Cytokinin promotes morphological and physiological processes including the tetrapyrrole biosynthetic pathway during plant development. Only a few steps of chlorophyll (Chl) biosynthesis, exerting the phytohormonal influence, have been individually examined. We performed a comprehensive survey of cytokinin action on the regulation of tetrapyrrole biosynthesis with etiolated and greening barley seedlings. Protein contents, enzyme activities and tetrapyrrole metabolites were analyzed for highly regulated metabolic steps including those of 5-aminolevulinic acid (ALA) biosynthesis and enzymes at the branch point for protoporphyrin IX distribution to Chl and heme. Although levels of the two enzymes of ALA synthesis, glutamyl-tRNA reductase and glutamate 1-semialdehyde aminotransferase, were elevated in dark grown kinetin-treated barley seedlings, the ALA synthesis rate was only significantly enhanced when plant were exposed to light. While cytokinin do not stimulatorily affect Fe-chelatase activity and heme content, it promotes activities of the first enzymes in the Mg branch, Mg protoporphyrin IX chelatase and Mg protoporphyrin IX methyltransferase, in etiolated seedlings up to the first 5 h of light exposure in comparison to control. This elevated activities result in stimulated Chl biosynthesis, which again parallels with enhanced photosynthetic activities indicated by the photosynthetic parameters F(V)/F(M), J (CO2max) and J (CO2) in the kinetin-treated greening seedlings during the first hours of illumination. Thus, cytokinin-driven acceleration of the tetrapyrrole metabolism supports functioning and assembly of the photosynthetic complexes in developing chloroplasts.

SummaryThe barley line albostrians exhibits a severe block in chloroplast development as a result of a mutationally induced lack of plastid ribosomes. White leaves of this mutant contain undifferentiated plastids, possess only traces of chlorophyll (Chl), and are photosynthetically inactive. Chl de®ciency, combined with a continuous heme requirement, should lead to drastic changes in the tetrapyrrole metabolism in white versus green leaves. We analyzed the extent to which the synthesis rate of the pathway and the porphyrin distribution toward the Chl-and heme-synthesizing bifurcation is altered in the white tissue of albostrians. Expression and activity of several distinctively regulated enzymes, such as glutamyl-tRNA glu reductase, glutamate 1-semialdehyde aminotransferase, Mg-and Fe-chelatase, and Chl synthetase, were altered in white mutant leaves in comparison to control leaves. A drastic loss in the rate-limiting formation of 5-aminolevulinate and in the Mg-chelatase and Mg-protoporphyrin IX methyltransferase activity, as well as an increase in Fe-chelatase activity, accounts for a decrease in the metabolic¯ux and the re-direction of metabolites. It is proposed that the tightly balanced control of activities in the pathway functions by different metabolic feedback loops and in response to developmental state and physiological requirements. This data supports the idea that the initial steps of Mg-porphyrin synthesis contribute to plastid-derived signaling toward the nucleus. The barley mutant albostrians proved to be a valuable system for studying regulation of tetrapyrrole biosynthesis and their involvement in the bi-directional communication between plastids and nucleus.