Table S1 Primers used in this paper.

Gene name / 5’-Forward primer-3’; 5’-Reverse primer-3’
PSY1 / GTGACCGAGCTCAGCCAG; TTAATCGGAAGGCAGATGCT
PSY2 / GGGCGTTGCGCATCTAGA; CAAGAAATCGTGCGAAATCA
PSY3 / TGGCTCTGCCTAAAGCTTACTACA; GCTAGAGCAGTGCCTCACTGAA
ZEBRA2 / CCGTCATGTTCGGCTTCTC; AAACTGAGGCTTTGACACCC
PDS / TCCTCTTGTTTTTGCAGACG; ATTTAAGGGTGCAGGCAATG
ZDS / AATGGAGGCAATGGGATACA; CCAGCATGTGTCATTTGAGG
LycB / CCTCGTCCAGTACGACAAGC; ATGCAGATCCTCACCTCACC
LycE / CAGCATCTGGTAGGCTTCTA; ACCATTCCTCCTCGTATAC
VxDE / ATGCCAAAATCTCTGCTCGT; ACAAACCGCTGTATGGCTGT
HEMA / CGCTATTTCTGATGCTATGGGT; TCTTGGGTGATGATTGTTTGG
POR / TGTACTGGAGCTGGAACAACAA; GAGCACAGCAAAATCCTAGACG
YGL1 / TCTTGGTGCGAGCTACATTG; GCTTGCCTGAACTGAAAAGG
CAO / GATCCATACCCGATCGACAT;CGAGAGACATCCGGTAGAGC
NYC1 / CATGCAACACCAACAAAAGG; CGGATTCTATCAGCCTCTGC
CAB / CCGTCAACAACAACGCCTGG; ACATCATCAACTTCTTCATC
RS / CTAACTAACTACGTGGCTATG; CGATGCTTGATCTTAGCTTA
FZ / AAAGGACATAACCTTGCAAG; AGTTTTCCTATTGAACCGTG
SA / GATTTGAGGAGTGGTATCTC; TGTGTTTGTGGATCTGACTC
ACTIN1 / ACATCGCCCTGGACTATGACCA; GTCGTACTCAGCCTTGGCAAT
ProZEB2 / AAGCTTGTTCCAGCACAAGACCCATT(HindIII);GTCGACGAGCTGGGTGGGGTTTTTAT(SalI)
RNAi / GGTTCATTTCCACCTACCTG; CCAACTGGATAGTTAATTCCC

Table S2 Relative abundance of individual carotenoid in the light-grown complemented (Comp.) line or zebra2-1 mutant compared with the wild type (WT) plant. The relative carotenoid abundance was shown as the ratio of peak area (between the Comp. and WT or between zebra2-1 mutant and WT) which was derived from the HPLC chromatogram recorded at 430 nm of the light-grown WT, zebra2-1 mutant and Comp. lines.

Compound / Peak area ratio
Comp./WT / zebra2-1/WT
neoxanthin / 0.85±0.13 / 1.07±0.21
violaxanthin / 0.83±0.11 / 0.83±0.16
lutein / 0.86±0.12 / 0.46±0.09
α-carotene / 0.78±0.08 / 0.30±0.05
β-carotene isomer 1 / 0.82±0.24 / 1.02±0.17
β-carotene isomer 2 / 0.83±0.15 / 1.33±0.13
Zeaxanthin / 0.77±0.14 / 3.82±0.11

Table S3 Relative abundance of individual carotenoid in the etiolated complemented (Comp.) line or zebra2-1 mutant compared with the wild type (WT) line. The relative carotenoid abundance was shown as the ratio of peak area (between the Comp. and WT or between the zebra2-1 mutant and WT) which was derived from the HPLC chromatogram recorded at 430 nm of etiolated WT, zebra2-1 mutant, and Comp. lines.

Compound / Peak area ratio
Comp./WT / zebra2-1/WT
neoxanthin / 1.00±0.12 / -
violaxanthin / 1.00±0.08 / -
lutein / 0.80±0.11 / -
ζ-carotene isomer 1 / 1.01±0.12 / ∞
ζ-carotene isomer 2 / 1.00±0.13 / 44.67±3.12
prolycopene / - / ∞
cis-lycopene isomer 1 / 1.09±0.20 / 11.68±1.08
neurosporene isomer 1 / 0.98±0.13 / ∞
cis-lycopene isomer 2 / 0.93±0.18 / 18847.48±1465.43
neurosporene isomer 2 / 1.05±0.19 / 11844.81±1556.98

Table S4 Relative abundance of individual carotenoid in mature stems from the complemented (Comp.) line or zebra2-1 mutant compared with the wild type (WT) line. The relative carotenoid abundance was shown as the ratio of peak area (between the Comp. and WT or between the zebra2-1 mutant and WT) which was derived from the HPLC chromatogram recorded at 430 nm of mature stems from WT, zebra2-1 mutant, and Comp. lines.

Compound / Peak area ratio
zebra2-1/WT
ζ-carotene isomer 1 / 8.87±0.96
ζ-carotene isomer 2 / 1081.17±141.11
prolycopene / ∞
cis-lycopene isomer 1 / 27±0.07
neurosporene isomer 1 / 37.34±4.82
cis-lycopene isomer 2 / ∞
neurosporene isomer 2 / 108.11±9.50

Table S5 Relative abundance of individual carotenoid in leaves from the zebra2-1 mutant under field light condition or shaded compared with the wild type (WT) line. The relative carotenoid abundance was shown as the ratio of peak area (between the zebra2-1 mutant and WT or the shaded zebra2-1 mutant and WT) which was derived from the HPLC chromatogram recorded at 460 nm of leaves from the WT and the zebra2-1 mutant (either under field light condition or shaded) plants.

Compound / Peak area ratio
zebra2-1/WT / shaded zebra2-1/WT
neoxanthin / 0.70±0.16 / 1.01±0.35
violaxanthin / 0.53±0.08 / 0.83±0.13
lutein / 0.23±0.09 / 0.38±0.12
β-carotene isomer 1 / 0.94±0.09 / 0.99±0.22
β-carotene isomer 2 / 0.51±0.08 / 0.89±0.14
zeaxanthin / 12.21±0.14 / 8.65±0.09

Fig. S1 Phenotypes of the wild type (WT), zebra2-1 mutant, complementary transgenic (Comp.) and RNAi transgenic (RNAi) seedlings at tillering stage.

Fig. S2 The CRTISO transcripts of different sizes detected in the wild type, zebra2-1mutant and complemented plants. The specific 326-bptranscriptin the wild type (lane 1), 302-bptranscript from zebra2-1 mutant (lane 2), andboth 326-bp and 302-bp transcripts in the complemented line (lane 3) were detected by semi-quantitative RT-PCR.

Fig. S3CRTISO expression in the wild type, zebra2 mutant, complemented (Comp.) and RNAi (RNAi) lines. Quantitative RT-PCR was performed to quantify relative gene expression levels from at least biological triplicate for those lines.

Fig. S4 Absorbance spectra of several carotenoids in this study. (1) lycopene; (2) neoxanthin; (3) violaxanthin; (4) β-carotene; (5) α-carotene; (6) zeaxanthin; (7) lutein; (8) prolycopene.