Characterisation of antioxidants in photosynthetic and non‐photosynthetic leaf tissues of variegated Pelargonium zonale plants

Abstract

Hydrogen peroxide is an important signalling molecule, involved in regulation of numerous metabolic processes in plants. The most important sources of H2 O2 in photosynthetically active cells are chloroplasts and peroxisomes. Here we employed variegated Pelargonium zonale to characterise and compare enzymatic and non-enzymatic components of the antioxidative system in autotrophic and heterotrophic leaf tissues at (sub)cellular level under optimal growth conditions. The results revealed that both leaf tissues had specific strategies to regulate H2 O2 levels. In photosynthetic cells, the redox regulatory system was based on ascorbate, and on the activities of thylakoid-bound ascorbate peroxidase (tAPX) and catalase. In this leaf tissue, ascorbate was predominantly localised in the nucleus, peroxisomes, plastids and mitochondria. On the other hand, non-photosynthetic cells contained higher glutathione content, mostly located in mitochondria. The enzymatic antioxidative system in non-photosynthetic cells relied on the ascorbate-glutathione cycle and both Mn and Cu/Zn superoxide dismutase. Interestingly, higher content of ascorbate and glutathione, and higher activities of APX in the cytosol of non-photosynthetic leaf cells compared to the photosynthetic ones, suggest the importance of this compartment in H2 O2 regulation. Together, these results imply different regulation of processes linked with H2 O2 signalling at subcellular level. Thus, we propose green-white variegated leaves as an excellent system for examination of redox signal transduction and redox communication between two cell types, autotrophic and heterotrophic, within the same organ.

Isolation of high-quality RNA from recalcitrant leaves of variegated and resurrection plants

“…In the present study, we aimed to evaluate and establish the optimal protocols for RNA extraction from challenging, phenolic-rich variegated leaves of P. zonale and desiccated leaves of R. serbica, two model systems in plant physiology with unsequenced genomes (Veljovic Jovanovic et al 2006;Tcherkez et al 2012;Abadie et al 2015;Vidović et al 2016a).…”

“…1) has proven to be a suitable model system to examine the carbon and nitrogen allocation between the source (photosynthetically active tissue) and the sink (non-photosynthetically active tissue) under different environmental stimuli providing the equal microenvironment conditions within the same organ, unlike common shoot/root relation studies (Vidović et al 2015). Moreover, we have recently established this system as an excellent model for studying the link between intensive photosynthetically dependent ROS generation in green and antioxidative response in white leaf sectors of P. zonale (Vidović et al 2016a, b). Both tissue types, particularly photosynthetically active one, contain high amounts of polyphenols including p-hydroxybenzoic and p-coumaric acids, catechin, kaempferol, and quercetin (Vidović et al 2015).…”

Evaluation of procedures for assessing anti- and pro-oxidants in plant samples

“…GSH, reduced glutathione; GSSG, oxidized glutathione. The GSH and GSSG contents were determined according to Vidović et al 46 …”

“…As an alternative to isolating subcellular fractions, a recently developed immunocytochemical method in combination with transmission electron microscopy has enabled the examination of the distribution of antioxidants such as ascorbate or glutathione at the subcellular level in situ. 26,[44][45][46] When combined with quantitative analyses of leaf extracts, this technique allows calculations of ascorbate or glutathione concentrations in plastids, mitochondria, peroxisomes, vacuoles, cytosol and apoplast. 47 It should be noted that the primary antibodies against ascorbate and glutathione do not differentiate between their reduced and oxidized forms.…”

“…85 Dehydroascorbate can be measured using o-phenylenediamine derivatization to yield a uorescent product, allowing high sensitivity HPLC detection. 40,42,46 A frequently applied approach is to reduce DHA with thiol compounds, such as dithiothreitol (DTT) or GSH and measure it as ASC. 37,71,75,78 The weak point of this approach is optimizing sample incubation with DTT.…”