Bożena Szal

Effect of mitochondrial genome rearrangement on respiratory activity, photosynthesis, photorespiration and energy status of MSC16 cucumber (Cucumis sativus) mutant.

The effects of changes in mitochondrial DNA in cucumber (Cucumis sativus L.) mosaic mutant (MSC16) on respiration, photosynthesis and photorespiration were analyzed under non-stressed conditions. Decreased respiratory capacity of complex I in MSC16 mitochondria was indicated by lower respiration rates of intact mitochondria with malate and by rotenone-inhibited NADH or malate oxidation in the presence of alamethicin. Moreover, blue native PAGE indicated decreased intensity of protein bands of respiratory chain complex I in MSC16 leaves. Concerning the redox state, complex I impairment could be compensated to some extent by increased external NADH dehydrogenases (ND(ex)NADH) and alternative oxidase (AOX) capacity, the latter presenting differential expression in the light and in the dark. Although MSC16 mitochondria have a higher AOX protein level and an increased capacity, the AOX activity measured in the dark conditions by oxygen discrimination technique is similar to that in wild-type (WT) plants. Photosynthesis induction by light followed different patterns in WT and MSC16, suggesting changes in feedback chloroplast DeltapH caused by different adenylate levels. At steady-state, net photosynthesis was only slightly impaired in MSC16 mutants, while photorespiration rate (PR) was significantly increased. This was the result of large decreases in both stomatal and mesophyll conductance to CO2, which resulted in a lower CO2 concentration in the chloroplasts. The observed changes on CO2 diffusion caused by mitochondrial mutations open a whole new view of interaction between organelle metabolism and whole tissue physiology. The sum of all the described changes in photosynthetic and respiratory metabolism resulted in a lower ATP availability and a slower plant growth.

Chilling stress and mitochondrial genome rearrangement in the MSC16 cucumber mutant affect the alternative oxidase and antioxidant defense system to a similar extent

The mosaic MSC16 cucumber (Cucumis sativus L.) mutant, which houses a rearranged mitochondrial genome, has altered respiratory chain activity, with a dysfunctional Complex I, increased external NADH dehydrogenases (ND(ex)) activity, and a higher alternative oxidase (AOX) capacity and AOX protein level. In the present study, changes in oxidative defense metabolism resulting from the respiratory chain dysfunction in the MSC16 mutant were compared with those induced by chilling. Chilling increased the enzymatic and non-enzymatic antioxidant defense systems in the wild-type (WT) but not in MSC16, which displays elevated antioxidant defenses as a result of the mitochondrial mutation. The high AOX capacity and protein level in MSC16 were unchanged as a result of chilling, whereas chilling increased these parameters in WT leaves. In mitochondria isolated from WT plants, superoxide was produced to a similar extent in the matrix and the intermembrane space, but in MSC16 mitochondria superoxide was produced largely within the intermembrane space. Mitochondria isolated from both genotypes after chilling showed increased superoxide production within the intermembrane space. Cytochemical detection revealed an increased abundance of H2O2 in the mitochondrial membrane in mesophyll cells of MSC16 leaves. The mitochondrial mutation also resulted in changes in the antioxidative defense system, including AOX, which were similar to those observed following chilling. The results presented here support the hypothesis that AOX is an effective marker of the cellular reprogramming resulting from stress. Moreover, we propose a role for reactive oxygen species (ROS) generated within the mitochondria in signal transduction.

Changes in energy status of leaf cells as a consequence of mitochondrial genome rearrangement.

The MSC16 cucumber (Cucumis sativus L.) mutant with lower activity of mitochondrial Complex I was used to study the influence of mitochondrial metabolism on whole cell energy and redox state. Mutant plants had lower content of adenylates and NADP(H) whereas the NAD(H) pool was similar as in wild type. Subcellular compartmentation of adenylates and pyridine nucleotides were studied using the method of rapid fractionation of protoplasts. The data obtained demonstrate that dysfunction of mitochondrial respiratory chain decreased the chloroplastic ATP pool. No differences in NAD(H) pools in subcellular fractions of mutated plants were observed; however, the cytosolic fraction was highly reduced whereas the mitochondrial fraction was more oxidized in MSC16, as compared to WTc. The NADP(H) pool in MSC16 protoplasts was greatly decreased and the chloroplastic NADP(H) pool was more reduced, whereas the extrachloroplastic pool was much more oxidized, than in WTc protoplast. Changes in nucleotides distribution in cucumber MSC16 mutant were compared to changes found in tobacco (Nicotiana sylvestris) CMS II mitochondrial mutant. In contrast to MSC16 cucumber, the content of adenylates in tobacco mutant was much higher than in tobacco wild type. The differences were more pronounced in leaf tissue collected after darkness than in the middle of the photoperiod. Results obtained after tobacco protoplast fractionating showed that the increase in CMS II adenylate content was mainly due to a higher level in extrachloroplast fraction. Both mutations have a negative effect on plant growth through perturbation of chloroplast/mitochondrial interactions.

Long‐term ammonium nutrition of Arabidopsis increases the extrachloroplastic NAD(P)H/NAD(P)+ ratio and mitochondrial reactive oxygen species level in leaves but does not impair photosynthetic capacity

Ammonium nutrition has been suggested to be associated with alterations in the oxidation-reduction state of leaf cells. Herein, we show that ammonium nutrition in Arabidopsis thaliana increases leaf NAD(P)H/NAD(P)(+) ratio, reactive oxygen species content and accumulation of biomolecules oxidized by free radicals. We used the method of rapid fractionation of protoplasts to analyse which cellular compartments were over-reduced under ammonium supply and revealed that observed changes in NAD(P)H/NAD(P)(+) ratio involved only the extrachloroplastic fraction. We also showed that ammonium nutrition changes mitochondrial electron transport chain activity, increasing mitochondrial reactive oxygen species production. Our results indicate that the functional impairment associated with ammonium nutrition is mainly associated with redox reactions outside the chloroplast.

The effect of phosphate deficiency on membrane phospholipid composition of bean (Phaseolus vulgaris L.) roots

Plasma membranes were isolated from roots of bean (Phaseolus vulgaris L.) plants cultured on phosphate sufficient or phosphate deficient medium. The phospholipid composition of plasma membranes was analyzed and compared with that of the microsomal fraction. Phosphate deficiency had no influence on lipid/protein ratio in microsomal as well as plasma membrane fraction. In phosphate deficient roots phospholipid content was lower in the plasma membrane, but did not change in the microsomal fraction. Phosphatidylcholine and phosphatidylethanolamine were two major phospholipids in plasmalemma and microsomal membranes (80 % of the total). After two weeks of phosphate starvation a considerable decrease (about 50 %) in phosphatidylcholine and phosphatidylethanolamine in microsomal membranes was observed. The decline in two major phospholipids was accompanied by an increase in phosphatidic acid and lysophosphatidylcholine content. The effect of alterations in plasma membrane phospholipids on membrane function e.g. nitrate uptake is discussed.

Dissecting the metabolic role of mitochondria during developmental leaf senescence

During developmental leaf senescence in Arabidopsis, mitochondria simultaneously maintain primary energy processes and are the site of a number of catabolic processes, thus ensuring effective nutrient reallocation. The functions of mitochondria during leaf senescence, a type of programmed cell death aimed at the massive retrieval of nutrients from the senescing organ to the rest of the plant, remain elusive. Here, combining experimental and analytical approaches, we showed that mitochondrial integrity in Arabidopsis (Arabidopsis thaliana) is conserved until the latest stages of leaf senescence, while their number drops by 30%. Adenylate phosphorylation state assays and mitochondrial respiratory measurements indicated that the leaf energy status also is maintained during this time period. Furthermore, after establishing a curated list of genes coding for products targeted to mitochondria, we analyzed in isolation their transcript profiles, focusing on several key mitochondrial functions, such as the tricarboxylic acid cycle, mitochondrial electron transfer chain, iron-sulfur cluster biosynthesis, transporters, as well as catabolic pathways. In tandem with a metabolomic approach, our data indicated that mitochondrial metabolism was reorganized to support the selective catabolism of both amino acids and fatty acids. Such adjustments would ensure the replenishment of α-ketoglutarate and glutamate, which provide the carbon backbones for nitrogen remobilization. Glutamate, being the substrate of the strongly up-regulated cytosolic glutamine synthase, is likely to become a metabolically limiting factor in the latest stages of developmental leaf senescence. Finally, an evolutionary age analysis revealed that, while branched-chain amino acid and proline catabolism are very old mitochondrial functions particularly enriched at the latest stages of leaf senescence, auxin metabolism appears to be rather newly acquired. In summation, our work shows that, during developmental leaf senescence, mitochondria orchestrate catabolic processes by becoming increasingly central energy and metabolic hubs.

Influence of mitochondrial genome rearrangement on cucumber leaf carbon and nitrogen metabolism

The MSC16 cucumber (Cucumis sativus L.) mitochondrial mutant was used to study the effect of mitochondrial dysfunction and disturbed subcellular redox state on leaf day/night carbon and nitrogen metabolism. We have shown that the mitochondrial dysfunction in MSC16 plants had no effect on photosynthetic CO2 assimilation, but the concentration of soluble carbohydrates and starch was higher in leaves of MSC16 plants. Impaired mitochondrial respiratory chain activity was associated with the perturbation of mitochondrial TCA cycle manifested, e.g., by lowered decarboxylation rate. Mitochondrial dysfunction in MSC16 plants had different influence on leaf cell metabolism under dark or light conditions. In the dark, when the main mitochondrial function is the energy production, the altered activity of TCA cycle in mutated plants was connected with the accumulation of pyruvate and TCA cycle intermediates (citrate and 2-OG). In the light, when TCA activity is needed for synthesis of carbon skeletons required as the acceptors for NH4+ assimilation, the concentration of pyruvate and TCA intermediates was tightly coupled with nitrate metabolism. Enhanced incorporation of ammonium group into amino acids structures in mutated plants has resulted in decreased concentration of organic acids and accumulation of Glu.

Extra-Cellular But Extra-Ordinarily Important for Cells: Apoplastic Reactive Oxygen Species Metabolism

Reactive oxygen species (ROS), by their very nature, are highly reactive, and it is no surprise that they can cause damage to organic molecules. In cells, ROS are produced as byproducts of many metabolic reactions, but plants are prepared for this ROS output. Even though extracellular ROS generation constitutes only a minor part of a cell’s total ROS level, this fraction is of extraordinary importance. In an active apoplastic ROS burst, it is mainly the respiratory burst oxidases and peroxidases that are engaged, and defects of these enzymes can affect plant development and stress responses. It must be highlighted that there are also other less well-known enzymatic or non-enzymatic ROS sources. There is a need for ROS detoxification in the apoplast, and almost all cellular antioxidants are present in this space, but the activity of antioxidant enzymes and the concentration of low-mass antioxidants is very low. The low antioxidant efficiency in the apoplast allows ROS to accumulate easily, which is a condition for ROS signaling. Therefore, the apoplastic ROS/antioxidant homeostasis is actively engaged in the reception and reaction to many biotic and abiotic stresses.

The Role of Reactive Oxygen Species Under Ammonium Nutrition

The use of ammonium as the sole nitrogen source leads to growth inhibition in most crop plant species; however, a comprehensive understanding of this phenomenon has yet to be developed. An accumulating amount of evidence indicates that ammonium toxicity is caused by impaired redox homeostasis, which increases reactive oxygen species (ROS) production. We describe the connection between NH4 + nutrition and ROS-producing reactions and analyse the changes in antioxidant systems under ammonium supply. In this review, the function of mitochondria as reductant sinks under NH4 + supply is discussed, and a central role in ROS production and signalling is proposed for these organelles.

Short‐term ammonium supply induces cellular defence to prevent oxidative stress in Arabidopsis leaves

Plants can assimilate nitrogen from soil pools of both ammonium and nitrate, and the relative levels of these two nitrogen sources are highly variable in soil. Long-term ammonium nutrition is known to cause damage to Arabidopsis that has been linked to mitochondrial oxidative stress. Using hydroponic cultures, we analysed the consequences of rapid shifts between nitrate and ammonium nutrition. This did not induce growth retardation, showing that Arabidopsis can compensate for the changes in redox metabolism associated with the variations in nitrogen redox status. During the first 3 h of ammonium treatment, we observed distinct transient shifts in reactive oxygen species (ROS), low-mass antioxidants, ROS-scavenging enzymes, and mitochondrial alternative electron transport pathways, indicating rapid but temporally separated changes in chloroplastic, mitochondrial and cytosolic ROS metabolism. The fast induction of antioxidant defences significantly lowered intracellular H2 O2 levels, and thus protected Arabidopsis leaves from oxidative stress. On the other hand elevated extracellular ROS production in response to ammonium supply may be involved in signalling. The response pattern displays an intricate plasticity of Arabidopsis redox metabolism to minimise stress in responses to nutrient changes.