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
Abstract:
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; UltrastructureSource:
University of Belgrade, Faculty of Biology, 2012, 1-147URI
http://eteze.bg.ac.rs/application/showtheses?thesesId=43https://fedorabg.bg.ac.rs/fedora/get/o:2416/bdef:Content/download
http://vbs.rs/scripts/cobiss?command=DISPLAY&base=70036&RID=41541135
http://nardus.mpn.gov.rs/123456789/2013
https://radar.ibiss.bg.ac.rs/handle/123456789/2428