Branka D. Živanović

Linking oxidative and salinity stress tolerance in barley: can root antioxidant enzyme activity be used as a measure of stress tolerance?

AimsA causal relationship between salinity and oxidative stress tolerance and a suitability of using root antioxidant activity as a biochemical marker for salinity tolerance in barley was investigated.MethodsNet ion fluxes were measured from the mature zone of excised roots of two barley varieties contrasting in their salinity tolerance using non-invasive MIFE technique in response to acute and prolonged salinity treatment. These changes were correlated with activity of major antioxidant enzymes; ascorbate peroxidase, catalase, and superoxide dismutase.ResultsIt was found that genotypic difference in salinity tolerance was largely independent of root integrity, and observed not only for short-term but also long-term NaCl exposures. Higher K+ retention ability (and, hence, salinity tolerance) positively correlated with oxidative stress tolerance. At the same time, antioxidant activities were constitutively higher in a sensitive but not tolerant variety, and no correlation was found between SOD activity and salinity tolerance index during large-scale screening.ConclusionAlthough salinity tolerance in barley correlates with its oxidative stress tolerance, higher antioxidant activity at one particular time does not correlate with salinity tolerance and, as such, cannot be used as a biochemical marker in barley screening programs.

Rutin, a flavonoid with antioxidant activity, improves plant salinity tolerance by regulating K+ retention and Na+ exclusion from leaf mesophyll in quinoa and broad beans

The causal relationship between salinity and oxidative stress tolerance is well established, but specific downstream targets and the role of specific antioxidant compounds in controlling cellular ionic homeostasis remains elusive. In this work, we have compared antioxidant profiles of leaves of two quinoa genotypes contrasting in their salt tolerance, with the aim of understanding the role of enzymatic and non-enzymatic antioxidants in salinity stress tolerance. Only changes in superoxide dismutase activity were correlated with plant adaptive responses to salinity. Proline accumulation played no major role in either osmotic adjustment or in the tissue tolerance mechanism. Among other non-enzymatic antioxidants, rutin levels were increased by over 25 fold in quinoa leaves. Exogenous application of rutin to glycophyte bean leaves improved tissue tolerance and reduced detrimental effects of salinity on leaf photochemistry. Electrophysiological experiments revealed that these beneficial effects were attributed to improved potassium retention and increased rate of Na+ pumping from the cell. The lack of correlation between rutin-induced changes in K+ and H+ fluxes suggest that rutin accumulation in the cytosol scavenges hydroxyl radical formed in response to salinity treatment thus preventing K+ leak via one of ROS-activated K+ efflux pathways, rather than controlling K+ flux via voltage-gated K+-permeable channels.

The Effects of Photoperiod, Glucose and Gibberellic Acid on Growth In Vitro and Flowering of Chenopodium Murale

In vitro culture of long-day plant Chenopodium murale L was established. The effects of photoperiod, glucose and gibberellic acid (GA3) on flowering and growth in vitro were investigated. Oscillatory changes of photoperiodic sensitivity were noticeable with regard to plant age. The plants induced at the phase of the 1st and the 3rd pair of leaves flowered to higher degree than those induced at the phase of 2nd pair. Plants induced at the phase of the 1st pair of leaves flowered to 17 % on 5 % glucose-containing medium and the addition of 5 mg dm-3 GA3 resulted in maximum flowering (43 %). Neither glucose nor GA3 were able to compensate for photoperiodic requirements for flowering. Hypocotyl growth was decreased and the 1st internode elongation and development of leaves were increased due to inductive photoperiodic conditions, as compared to non-inductive ones.

Alternative respiration of fungus Phycomyces blakesleeanus.

Respiratory characteristics of germinating spores, developing mycelium and mitochondria of the fungus Phycomyces blakesleeanus were investigated by means of oxygen Clark-type electrode. The effects of respiratory inhibitors and metabolic compounds on oxygen consumption were tested. It was demonstrated that P. blakesleeanus apart of cyanide-sensitive respiration, CSR, possess alternative respiration, (cyanide-resistant respiration, CRR) which is constitutive and whose capacity decreases during development. Maximum is observed for activated spores where CRR capacity is significantly greater than CSR. After treatment with antimycin A, a third type of respiration insensitive to antimycin A and low concentration of SHAM (sufficient for inhibition of CRR), but sensitive to cyanide and high concentration of SHAM, has been expressed.

Ca2+ and H+ Ion Fluxes near the Surface of Gravitropically Stimulated Phycomyces Sporangiophore

Abstract: The single‐celled fungus Phycomyces blakesleeanus forms vertically oriented sporangiophores from hyphae, which display a negative gravitropic response. Longitudinal growth and gravitropic bending of these sporangiophores were measured with noninvasive H+ and Ca2+ ion‐selective microelectrode measurements. The directions of H+ and Ca2+ fluxes, recorded at different locations of sporangiophores, were opposite when the sporangiophores were kept in vertical position. Ca2+ fluxes were in most experiments positive (efflux), while H+ fluxes were negative (influx). The direction of ion fluxes depended on developmental stage of sporangiophores and changed with gravistimulation.

Effect of darkness on growth and flowering of Chenopodium rubrum and C. murale plants in vitro

Chenopodium rubrum, a short-day plant, and C. murale, a long-day plant, were grown in vitro in continuous darkness. Control C. rubrum plants exposed to continuous darkness for 15 d at cotyledonary phase, did not flower, while 80 % of plants flowered on the medium with 5 % glucose and 10 mg dm−3 GA3. Control C. murale plants exposed to continuous darkness for 10 d at the age of 4th pair of leaves, did not flower, while GA3 (1 – 5 mg dm−3) stimulated flowering up to 65 %.

Filter strip as a method of choice for apoplastic fluid extraction from maize roots.

Apoplastic fluid was extracted from maize (Zea mays L.) roots using two procedures: collection from the surface of intact plant roots by filter paper strips (AF) or vacuum infiltration and/or centrifugation from excised root segments (AWF). The content of cytoplasmic marker (glucose-6-phosphate, G-6-P) and antioxidative components (enzymes, organic acids, phenolics, sugars, ROS) were compared in the extracts. The results obtained demonstrate that AF was completely free of G-6-P, as opposed to AWF where the cytoplasmic constituent was detected even at mildest centrifugation (200×g). Isoelectric focusing of POD and SOD shows the presence of cytoplasmic isoforms in AWF, and HPLC of sugars and phenolics a much more complex composition of AWF, due to cytoplasmic contamination. Organic acid composition differed in the two extracts, much higher concentrations of malic acid being registered in AF, while oxalic acid due to intracellular contamination being present only in AWF. EPR spectroscopy of DEPMPO spin trap in the extracts showed persistent generation of hydroxyl radical adduct in AF. The results obtained argue in favor of the filter strip method for the root apoplastic fluid extraction, avoiding the problems of cytoplasmic contamination and dilution and enabling concentration measurements in minute regions of the root.

Changes in Chenopodium rubrum Seeds with Aging

Abstract: We studied antioxidative system, germination, growth, and flowering in vitro in Chenopodium rubrum seeds of different ages. Peroxidase, superoxide dismutase, and catalase activity, as well as glutathione status, were determined in 2.5‐h imbibed seeds. Germination was tested under controlled conditions. Growth and flowering of plants were tested in vitro. The enzyme activities and glutathione content were higher in younger seeds. Germination declines with seed age. Plants derived from older seeds were smaller, and flowering percentage was lower compared to plants derived from younger seeds. Gibberellic acid reduced the difference in growth and flowering between plants derived from seeds different in age.

A New Model System for Investigation of Ionic Channels in Filamentous Fungi: Evidence for Existence of Two K+-Permeable Ionic Channels in Phycomyces blakesleeanus

Abstract: A technique was developed to obtain viable cytoplasmic droplets, enabling the formation of a gigaohm seal. Such cytoplasmic droplets can be used for characterization of plasma membrane ion channels from filamentous fungi by patch‐clamp technique. Two K+ ionic channels are characterized with a conductance of 43 pS and 74 pS.

Photoperiodic induction of flowering in Chenopodium rubrum L. might be controlled by an oscillatory mechanism

Summary Long-term recordings of bioelectric potential difference across intact Chenopodium rubrum L. plants have been performed by subjecting them to different conditions for flowering during 10 days. Switching the light-on/off caused transient changes of the potential difference and the photosynthetic process was the major generator of those transients. Besides these transients, with average amplitude of 32 mV and duration of 22 min, self-sustained oscillations of the potential difference were also observed predominantly in the light-on period. The form of the individual spontaneous transients was usually similar to the light-induced transients, lasting 10–15 min, with an average amplitude of 6–7 mV. As the age of the plant increases, the frequency of the oscillations increased to more than four spikes per hour for some induced plants. With induced plants the oscillations were more ordered and of higher frequency. The results obtained point to the possibility that a frequency-controlled oscillatory bioelectric mechanism might be one of the initial steps in flowering control.