Galina Brychkova

A critical role for ureides in dark and senescence-induced purine remobilization is unmasked in the Atxdh1 Arabidopsis mutant.

The remobilization of metabolites during stress and senescence plays an important role in optimal plant adaptation to the environment. The plant molybdenum co-factor (MoCo) and flavin-containing enzyme xanthine dehydrogenase (XDH; EC 1.2.1.37) are pivotal for purine remobilization, and catalyze the conversion of the purine catabolic products hypoxanthine and xanthine to uric acid, which is subsequently degraded to the ureides allantoin and allantoate. We observed that in wild-type plants conditions of extended darkness or increasing leaf age caused induction of transcripts related to purine catabolism, resulting in marked accumulation of the purine catabolic products allantoin and allantoate. In contrast, Arabidopsis mutants of XDH, Atxdh1, accumulated xanthine and showed premature senescence symptoms, as exemplified by enhanced chlorophyll degradation, extensive cell death and upregulation of senescence-related transcripts. When dark-treated mutant lines were re-exposed to light, they showed elevated levels of reactive oxygen species (ROS) and a higher mortality rate compared with wild-type plants. Interestingly, the level of ROS and mortality could be attenuated by the addition of allantoin and allantoate, suggesting that these metabolites can act as scavengers of ROS. The results highlight a crucial need for the controlled maintenance of ureide levels mediated by AtXDH1 activity during dark stress and ageing, and point to the dual functionality of ureides as efficient stores of nitrogen and as cellular protectants. Thus, the regulation of ureide levels by Atxdh1 has general implications for optimal plant survival during nutrient remobilization, such as occurs during normal growth, dark stress and senescence.

Sulfite Reductase Protects Plants Against Sulfite Toxicity

Summary: Sulfite oxidase and the key elements of the sulfite network enzymes that include sulfite reductase, UDP-sulfoquinovose synthase, β-mercaptopyruvate sulfurtransferases, and adenosine-5′-phosphosulfate reductase has a important role in maintaining sulfite homeostasis, where sulfite appears to act as an orchestrating signal molecule. Plant sulfite reductase (SiR; Enzyme Commission 1.8.7.1) catalyzes the reduction of sulfite to sulfide in the reductive sulfate assimilation pathway. Comparison of SiR expression in tomato (Solanum lycopersicum ‘Rheinlands Ruhm’) and Arabidopsis (Arabidopsis thaliana) plants revealed that SiR is expressed in a different tissue-dependent manner that likely reflects dissimilarity in sulfur metabolism between the plant species. Using Arabidopsis and tomato SiR mutants with modified SiR expression, we show here that resistance to ectopically applied sulfur dioxide/sulfite is a function of SiR expression levels and that plants with reduced SiR expression exhibit higher sensitivity than the wild type, as manifested in pronounced leaf necrosis and chlorophyll bleaching. The sulfite-sensitive mutants accumulate applied sulfite and show a decline in glutathione levels. In contrast, mutants that overexpress SiR are more tolerant to sulfite toxicity, exhibiting little or no damage. Resistance to high sulfite application is manifested by fast sulfite disappearance and an increase in glutathione levels. The notion that SiR plays a role in the protection of plants against sulfite is supported by the rapid up-regulation of SiR transcript and activity within 30 min of sulfite injection into Arabidopsis and tomato leaves. Peroxisomal sulfite oxidase transcripts and activity levels are likewise promoted by sulfite application as compared with water injection controls. These results indicate that, in addition to participating in the sulfate assimilation reductive pathway, SiR also plays a role in protecting leaves against the toxicity of sulfite accumulation.

An Essential Role for Tomato Sulfite Oxidase and Enzymes of the Sulfite Network in Maintaining Leaf Sulfite Homeostasis

Little is known about the homeostasis of sulfite levels, a cytotoxic by-product of plant sulfur turnover. By employing extended dark to induce catabolic pathways, we followed key elements of the sulfite network enzymes that include adenosine-5′-phosphosulfate reductase and the sulfite scavengers sulfite oxidase (SO), sulfite reductase, UDP-sulfoquinovose synthase, and β-mercaptopyruvate sulfurtransferases. During extended dark, SO was enhanced in tomato (Solanum lycopersicum) wild-type leaves, while the other sulfite network components were down-regulated. SO RNA interference plants lacking SO activity accumulated sulfite, resulting in leaf damage and mortality. Exogenous sulfite application induced up-regulation of the sulfite scavenger activities in dark-stressed or unstressed wild-type plants, while expression of the sulfite producer, adenosine-5′-phosphosulfate reductase, was down-regulated. Unstressed or dark-stressed wild-type plants were resistant to sulfite applications, but SO RNA interference plants showed sensitivity and overaccumulation of sulfite. Hence, under extended dark stress, SO activity is necessary to cope with rising endogenous sulfite levels. However, under nonstressed conditions, the sulfite network can control sulfite levels in the absence of SO activity. The novel evidence provided by the synchronous dark-induced turnover of sulfur-containing compounds, augmented by exogenous sulfite applications, underlines the role of SO and other sulfite network components in maintaining sulfite homeostasis, where sulfite appears to act as an orchestrating signal molecule.

The determination of sulfite levels and its oxidation in plant leaves

Sulfur is the sixth most abundant element in life and an important building block of proteins and cellular metabolites. Plants like bacteria can synthesize their sulfur-containing biomolecules from sulfate, where sulfite is an intermediate of the sulfur assimilation pathway. Above a certain threshold SO(2)/sulfite is cytotoxic and is rapidly metabolized to avoid damage. However, the existing data show considerable differences in basal sulfite levels both between species and apparent discrepancies in measured levels in the same species. In order to resolve this question we employed a sulfite detection method using chicken sulfite oxidase and developed an independent enzymatic assay, based on the specific detection of sulfite by sulfite reductase and compared those measurements to a modified colorimetric fuchsin-based method, specific for sulfite detection. We show here that when properly used the sulfite levels detected by the three methods can yield identical results. Furthermore, to examine the capacity of the plant to detoxify sulfite we injected sub-lethal sulfite solutions (yet, several folds higher than the basal levels) into Arabidopsis and tomato leaves and monitored the excess sulfite turnover. Within 3h of sulfite injection, more than 80% of the injected sulfite in Arabidopsis and 91% in tomato were oxidized to sulfate, demonstrating the high capacity of the sulfite oxidation mechanism/s in plants.

Formation of xanthine and the use of purine metabolites as a nitrogen source in Arabidopsis plants.

In our recent paper in Plant J1, we described the remobilization of purine metabolites during natural and dark induced senescence in wild type and Atxdh1 mutant lines impaired in xanthine dehydrogenase (XDH), a pivotal enzyme in the purine catabolism pathway. In the light of these observations and additional evidence shown here, we discuss the probable pathways leading to xanthine synthesis in Arabidopsis plants during senescence and the role that purine metabolites play as an ongoing source of nitrogen in plant growth. Addendum to: Brychkova G, Alikulov Z, Fluhr R, Sagi M. A critical role for ureides in dark and senescence-induced purine remobilization is unmasked in the Atxdh1 Arabidopsis mutant. Plant J 2008; 54:496–509.

Impairment in Sulfite Reductase Leads to Early Leaf Senescence in Tomato Plants

Insufficient sulfite reductase activity activates the upstream components of the sulfate reduction pathway in tomato plants, resulting in accumulation of toxic sulfite and early leaf senescence. Sulfite reductase (SiR) is an essential enzyme of the sulfate assimilation reductive pathway, which catalyzes the reduction of sulfite to sulfide. Here, we show that tomato (Solanum lycopersicum) plants with impaired SiR expression due to RNA interference (SIR Ri) developed early leaf senescence. The visual chlorophyll degradation in leaves of SIR Ri mutants was accompanied by a reduction of maximal quantum yield, as well as accumulation of hydrogen peroxide and malondialdehyde, a product of lipid peroxidation. Interestingly, messenger RNA transcripts and proteins involved in chlorophyll breakdown in the chloroplasts were found to be enhanced in the mutants, while transcripts and their plastidic proteins, functioning in photosystem II, were reduced in these mutants compared with wild-type leaves. As a consequence of SiR impairment, the levels of sulfite, sulfate, and thiosulfate were higher and glutathione levels were lower compared with the wild type. Unexpectedly, in a futile attempt to compensate for the low glutathione, the activity of adenosine-5′-phosphosulfate reductase was enhanced, leading to further sulfite accumulation in SIR Ri plants. Increased sulfite oxidation to sulfate and incorporation of sulfite into sulfoquinovosyl diacylglycerols were not sufficient to maintain low basal sulfite levels, resulting in accumulative leaf damage in mutant leaves. Our results indicate that, in addition to its biosynthetic role, SiR plays an important role in prevention of premature senescence. The higher sulfite is likely the main reason for the initiation of chlorophyll degradation, while the lower glutathione as well as the higher hydrogen peroxide and malondialdehyde additionally contribute to premature senescence in mutant leaves.

A Novel In-gel Assay and an Improved Kinetic Assay for Determining In Vitro Sulfite Reductase Activity in Plants

Sulfite reductase (SiR; EC 1.8.7.1), an essential enzyme in the sulfate reduction pathway, catalyzes the reduction of sulfite to sulfide, as an intermediate for cysteine biosynthesis. The commonly used kinetic assay for the detection of in vitro SiR activity in plants is based on a coupled reaction, in which the sulfide produced is converted to cysteine through the presence, in the assay medium, of O-acetylserine sulfhydralase (EC 2.5.1.47) and its substrate, O-acetylserine. An improved kinetic assay for SiR activity in crude desalted protein extracts was developed. The improvement was based on pre-treatment of the protein with tungstate, which improved SiR activity in Arabidopsis and tomato leaf by 29 and 12%, respectively, and the addition of NADPH to the reaction medium, which increased SiR activity by 1.6- and 2.8-fold in Arabidopsis and tomato, respectively, in comparison with the current protocols. Despite the availability and reliability of the kinetic assay, there is currently no assay that enables the direct detection of SiR in relatively large numbers of samples. To meet this need, we developed a novel in-gel assay to detect SiR activity in crude extracts. The method is based on the detection of a brownish-black precipitated band of lead sulfide, formed by the reaction of lead acetate with sulfide. The in-gel assay for SiR activity is reliable, sensitive and technically simpler than the kinetic assay, and opens up the possibility for detecting active SiR isoenzymes and splice variants.

Smallholder Farmers and Climate Smart Agriculture Technology and Labor-productivity Constraints amongst Women Smallholders in Malawi

Abstract Climate change and variability present a major challenge to agricultural production and rural livelihoods, including livelihoods of women smallholder farmers. There are significant efforts underway to develop, deploy, and scale up Climate-Smart Agricultural (CSA) practices and technologies to facilitate climate change adaptation for farmers. However, there is a need for gender analysis of CSA practices across different farming and cultural systems to facilitate adoption by, and livelihood improvements for, women smallholder farmers. Climate change poses challenges for maintaining and improving agricultural and labor productivity of women smallholder farmers. The labor productivity of many women smallholders is constrained by lack of access to labor-saving technologies and the most basic of farm tools. Poorer smallholders face a poverty trap, due to low agricultural and labor productivity, from which they cannot easily escape without access to key resources such as rural energy and labor-saving technologies. In Malawi, the agricultural system is predominantly rainfed and largely composed of smallholders who remain vulnerable to climate change and variability shocks. Despite the aspirations of women smallholders to engage in CSA, our research highlights that many women smallholders have either limited or no access to basic agricultural tools, transport, and rural energy. This raises the question of whether the future livelihood scenarios for such farmers will consist of barely surviving or “hanging in”; or whether such farmers can “step up” to adapt better to future climate constraints; or whether more of these farmers will “step out” of agriculture. We argue that for women smallholder farmers to become more climate change resilient, more serious attention to gender analysis is needed to address their constraints in accessing basic agricultural technologies, combined with participatory approaches to develop and adapt CSA tools and technologies to their needs in future climates and agro-ecologies.

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

AAO4 plays a critical role in delaying silique senescence by catalyzing aldehyde detoxification and generates both hydrogen peroxide and superoxide. 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.

Molybdenum application enhances adaptation of crested wheatgrass to salinity stress

Crested wheatgrass (Agropyron cristatum) is a novel halophyte crop for sustainable agriculture in Northern Kazakhstan. This study investigated the effect of molybdenum (Mo) as molybdate (Na2MoO4·2H2O) and its chemical antagonist tungsten (W) as tungstate (Na2WO4·2H2O) on plant response to salinity treatment. The results showed that the treatment of A. cristatum with Mo significantly improved plant health, as opposed to the W application, which negatively correlated with root and shoot development. Indeed, Mo prevented oxidative damage to plant tissues subjected to salinity stress through increased activities of the three Mo-containing enzymes, nitrate reductase, aldehyde oxidase and xanthine dehydrogenase. Contrarily, treatment with tungsten negatively affected these enzymes’ activities, resulting in increased sensitivity to salt stress. Hence, our results suggested that the Mo-treatment might play an important role in the process of halophyte plant A. cristatum adaptation to salt stress.