This trend fits well to the observation that both TFs are strongly upregulated in the night and extended night, while bZIP11 is downregulated under these conditions (see Figure 5 figure supplement 3).
The expression of all three genes increased steadily during a 4 hr extended night treatment, but the increase was delayed in the bzip63 mutant as compared to the wt.
The wt construct showed strong phosphorylation in the light (bands 6 and 7) and an even stronger phosphorylation in extended night (band 8), as well as reduced phosphorylation in the presence of sucrose (bands 1, 2, and 4).
However, we could use the line for Phos-tag gels and we here we saw indeed a striking effect: Compared to the triple mutant, that did not show any change in the phosphorylation pattern in response to starvation, the S294/S300 double mutant did still show a strong shift in the extended night.
The extended night treatment represents a starvation condition for the plant.
Mr. Kofman seems to be aiming at low-brow absurdism, something between Richard Foreman's Ontological-Hysteric Theater and an extended "Saturday Night Live" skit, and the dialogue works overtime trying to be enigmatic.
After six hours of extended night (EN), we observed a clearly impaired induction of ASN1 (DIN6) and ProDH.
To further substantiate the relevance of S29 phosphorylation in vivo, we compared the phosphorylation patterns of the S29/S294/S300A mutant with a S294/S300A mutant in the light and after extended night treatments in the genomic complementation lines.
In the genomic complementation line, the most phosphorylated form of bZIP63 was the strongest band under extended night conditions, which might be a result of the different bZIP63 amounts in relation to the endogenous kinase(s), and likely better reflects the phosphorylation state of the endogenous protein.
This could be S29, as we showed with a new S294/300A mutant that S29 is phosphorylated in extended night, but not in the light.
