Accumulation of plastid protein precursors under norflurazon-induced carotenoid deficiency and oxidative stress in barley

Young barley seedlings devoid of carotenoids were assayed for their ability to synthesize plastid heat and light stress proteins (HSP32, ELIP) under oxidative stress caused by norflurazon (NF). Dark-grown barley plantlets were grown from seeds imbibed in 10–4 M NF, an inhibitor of carotenoid biosynthesis, then, where indicated, subjected to heat stress (at 42 °C in the dark for 2 h). Thereafter the plantlets were either illuminated under a moderate high light flux (300 μmol·m–2·s–1) or in low light (30 μmol·m–2·s–1). Western blot analysis indicated that seedlings treated with NF and heat shock contained in comparison with control seedlings two protein bands that could be detected with antibodies against small plastid HSPs: a mature protein HSP32 and an additional band of 36 kDa, while heat-shock-treated seedlings in the absence of NF contained merely HSP32. The amount of 36-kDa protein increased with time of incubation under high light and after 24 h incubation its level exceeded that of HSP32. The data (immunological similarity, differences in the molecular masses, peptide mapping and the disappearance of the 36-kDa band after removal of NF) suggested a precursor-product relationship between HSP32 and the 36-kDa protein. This 36-kDa precursor always associated with the membrane fraction while the mature protein resided partially in membranes, but predominantly in the soluble fraction of the cells. Similar effects of NF were shown on the accumulation of the ELIP precursor. Thus, the accumulation of plastid protein precursors in NF-treated plantlets was shown both for the chloroplast HSP32, localized in stroma, and for ELIP localized in thylakoid membranes. A possible mechanism for the accumulation of protein precursors is discussed.