Plastid-membrane-associated polyamines and thylakoid transglutaminases during etioplast-to-chloroplast transformation stimulated by kinetin

The amounts of polyamines (PAs) bound to etioplast membranes varied during chloroplast development in cucumber cotyledons ( Cucumis sativus L. cv. Racibór). Putrescine (PU) and spermidine (SD) levels increased in the early greening stage (6 h of light exposure) but decreased in the late greening stage (24 h) in the thylakoid-enriched fraction. In the highly enriched PSIIα fraction, the trend of changes in the amount of bound PAs was different: levels of SD and spermine (SM) increased in the late stage. In both fractions, their levels were additionally increased by kinetin treatment. In the presence of exogenous protein transglutaminase (TGase) substrate ( N ', N '-dimethylcasein) and 5 m M Ca²⁺, kinetin initially caused a marked increase in thylakoid transglutaminase (ThylTGase) activity (6 h), followed by a decrease at the end of greening. The radiometric assay showed that PU and SM binding to thylakoid proteins was very low, while SD binding was seven to eight times higher. Kinetin increased SD conjugation in the early greening stage by about 36%. When chloroplast membranes were fully organized, ThylTGase activity decreased. In etioplast membranes and during the early greening stage, the 77-kDa and 30-kDa bands were mainly immunodetected with antibodies raised against the animal TGase, which were in general slightly stronger for kinetin-treated than the control samples. At the end of greening, the level of 77-kDa ThylTGase dramatically decreased. ThylTGase activity was found to be Ca²⁺ dependent. PAs conjugated via ThylTGase, in addition to the PAs bound by all possible types of linkage, could represent an important component of the mechanism of stimulation of etioplast-to-chloroplast transformation by kinetin.