Kаrаktеrizаciја prоcеsа prоgrаmirаnе ćеliјskе smrti i sеnеscеnciје u listоvimа Nicotiana tabacum L. in vitro
Characterisation of programmed cell death and senescence in leaves of Nicotiana tabacum L. in vitro
Doctoral thesis (Published version)
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Leaf senescence constitutes the final stage of leaf development that results in the coordinated degradation of macromolcules and the subsequent nutrient relocation to other plant parts. Leaf senescence involves an integrated action at the cellular, tissue, organ and organism levels under the control of a highly regulated genetic program. During senescence, leaf cells undergo orderly changes in cell structure, metabolism and gene expression leading to cell death, recognized as a form of programmed cell death (PCD). In vitro grown Nicotiana tabacum L. has been chosen as a model plant system, since a single plant provides a large set of leaves in which there is a gradation of senescence. The dynamics of ultrastructural changes in leaf mesophyll cells, as well as metabolic, biochemical and molecular changes during leaf senescence were investigated. We determined the time sequence of senescence-related events in mesophyll cells during physiological ageing of leaves. Typical symptoms of autophagic PCD were detected in senescing leaves using cytological analysis and TUNEL assay. We showed that PCD-specific nuclear DNA fragmentation occurs during natural tobacco leaf senescence but that it starts early in leaf development, thus preceeding ultrastructural changes such as chloroplast degradation and chromatin condensation. Chlorophyll and soluble protein content were identified as reliable biomarkers for estimating the relative age of a particular leaf along the main plant axis. Using HPLC/MS more than 20 different cytokinins were identified and quantified in tobacco leaves. The correlation of senescence with decline in total cytokinin content and the levels of trans-Z was found. The progression of leaf senescence correlated with a gradual decrease in endogenous levels of both IAA and ABA. Immunohistological analysis showed that high levels of cytokinin benzyladenine accelerate senescence thus inducing PCD. The activities of different antioxidative enzymes were analyzed during physiological ageing of leaves. Progression of senescence is correlated with decrease in CAT activity and a concomitant increase in POD activity. Primary antioxidative protection in the course of ageing is provided by SOD, whereas POD are implicated in defense against detrimental ROS action in an initial phase of senescence. Molecular analysis of leaf senescence has revealed activation of genes encoding enzymes involved in conversion and remobilization of degradation products in senescing leaves. Quantitative RT-PCR showed that the expression of cysteine proteases responsible for amino acid remobilization, typically associated with leaf senescence and not found in other forms of PCD, was induced in senescent leaves. Induced expression of SAGs involved in nitrogen mobilization in senescing leaves, Gln1-3 и GDH2, contiguous with decrease in GS2 expression indicated that, during senescence, ammonia assimilation is progressively shifted from the chloroplasts to the cytosol of leaf mesophyll cells.
Keywords:Nicotiana tabacum L.; qRT-PCR; Antioxidative enzymes; Cytokinins; Leaf senescence; Nitrogen remobilization; Programmed cell death; TUNEL assay; Ultrastructure
Source:University of Belgrade, Faculty of Biology, 2012, 1-147