PMC

Carotenoid Biosynthetic Pathway in Higher Plants.
The pathway shows the primary steps found in most plant species. Arabidopsis mutations, ccr2, lut1, lut2, lut5, aba1, and npq1, are shown in italics. βLCY, β-cyclase; βOH, β-hydroxylase; εLCY, ε-cyclase; εOH, ε-hydroxylase; NCED, 9-cis-epoxycarotenoid dioxygenase; NXS, neoxanthin synthase; VDE, violaxanthin deepoxidase; ZE, zeaxanthin epoxidase; Z-ISO, ζ-carotene isomerase.

Carotenoid Accumulation in Arabidopsis Mutants.
(A) Lutein levels are expressed as a percentage of the total carotenoid pool relative to the wild type. Data are the average and se of three to six biological replicates of leaf tissues from 3- to 5-week-old plants.
(B) HPLC chromatogram of etiolated tissues from the wild type, ccr1, and ccr2. N, neoxanthin; V, violaxanthin; L, lutein; cis-L, cis-lycopene isomers; cis-N, cis-neurosporene isomers; ζ-C, ζ-carotene.

SDG8 Targets the CRTISO Promoter and Alters Expression of Neighboring Genes.
(A) Expression of genes neighboring CRTISO in 4-week-old leaf tissues.
(B) Lutein levels in leaf tissues from transgenic lines (3 to 53 independent lines per transgenic) harboring pMDC32:CaMV35S-CRTISO and pPZP200:CRTISO-CRTISO are expressed as a percentage of the total carotenoid pool relative to the wild type. The average and se are given.
(C) Schematic diagram showing genes neighboring CRTISO, position of PCR amplicons, and CRTISO overexpression binary vectors.

Mutations in EFS/CCR1/SDG8 Impair Lutein Biosynthesis.
(A) Location of mutations in SDG8 resulting in premature stop codons (ccr1-1, 1-5, 1-6, and 1-7), a splice variant (ccr1-2), a residue change in the SET domain (ccr1-4), and T-DNA insertions (SALK_026642 and SALK_065480).
(B) SDG8 conserved domains include a Cys-rich zinc finger motif (CW domain) and the SET domain that is invariably preceded by an AWS (associated with SET) domain and followed by a Cys-rich post-SET domain.
(C) Lutein levels in leaf tissues from efs, SALK_065480, and ccr1-1 are expressed as a percentage of the total carotenoid pool relative to the wild type. The average of 3 to 10 plants and se are given.

Shoot Branching and Auxin Transport Are Altered in ccr1.
(A) Rosette and cauline branching. Rosette branches (R) excluding the main primary floral bolt (MB) and cauline branches (C) (see inset) were counted, and the average ± se (n = 5) are given.
(B) Representative images of single and double mutants.
(C) Rosette and cauline branching in reciprocal grafts. Genotypes are annotated as scion/rootstock with Columbia wild-type plants expressing a constitutive 35S:β-glucuronidase (GUS) marker. Averages ± se (n = 8 to 20 for grafts and 20 to 23 for control plants) are given. D refers to a double ccr1-1 × max4-1 mutant.
(D) Transport of (³H)IAA in inflorescence stem sections. Average ± se (n = 3 to 4 independent pools each of three sections) are given.
(E) IAA content in primary inflorescence stems of 30-d-old plants, including cauline leaves and branches but excluding siliques. Data are averages ± se of three pools of eight plants.

Analysis of H3K4 Methylation of Chromatin Surrounding the Carotenoid Isomerase.
(A) The position of PCR amplicons, CH1, CH2, CH3, and CH4, used to quantify H3K4 methylation associated with CRTISO. Primer sequences are given in Supplemental Table 2 online.
(B) and (C) The level of H3K4me3 (B) and H3K4me2 (C) in CRTISO chromatin is presented as a ratio of mutant to wild type, following normalization using a region of the housekeeping gene S-ADENOSYL METHIONINE SYNTHASE. The predicted means (on the untransformed scale) of three independent experiments are given, and error bars represent the least significant difference (GenStat; analysis of variance). If mutant and wild-type error bars do not overlap, then their corresponding means can be considered as statistically significantly different at the 5% level (P < 0.05). For comparison, the dashed line indicates the level of no change in expression

Gene Expression in ccr1 and efs.
Leaf and root issues were pooled from independent plants, and RT-PCR used to quantify gene expression levels from at least two biological replicates were determined in mutant lines and normalized to the wild type. Standard error bars are displayed (n = 4). Abbreviations are given in Supplemental Table 2 online.
(A) Gene expression of carotenoid biosynthesis, strigolactone biosynthesis, and auxin transport proteins in wild-type and ccr1-4 leaf tissues (10 d old).
(B) Relative expression levels of CRTISO and εLCY in 4-week-old leaves from the early flowering mutants efs and ccr1-1. For comparison, the dashed line indicates the level of no change in expression.
(C) Relative expression levels of CRTISO and εLCY in 8-week-old leaf tissues from six ccr1 alleles. The average transcript abundance from one biological replicate is displayed.
(D) Gene expression in roots from 4-week-old wild-type and ccr1-4 plants growing on MSO media.