N. Wilhelmová

Leaf senescence and activities of the antioxidant enzymes

Senescence is a genetically regulated process that involves decomposition of cellular structures and distribution of the products of this degradation to other plant parts. Reactions involving reactive oxygen species are the intrinsic features of these processes and their role in senescence is suggested. The malfunction of protection against destruction induced by reactive oxygen species could be the starting point of senescence. This article reviews biochemical changes during senescence in relation to reactive oxygen species and changes in antioxidant protection.

Changes in antioxidative protection in bean cotyledons during natural and continuous irradiation-accelerated senescence

We employed continuous irradiation (CL) for induction of premature senescence caused by enhanced production of reactive oxygen species. As a model plant we used bean (Phaseolus vulgaris L. cv. Jantar) cotyledons because they have well defined and a quite short life span. Senescence of bean cotyledons induced by CL progressed more rapidly than natural senescence: the life span of CL cotyledons was 13 d compared to 16 d in controls (C). Chl content was significantly lower in 10- and 13-d-old CL plants than in C plants and the change with age was not statistically significant. Activities of all antioxidative enzymes declined either with senescence onset or during whole life span. Activity of antioxidative enzymes, except ascorbate peroxidase, was lower in CL plants compared to C plants. On the contrary, contents of non-enzymatic antioxidants β-carotene and ascorbate were higher in CL plants than in C plants. No significant difference, except in the youngest cotyledons, was observed in glutathione content.

Photosynthesis in leaves of Nicotiana tabacum L. infected with tobacco mosaic virus

In tobacco leaves inoculated with tobacco mosaic virus (TMV), changes in chlorophyll (Chl) and carotenoid contents, parameters of slow Chl fluorescence kinetics, i.e. the maximum quantum yield of photosystem (PS2) photochemistry Fv/Fm, the effective quantum yield of photochemical energy conversion in PS2 Φ2, ratio of quantum yields of photochemical and concurrent non-photochemical processes in PS2 Fv/F0, non-photochemical quenching (NPQ), and photochemical activities of isolated chloroplasts from systemically infected tobacco leaves were investigated. We compared two successive stages of infection, the first in the stage of vein clearing at 9th day post inoculation (dpi) and the second at 22nd dpi when two different regions, i.e. light- (LGI) or dark-green (DGI) islands in the infected leaf were apparent and symptoms were fully developed. These two different regions were measured separately. The Chl and carotenoid contents in infected leaves decreased with a progression of infection and were lowest in LGI in the second stage. Also the ratio of Chl a/b declined in similar manner. The maximum quantum yield of PS2 photochemistry Fv/Fm, was decreased in the following order: first stage, DGI, and LGI. The same is true for the ratio Fv/F0. The decrease of Φ2 in infected leaves declined as compared to their controls. On the contrary, NPQ increased in infected leaves, the highest value was found in the first infection stage. Photochemical activities of the whole electron transport chain in isolated chloroplasts dramatically declined with the progression of symptoms, the lowest value was in LGI. Similarly, but to a lesser extent, the activity of PS2 in isolated chloroplasts decreased in infected leaves. Generally, the most marked impairment of the photosynthetic apparatus was manifested in the LGI of infected leaves.

Photosynthesis and water relations in transgenic tobacco plants with T-DNA carrying gene 4 for cytokinin synthesis

Expression of the gene 4 for cytokinin synthesis in two clones of transgenic tobacco plants resulted in slightly higher(ca. 10 – 20 %) levels of endogenous cytokinins -zeatin, zeatin riboside and isopentenyladenosine. This was associated with changes in photosynthetic processes on chloroplast and leaf level. In comparison with nontransformed plantlets a higher degree of grana stacking was found in both clones of transgenic plants which indicated a higher proportion of photosystem 2 lightharvesting complex in thylakoids. This was supported by a decreased chlorophylla/b ratio in the separated grana fraction. The rate of leaf net photosynthetic rate (measured as CO2 uptake) wasca. 25 % higher in both clones than in nontransformed plants. Also the rates of dark and light respiration, and CO2 compensation concentration were higher in transgenic plants. On the other hand, parameters of water relations (water, osmotic and pressure potentials, stomatal and cuticular transpiration rates) were not significantly affected in transgenic tobacco plants.

Effects of exogenous nitric oxide on photosynthesis

Nitric oxide (NO) is an important signalling molecule with diverse physiological functions in plants. In plant cell, it is synthesised in several metabolic ways either enzymatically or nonenzymatically. Due to its high reactivity, it could be also cytotoxic in dependence on concentration. Such effects could be also mediated by NO-derived compounds. However, the role of NO in photosynthetic apparatus arrangement and in photosynthetic performance is poorly understood as indicated by a number of studies in this field with often conflicting results. This review brings a short survey of the role of exogenous NO in photosynthesis under physiological and stressful conditions, particularly of its effect on parameters of chlorophyll fluorescence.

High level of endogenous cytokinins in transgenic potato plantlets limits photosynthesis

Introduction of the gene for cytokinin synthesis into potato genome lead to a manifold increase in the level of cytokinins (zeatin, zeatin riboside, isbpentenyl-adenine, isopentenyladenosine) in plantlets grownin vitro.The increasing cytokinin level was associated with increasing tendency to teratoma formation, to decreasing leaf net photosynthetic rate and to increasing dark and light respiration rates and CO2 compensation concentration. During plantlet (or teratoma) ontogeny, net photosynthetic rate increased simultaneously with the decrease in cytokinin level. High level of endogenous cytokinins was associated also with lower photochemical activities of both photosystems in isolated chloroplasts.

Photosynthesis and protective mechanisms during ageing in transgenic tobacco leaves with over-expressed cytokinin oxidase/dehydrogenase and thus lowered cytokinin content

The content of cytokinins (CKs), the plant inhibitors of the final phase of plant development, senescence, is effectively controlled by irreversible degradation catalysed by cytokinin oxidase/dehydrogenase (CKX). In transgenic tobacco, denoted as AtCKX, with over-expressed CKX causing lowered CK content, we investigated changes in the time courses of chlorophyll (Chl) and xanthophyll (violaxanthin, antheraxanthin, zeaxanthin, neoxanthin, and lutein) contents. We also determined parameters of slow Chl fluorescence kinetics such as minimum Chl fluorescence yield in the darkadapted state F0, maximum quantum yield of PS2 photochemistry (Fv/Fm), maximum ratio of quantum yields of photochemical and concurrent non-photochemical processes in photosystem 2 (PS2), Fv/F0, non-photochemical quenching (NPQ), and effective quantum yield of photochemical energy conversion in PS2 (Φ2). We used three different developmental leaf stages, old, mature, and young, and compared this with time courses of these characteristics in leaves with natural CK levels. The parameters Fv/Fm, Fv/F0, and Φ2 were unchanged during ageing in AtCKX plants in contrast to control ones where a significant decrease in old leaves was found. In control plants F0 increased during ageing, but in the oldest leaf a considerable decrease was observed. This could indicate progressive damage to PS2 reaction centres and then detachment and rapid degradation of Chl. This is in agreement with time course of Chl content. NPQ decreased with age and was similar in both plant types. We observed a decline of xanthophyll contents in the oldest leaves in both plant types, but the contents were enhanced in AtCKX compared to control plants, especially of neoxanthin. The higher xanthophyll contents in the transgenic plants contribute to a better photoprotection and the fluorescence parameters indicated that photosynthetic apparatus was in better condition compared to control and it consequently postponed the onset of leaf senescence.

Changes in French bean cotyledon composition associated with modulated life-span

The onset of Phaseolus vulgaris L. cotyledon senescence and its characteristics were modulated by irradiance (higher or lower than standard) and by epicotyl decapitation. The cotyledon life-span of 16 d was not influenced by irradiance while decapitation prolonged the life-span to 28 d. The fresh mass of cotyledons, an indicator of organ viability, decreased in a similar manner in all non-decapitated plants, though it was relatively slower in plants grown under a low irradiance (LI). Three days after decapitation the fresh mass of cotyledons increased by one third, a slight decrease was observed on the 21st d, and it lasted until the end of the life span. Deducing from the fall of chlorophyll (Chl) concentration expressed per unit protein, senescence started after the 10th day in non-decapitated plants. Decapitation postponed the onset of senescence until the 21st day. Expression of Chl amount per unit dry mass did not detect any changes in LI plants, hence this parameter can not be used for the assessment of senescence. The measurements of Chl a and b concentrations indicated that the light-harvesting complexes (LHCs) proliferated during ageing and were rapidly destroyed at the onset of senescence. Changes of the concentrations of carotenoids supported the hypothesis of free radicals involvement in senescence. The bean cotyledons responded to free radical production induced under higher irradiance by increased β-carotene synthesis. Oxidative damage to galactolipids during senescence was documented by fluorescence measurements. The changes in cotyledon composition were correlated to morphologic changes observed by electron microscopy.

Zinc induces DNA damage in tobacco roots

We applied the alkaline version of the single-cell gel electrophoresis (comet assay) to seedlings of heterozygous tobacco (Nicotiana tabacum L. var. xanthi) treated with zinc acetate dihydrate (20 to 80 mM Zn2+ for 2 h or 2 to 12 mM Zn2+ for 24 h). A dose dependent increase in DNA damage expressed by the tail moment values were observed in nuclei isolated from the roots after 2 and 24 h Zn2+ treatments. In contrast, Zn2+ did not induce significant DNA damage to leaf nuclei, with the exception of 10 or 12 mM Zn2+ for 24 h. Somatic mutations, identified as dark green, yellow, and dark green/yellow double sectors on the pale green tobacco leaves were not detected after any Zn2+ treatments. The accumulation of Zn in roots and shoots was determined by inductively coupled plasma optical emission spectrometry and the Zn content in roots was about three times higher than in shoots.

Antioxidant protection during ageing and senescence in transgenic tobacco with enhanced activity of cytokinin oxidase/dehydrogenase

We studied changes in physiological parameters of whole leaves and in antioxidant protection of chloroplasts during ageing and senescence of tobacco (Nicotiana tabacum L. cv. Samsun NN) leaves with enhanced cytokinin oxidase/dehydrogenase activity (CKX) or without it (WT). Old leaves of CKX plants maintained higher pigment content and photosystem 2 activity compared to WT leaves of the same age. Chloroplasts of old CKX plants showed better antioxidant capacity represented by higher superoxide dismutase, dehydroascorbate reductase and glutathione reductase activities.