Anna M. Rychter

The relationship between phosphate status and cyanide-resistant respiration in bean roots

Bean (Phaseolus vulgaris L.) seedlings were cultured on complete or phosphate-deficient nutrient medium. After 14 days of culture on phosphate-deficient medium the visible symptoms of P(i) deficiency were observed only in the shoot, the fresh and dry weights of the roots were slightly higher than in control plants. The decreased P(i) content in the roots had little effect on total respiration rate but had an effect on the level of inhibition of respiration by cyanide. The high resistance of respiration to cyanide observed in P(i) -deficient roots was the result of the suppression of cytochrome path activity and an increased participation of the alternative, cyanide-resistant pathway. The cytochrome pathway activity increased when inorganic phosphate was supplied to P(i) -deficient roots for 1 or 3.5 h. It is speculated that the suppression of cytochrome pathway in P(i) -deficient roots may result from restriction of the phosphorylating capacity or a partial inhibition of cytochrome oxidase activity.

Changes in the concentration of phenolic compounds and exudation induced by phosphate deficiency in bean plants (Phaseolus vulgaris L.)

The effect of prolonged phosphate starvation of bean plants (Phaseolus vulgaris L.) on the concentration of phenolics and their exudation by roots was studied. Plants cultured on phosphate-deficient media maintained a steady concentration of total phenolics in the leaves, whereas in the leaves of plants grown on complete nutrient media the phenolic concentration decreased. After 18 days of culture, higher total phenolics and anthocyanin concentrations in phosphate-deficient leaves compared with control leaves were observed. The divergent trends in total phenolic concentrations between phosphate-deficient and control leaves corresponded to the changes in the activity of L-phenylalanine ammonia-lyase. In the roots, the concentration of total phenolics was lower in phosphate-deficient plants compared with control plants. However, after 18 days of culture of bean plants, the amount of exuded phenolics from phosphate-deficient roots was 5-times higher than that from the roots of control plants. The activity of L-phenylalanine ammonia-lyase was twice as high in the roots of phosphate-starved plants. Comparable rates in the exudation of phenolics by bean roots observed after 18 days of culture on nitrogen-deficient or phosphate-deficient medium may suggest a similar system of signal transduction for phenolics release. The results are discussed in relation to the possible functions of phenolics in nutrient uptake and as chemical signals in root-soil microbe interactions to enhance the plant adaptation to particular environmental conditions.

Assimilate translocation in bean plants (Phaseolus vulgaris L.) during phosphate deficiency

Summary A rapid decrease in inorganic phosphate content in bean plants ( Phaseolus vulgaris L. cv. Zlota Saxa) grown on phosphate deficient medium induced an increased translocation of assimilated carbon from the shoot to the root. The incorporation of l4 CO 2 to the leaves of the plants after 16 days of culture on phosphate deficient medium was about 14 % higher than in control plants grown on complete medium. The 14 C labelling in the roots of phosphate deficient plants (−P) was about 100% higher than in the control (+P). Most of the labelling was found in the water soluble fraction. The water soluble fraction was divided into sugar, amino acid and organic acid fractions. The sugar fraction was the largest fraction and its radioactivity was higher in the roots of phosphate deficient plants than in control roots. The radioactivity of amino acid and organic acid fractions was markedly lower in phosphate deficient compared with phosphate sufficient roots. The higher labelling of sugars in −P roots corresponded to the higher content of sucrose and glucose. Rather the higher glucose content in −P roots observed earlier during the culture than the increase in sucrose level suggested the enhanced hydrolysis of transported sucrose. However, the activities of acid and neutral invertases in +P and −P roots were very similar and the differences statistically insignificant. The greater assimilate transport and sugar accumulation in bean roots appears to be the early plant response to phosphate deficiency.

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.

Oxidative stress during phosphate deficiency in roots of bean plants (Phaseolus vulgaris L.)

Summary The oxidative stress symptoms were studied during phosphate deficiency. Prolonged phosphate starvation of bean plants ( Phaseolus vulgaris L.) and severe decrease of inorganic phosphate concentration resulted in increased lipid peroxidation and hydrogen peroxide concentration in root tissues. The ratio of reduced to total ubiquinone was also higher in whole roots and isolated mitochondria from the roots of phosphate-deficient plants. No effect of phosphate deficiency on ascorbate peroxidase and superoxide dismutase activities was detected. However, the activities of catalase and total peroxidase were higher in extracts of phosphate-deficient roots compared to control roots. These results indicate that phosphate starvation is an abiotic stress that imposes an oxidative stress in bean root cells. The role of alternative oxidase in stabilizing the reduction level of ubiquinone, and thus preventing active oxygen species formation, is discussed.

Differential response of antioxidant systems in leaves and roots of barley subjected to anoxia and post-anoxia.

Mitochondria and chloroplasts are the primary sources of reactive oxygen species in photosynthetic tissues. Mitochondria play a dual role in oxidative stress, being both ROS producers and integrators of the cell antioxidant defense systems. Anoxia followed by re-exposure to air may create conditions of elevated ROS generation in plants. The reactions of barley leaves and root antioxidant systems after anoxia were investigated. Alternative oxidase and low-mass antioxidants showed different patterns of change in leaves and roots in response to changing oxygen availability. Mitochondria from leaves showed a reduced AOX capacity and AOX protein level during post-anoxia, while the AOX capacity in root mitochondria increased. In leaf tissue, ascorbate and glutathione were immediately oxidized after restoration of aerobiosis due to the enhanced ROS production, whereas in roots ascorbate and glutathione became even more reduced. The capacity of antioxidant systems was not exceeded in either leaf or root tissue after anoxia; no signs of oxidative damage were observed and the respiratory activity of mitochondria was retained. We suggest that the leaf and root cell antioxidant systems react differentially to changes in oxygen concentration. In leaves, low-mass antioxidants play the major role in ROS detoxification, while in roots, increased AOX capacity supports the antioxidant systems, possibly by preventing ROS formation.

Changes of alternative oxidase activity, capacity and protein content in leaves of Cucumis sativus wild-type and MSC16 mutant grown under different light intensities

In vitro studies demonstrated that alternative oxidase (AOX) is biochemically regulated by a sulfhydryl-disulfide system, interaction with alpha-ketoacids, ubiquinone pool redox state and protein content among others. However, there is still scarce information about the in vivo regulation of the AOX. Cucumis sativus wild-type (WT) and MSC16 mutant plants were grown under two different light intensities and were used to analyze the relationship between the amount of leaf AOX protein and its in vivo capacity and activity at night and day periods. WT and MSC16 plants presented lower total respiration (V(t)), cytochrome oxidase pathway (COP) activity (v(cyt)) and alternative oxidase pathway (AOP) activity (v(alt)) when grown at low light (LL), although growth light intensity did not change the amount of cytochrome oxidase (COX) nor AOX protein. Changes of v(cyt) related to growing light conditions suggested a substrate availability and energy demand control. On the other hand, the total amount of AOX protein present in the tissue does not play a role in the regulation neither of the capacity nor of the activity of the AOP in vivo. Soluble carbohydrates were directly related to the activity of the AOP. However, although differences in soluble sugar contents mostly regulate the capacity of the AOP at different growth light intensities, additional regulatory mechanisms are necessary to fully explain the observed results.

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.

The influence of phosphate deficiency on photosynthesis, respiration and adenine nucleotide pool in bean leaves

The decrease in inorganic phosphate (Pi) content of 10-d-old Phaseolus vulgaris L. plants did not affect rates of photosynthesis (PN) and respiration (RD), leaf growth, and adenylate concentration. Two weeks of phosphate starvation influenced the ATP content and leaf growth more than PN and RD. The ATP concentration in the leaves of 15- and 18-d-old phosphate deficient (-P) plants after a light or dark period was at least half of that in phosphate sufficient (+P, control) plants. Similar differences were found in fresh and dry matter of leaves. However, PN declined to 50 % of control in 18-d-old plants only. Though the RD of -P plants (determined as both CO2 evolution and O2 uptake) did not change, an increased resistance of respiration to KCN and higher inhibition by SHAM (salicylhydroxamic acid) suggested a higher engagement of alternative pathway in respiration and a lower ATP production. The lower demand for ATP connected with inhibition of leaf growth may influence the ATP producing processes and ATP concentration. Thus, the ATP concentration in the leaves depends stronger on Pi content than on PN and RD.

Low phosphate nutrition alters bean plants' ability to assimilate and translocate nitrate

Abstract Bean plants (Phaseolus vulgaris L.) were cultured for 10 or 18 days on phosphate sufficient (+P) or phosphate deficient (‐P) nutrient medium. Nitrate and phosphate distribution between shoot and root, nitrate uptake, and nitrate reductase activity (NR activity, in vivo and in vitro) in root and leaves was estimated. The decrease in Pi concentration in leaves and roots led to decreased rate of NO3 uptake and increased NO3 accumulation in roots, accompanied by alterations in NO3 distribution between shoot and roots. Nitrate reductase activity estimated in vitro was twice higher than estimated in vivo and both in +P and ‐P plants was lower in the roots than in the shoots. The decrease of NR activity in ‐P plants was more pronounced in the roots and after 2 weeks of phosphate starvation it was about 40% lower as compared with the control. The depression in nitrate uptake may be the result of feedback inhibition due to accumulation of nitrate in the roots. The increased NO3 concentration in root tissu...