Supplemental Data

Files in this Data Supplement:

• Supplemental Figure 1 - Figure S1. Southern blot analysis of P. patens genomic DNA. The blot was hybridized with the DNA binding domain of PpGlk1. There is an internal HindIII site in the PpGlk1 gene.
• Supplemental Figure 2 - Figure S2. Northern blot analysis of PpGlk gene expression in protonema during a 24 hour diurnal cycle. Protonemal tissue was harvested at the times indicated during the 8th day after fragmentation and regeneration. Blots were hybridized with gene-specific fragments of A) PpGlk1 and B) PpGlk2 . Hybridization signal was quantified with a phosphorimager, standardized to the level of ethidium bromide fluorescence of the 26S ribosomal RNA (measured using a Kodak EDAS-290 camera) and presented as relative arbitrary units.
• Supplemental Figure 3 - Figure S3. Generation of PpGlk1:uidA and PpGlk2:uidA fusion lines. A) Schematic diagram to show the structure of PpGlk1, PpGlk2 and the PpGlk:uidA transformation constructs. The sequences flanking PpGlk genes are shown with black lines and coding regions with boxes (red box = PpGlk DBD; orange box = PpGlk GCT box; blue box = uidA and grey box = kanamycin resistance cassette ? 35S:nptII). B) Schematic representation of endogenous PpGlk loci showing the DBD and GCT boxes as in (A). Sph1 restriction sites are marked by green triangles and restriction fragment sizes are indicated in bp. The position of fragments used for hybridization in (C) is marked below the gene with purple and orange bars. These regions are conserved between the two genes and thus either PpGlk1 or PpGlk2 fragments can be used to detect both. C) Southern blot analysis of the PpGlk loci in PpGlk1:uidA and PpGlk2:uidA lines. SphI-genomic digests of wild-type (WT), PpGlk1:uidA-1 to -3 and PpGlk2:uidA-1 to -3 were hybridized first with the PpGlk1 5? region [purple bar in (B)] (left hand blot). The 8.5 kb PpGlk1 fragment shifted to 12 kb in the PpGlk1:uidA lines and the 1.4 kb PpGlk2 fragment shifted to 5.2 kb in the PpGlk2:uidA lines, indicating gene replacement in both cases. The blots were subsequently hybridized with the PpGlk1 3? region [orange bar in (B)] (right hand blot). Since the SphI sites are located downstream of uidA and 35S:nptII in both constructs, PpGlk1 and PpGlk2 fragments should be the same size as wild-type even after gene replacement. However, in PpGlk2:uidA-1, PpGlk2:uidA-2 and all three PpGlk1:uidA lines, the hybridization pattern indicated the presence of repeats consisting of 35S:nptII and the 3? PpGlk homologous sequences. This prediction was confirmed by hybridization to different regions of the construct, by PCR analysis and by repeated Southern blot analysis using NcoI- and EcoRI- genomic digests (data not shown). D) Schematic representation of PpGlk:uidA loci, deduced from Southern blots in (C). DNA sequences are indicated by lines and boxes as in (A).
• Supplemental Figure 4 - Figure S4. Generation of Ppglk single and double mutants. A) Schematic diagram to show the structure of PpGlk1, PpGlk2 and the targeting constructs for both genes (grey box = antibiotic resistance cassette; red box = PpGlk DBD; orange box = PpGlk GCT box). B) Schematic representation of endogenous PpGlk loci showing the DBD and GCT boxes as in (A). NcoIrestriction sites are marked by green triangles and restriction fragment sizes are indicated in bp. The position of the fragment used for hybridization in (C) is marked below the genes with a purple bar. This region is conserved between the two genes and thus either PpGlk1 or PpGlk2 fragments can be used to detect both. C) Southern blot analysis of the Ppglk loci in Ppglk single mutants and double mutants. NcoI digests were hybridized to a PpGlk1 fragment [purple bar in (B)]. In all three Ppglk1 lines, the 1.1 kb PpGlk1 fragment was shifted to 1.8 kb due to gene replacement. Two other fragments were observed at 2.3 kb and 4.5 kb. These fragments corresponded to the sizes of construct repeats and construct-vector repeats respectively. The 2 kb PpGlk2 fragment was the same as wild-type in Ppglk1 mutant lines but was shifted to 4 kb in Ppglk2 mutant lines. The 3 kb fragment in Ppglk2-1 and the 3.3 kb fragment in Ppglk2-2 and Ppglk2-3 corresponded to the expected sizes for construct-vector repeats and construct repeat(s) respectively. The Ppglk1-1;glk2-4 double mutant had the same hybridization pattern as Ppglk1-1 except that the PpGlk2 fragment was shifted to 4 kb in the double mutant. Similarly, in the Ppglk1-5;glk2-1 double mutant, the 3 kb and 4 kb fragments were as in Ppglk2-1 but the PpGlk1 fragment was shifted to 1.8 kb. D) Schematic representation of Ppglk1 and Ppglk2 loci deduced from Southern blots in (C). DNA sequences are indicated by lines and boxes as in (A). Blue lines indicate the presence of vector plasmid.
• Supplemental Figure 5 - Figure S5. Thylakoid stacking in Ppglk double mutants. Representative transmission electron micrographs of WT (A, C, E), Ppglk1-5;glk2-1 double mutant (B, D) and Ppglk1-1;glk2-4 double mutant (F) chloroplasts. A) and D) correspond to Figs. 5A & B respectively in the main paper. The number of appressed membrane layers in each region is indicated in yellow. The average number of layers per stack is 6.8 ± 2.14 in WT (n = 84) and 3.35 ± 0.72 in double mutants (n = 106). Scale bar = 1 μm
• Supplemental Figure 6 - Figure S6. PpGlk1 transgenes in Arabidopsis. BamHI digests of DNA from Atglk1;glk2 double mutants, and from double mutant lines transformed with 35S:PpGlk1, hybridized with a fragment of the PpGlk1 gene that does not hybridize to the Arabidopsis GLK genes. Plants 8.2, 8.15 and 8.14 are segregants in the T3 or T4 generation of transformed line 8. Two transgenes have segregated, one with 8.2 and one with 8.15. 8.14 has segregated away from both transgenes. Plants 12.4 and 12.3 represent a similar scenario in a second independently transformed line. In this case, 12.4 contains the transgene whereas 12.3 does not.
• Supplemental Data 1
• Supplemental Data 2