Guanylyl cyclase activity during photoperiodic flower induction in Pharbitis nil

It is known that the level of cGMP is modulated in plant cells in response to a number of stimuli but intracellular events dependent on cGMP metabolism are not clear. Guanylyl cyclases (GCs) are enzymes which are responsible for synthesis of cGMP in eukaryotic and prokaryotic cells. To collect evidence for the participation of cGMP in light signal transduction we isolated enzyme with guanylyl cyclase activity from Pharbitis nil and analysed its level and activity during photoperiodic flower induction. Soluble proteins were isolated from seedlings of a model short-day plant P. nil, partly purified and identified by in vivo and in vitro enzyme assay. In green plants enzyme activity amounted to 484 nmol cGMP/min/mg protein, whereas in etiolated plants it was three times lower (158 nmol cGMP/min/mg protein). Analyse cyclase consists of a single polypeptide of Mr 40 kDa. In order to determine if changes in guanylyl cyclase activity occurred in response to a long, inductive night, we measured enzyme activity in 4-h intervals and observed its increase at 4, 8 and 16 h of darkness. This pattern also fits well with changes in the endogenous cGMP level during a 16 h long flower inductive night. Immunocytochemical analysis confirmed these observations and revealed that changes in the GC level during light/dark conditions appeared. During 16 h long inductive night the strongest signal was observed in cotyledons after 4 and 16 h of the darkness. A high level of fluorescence was generally distributed in mesophyll, however, it was also observed in guard cells. Staining was apparently absent in the veins and cotyledon body. Furthermore, the location inside the cell was analysed. The protein was immunolocalized preferentially in the cytosol, chloroplasts and peroxysomes. Taken together, these data demonstrate in Pharbitis nil the presence of an enzyme which is able to convert GTP to cGMP. Because its level and activity are affected by light we believe that GC/cGMP play a substantial role in light/dark dependent process in plants, such as photoperiodic flower induction.