shown in well-watered plants.

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.

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.

ABSTRACT The role of the cytosolic O-acetylserine-(thiol) lyase A (OASTLA), chloroplastic OASTLB and mitochondrion OASTLC in plant resistance/sensitivity to selenate was studied in Arabidopsis plants. Impairment in OASTLA and B resulted in reduced biomass, chlorophyll and soluble protein levels compared with impaired OASTL C and Wild-Type treated with selenate. The lower organic-Se and protein-Se levels followed by decreased organic-S, S in proteins and total glutathione in oastlA and oastlB compared to Wild-Type and oastlC are indicative that Se accumulation is not the main cause for the stress symptoms, but rather the interference of Se with the S-reduction pathway. The increase in sulfite oxidase, adenosine 5′-phosphosulfate reductase, sulfite reductase and OASTL activity levels, followed by enhanced sulfite and sulfide, indicate a futile anabolic S-starvation response to selenate-induced organic-S catabolism in oastlA and oastlB compared to Wild-Type and oastlC . Additionally, the catabolic pathway of L-cysteine degradation was enhanced by selenate, and similar to L-cysteine producing activity, oastlA and B exhibited a significant decrease in L-cysteine desulfhydrase (DES) activity, compared with WT, indicating a major role of OASTLs in L-cysteine degradation. This notion was further evidenced by sulfide dependent DES in-gel activity, immunoblotting, immunoprecipitation with specific antibodies and identification of unique peptides in activity bands generated by OASTLA, B and C. Similar responses of the OASTLs in Seleno-Cysteine degradation was demonstrated in selenate stressed plants. Notably, no L-cysteine and L-Seleno-Cysteine DES activity bands but those related to OASTLs were evident. These results indicate the significance of OASTLs in degrading L-cysteine and L-SelenoCysteine in Arabidopsis . Summary The cytosolic OASTLA and chloroplastic OASTLB have significantly higher desulfhydrase activity rates than the cytosolic DES1 and are able to degrade L-Cys and L-SeCys to sulfide and selenide, respectively in Arabidopsis .