## 3 Results and Discussion
### The Synechocystis Z-ISO Is Required for Carotenoid Biosynthesis in Darkness In Vivo  
We next examined the role of Slr1599 in _Synechocystis_ carotenoid biosynthesis in vivo. A mutant lacking slr1599 was generated by the partial replacement of the open reading frame with an erythromycinomycin resistant cassette (Figure 4A). Under photoautotrophic conditions (constant illumination), the \(\Delta\)slr1599 mutant grew similarly to the WT, and the absorbance spectra of both strains were almost identical (Figure 4B). The production of \(\beta\)-carotene was used to indicate Z-ISO activity; \(\beta\)-carotene levels were similar in the WT and \(\Delta\)slr1599 strains grown under constant illumination, indicating that the non-enzymatic photo-isomerization of the 15,15' _cis_ double bond of 9,15,9'-tri-_cis_-\(\zeta\)-carotene can occur in the absence of Slr1599 (Figure 4C). This is analogous to the case with the _Synechocystis_\(\Delta\)_crtH_ mutant, where the loss of CRT-ISO does not result in a significant carotenoid defect in the light due to the photolability of the _cis_ double bonds of 7,9,7',9'-tetra-_cis_-lycopene, such that all-_trans_-lycopene is formed non-enzymatically [23]. Figure 3: Slr1599 isomerizes 9,15,9′-tri-_cis_-\(\zeta\)-carotene when produced in _E. coli._ (**A**) RP-HPLC analysis of carotenoids extracted from dark-grown _E. coli_ cells containing pAC-ZETApi and either empty pCDFDuetTm-1 (labeled PDS), pCDFDuetTm-1 containing _Synechocystis_ crtQ (PDS, ZDS), pCDFDuetTm-1 containing _Synechocystis_ str1599 (PDS, Slr1599), or pCDFDuetTm-1 containing the _Z-ISO_ gene (PDS, _ZmZ_-ISO). Eluted pigments were detected by monitoring absorbance at 425 nm. (**B**) RP-HPLC profile of carotenoids extracted from dark-grown _E. coli_ cells harboring empty pCDFDuetTm-1 (PDS), pCDFDuetTm-1 containing _Synechocystis_ crtQ (PDS, ZDS), pAC-ZETApi and pCDFDuetTm-1 containing both _crtQ_ and slr1599 (PDS, ZDS, Slr1599) or pAC-ZETApi and pCDFDuetTm-1 containing both _crtQ_ and the maize Z-ISO gene (PDS, ZDS, _ZmZ_-ISO). Eluted pigments were detected by monitoring absorbance at 400 nm. (**C**) Absorbance spectra of the numbered carotenoid species in panels (A) and (B). Peaks 1 and 8 = 9,15,9′-tri-_cis_-\(\zeta\)-carotene; peak 2 = alternative central _cis_-isomer of \(\zeta\)-carotene [44]; peak 3 = 9,9′-di-_cis_-\(\zeta\)-carotene; peak 4 = all-_trans_\(\zeta\)-carotene [44]; peaks 5–7 = _cis_-lycopene isomers [23]. _Synechocystis_ cannot grow in complete darkness even when supplemented with glucose due to its requirement for at least a short (\(\sim\)5 min) daily pulse of light [30]. The pulse of light that permits LAHG is not sufficient to photo-isomerize the _cis_-bonds in 7,9,7,\({}^{\prime}\),9\({}^{\prime}\)-tetra-_cis_-lycopene and so a \(\Delta\)_crtH_ mutant cannot produce carotenoids under these conditions [23]. We tested the \(\Delta\)sIr1599 mutant under the same LAHG conditions and observed an almost complete loss of \(\beta\)-carotene accompanied by a significant accumulation of \(\zeta\)-carotene (Figure 4C), showing that unlike the WT, the mutant is unable to convert 9,15,9\({}^{\prime}\)-tri-_cis_-\(\zeta\)-carotene to 9,9\({}^{\prime}\)-di-_cis_-\(\zeta\)-carotene in the absence of prolonged illumination. The defect in carotenoid biosynthesis in the \(\Delta\)sIr1599 strain under LAHG is reflected by the loss of carotenoid absorbance at \(\sim\)475 nm and the concomitant appearance of absorbance features in the 350-430 nm region of the absorbance spectrum due to the accumulation of 9,15,9\({}^{\prime}\)-tri-_cis_-\(\zeta\)-carotene (Figure 4B). We ruled out a polar effect of the disruption of slr1599 by the complementation of the \(\Delta\)slr1599 mutant; the _in trans_ expression of slr1599 or the _Arabidopsis Z-ISO_ gene (At1g10830 lacking its predicted 58 aa N-terminal chloroplast transit peptide) under the control of the native _psbAII_ promoter restored \(\beta\)-carotene biosynthesis under LAHG (Figure S1).