Induzione delle Heat shock proteins (Hsp), Programmed Cell Death (PCD) e analisi proteomica di campioni vegetali sottoposti a stress abiotici

Abstract

Plants are constantly under stress, this may negatively affect their development and growth. The effects of stress can be visible as the reduction in rate of growth, and not evident as changes in gene expression and cellular metabolism. Many crops (especially cereals) are sensitive to salts during the development, retarding germination and plant development, and later affecting seed and/or biomass production. In this work, the effects of abiotic stress, particularly salts stress, heavy metal stress, and pollution, have been investigated, respectively in barley (hordeum vulgare), in Bryophyta (Leptodictyum riparium, Conocephalum conicum, Leptodon smithii ), and in holm (Quercus ilex). The Heat Shock Proteins (HSP) play a primary role in the response to stress, counteracting the toxic effects on proteins and enzymes, protecting them from misfolding and proteolytic pathways. Western blotting analysis in roots of barley plant, grown in presence of 150 mM NaCl, showed that 6 h after the salt stress the levels of the HSP70 increased, and after 48-72 h the Hsp70 levels returned to the initial levels. The salt stress, provokes damages to structure and organisation, in fact in barley after 8 hours of salt stress DNA laddering was observed. This phenomenon can be ascribed to the increase of specific endonuclease activity, causing the formation of SNA oligonucleosomal fragments that differed less than 200 bp, typical of Programmed Cell Death (PCD). Western blotting analysis of the effects of heavy metal stress on samples of Leptodictyum riparium and Conocephalum conicum, showed a time and dose dependent increase in the levels of Hsp70, while the effects of anthropic pollution on Leptodon smithii showed that Hsp70 levels increased in the samples collected in urban areas exposed to high levels of pollution. The effects of salt-stress on the growth and protein pattern in the roots of two barley cultivars, Nure and Tidone, were investigated. Salt stress was induced with different salt concentration (50, 150 and 300 mM NaCl) in hydroponic cultures for different periods of time (8 and 12 hrs). Root samples were analyzed by to two-dimensional SDS gel electrophoresis. Protein spots differing in abundance were identified, suggesting differences in response to salt stress in the two cultivars. After 8 h of exposure to 300 mM NaCl, Nure showed noticeable changes in protein patterns with respect to Tidone. Therefore it seems that Nure can adapt to increased salt stress by selective changes in protein expression in the roots. Some of the protein spots involved in stress response by mass spectrometry (MALDI-TOF) have been identified, and among the protein up and down-regulated it has been identified BLT63, an elongation factor. The down-regulation of this protein in Tidone and the up-regulation of this protein in Nure, during the salt stress, could explain the differences of growth between these two different cultivars.