(Appendices)

In Planta Cleavage of Carotenoids by Arabidopsis Carotenoid Cleavage Dioxygenase 4 in Transgenic Rice Plants

Mi-Hee Song1,2,†, Sun-Hyung Lim2,†, Jae Kwang Kim3,Eun Sung Jung4, K.M. Maria John4, Min-Kyoung You5, Sang-Nag Ahn1,Choong Hwan Lee4,*,Sun-Hwa Ha5,*

Appendix A. Supplementary Tables
Table S1. Carotenoid content and composition in the leaves of the transgenic plants
Plants / Carotenoid content (%)
α-Carotene / β-Carotene / Lutein / Zeaxanthin / Antheraxanthin / Violaxanthin / Total
NT / 1.83±0.54 (1.0) / 84.47±7.85 (48.0) / 82.76±11.58 (47.0) / 1.26±0.33 (0.7) / 1.29±0.23 (0.7) / 4.43±0.49 (2.5) / 176.04±20.00
T-1-3 / 0.87±0.06 (0.7) / 60.27±4.19 (47.0) / 59.08±5.88 (46.0) / 2.12±0.08 (1.7) / 2.25±0.44 (1.8) / 3.72±1.06 (2.9) / 128.31±3.33
T-1-4 / 0.79±0.01 (0.6) / 56.06±4.76 (44.2) / 60.96±4.44 (48.0) / 2.59±0.30 (2.0) / 2.74±0.22 (2.2) / 3.84±0.26 (3.0) / 126.97±9.98
T-1-5 / 0.86±0.07 (0.7) / 64.02±3.58 (48.7) / 58.90±5.47 (44.8) / 2.38±0.19 (1.8) / 2.14±0.20 (1.6) / 3.22±0.43 (2.5) / 131.52±9.93
T-5-4 / 0.91±0.01 (0.7) / 64.23±0.35 (48.9) / 58.53±2.12 (44.6) / 2.26±0.07 (1.7) / 2.12±0.09 (1.6) / 3.23±0.17 (2.5) / 131.28±2.79
T-5-5 / 0.83±0.03 (0.6) / 65.57±3.36 (50.8) / 55.68±1.03 (43.1) / 1.84±0.04 (1.4) / 1.76±0.03 (1.4) / 3.52±0.29 (2.7) / 129.21±4.20
T-5-6 / 1.34±0.02 (1.0) / 65.07±1.19 (47.8) / 62.42±7.23 (45.8) / 2.45±0.28 (1.8) / 1.86±0.31 (1.4) / 3.15±0.69 (2.3) / 136.28±9.67

Data are expressed as mean (g/gflesh weight) SD from three independent experiments.

Table S2.Primer sets used for quantitative real-time (qRT)-PCR to confirm the expression levels of three endogenous rice CCD genes (OsCCD7, 8a, and 8b) in rice leaf tissues of two independent lines (T-1 and T-5) of AtCCD4-transgenic rice plants shown in Figure S2.
Gene / Accession number
or Locus ID / Primer sequence (forward/reverse) / Productsize (bp)
AtCCD4 / At4g19170 / 5'-GGATTCAATATCATTCACGCTATT-3'/
5'-TCTTCACCTTCTCCACCAA-3' / 114
OsCCD7 / Os04g0550600
(Os04g46470) / 5'-TGGTGGCTATGTTCTTCTTG-3'/
5'-GCATTCTCTGTCCCTATCTTC-3' / 93
OsCCD8a / Os01g0566500
(Os01g38580) / 5'-TCATGGCGATCCACTTCAT-3'/
5'-GTCACCGTAGTGCTCACA-3' / 86
OsCCD8b / Os01g0746400
(Os01g54270) / 5'-AACTGCCTTCATTACTCCTG-3'/
5'-CGACATACGCCTCTATATTACA-3' / 140
OsUBQ5 / Os01g0328400
(Os01g22490) / 5'-GAAGTAAGGAAGGAGGAGGA-3'/
5'-AAGGTGTTCAGTTCCAAGG-3' / 100

Appendix B. Supplementary figures

Fig. S1.Expression analysis of transgenic plants using the semi-quantitative RT-PCR. Sample No. of 1, 2, 3, 5, 6 and 7 indicated the independent transgenic rice plants harboring pPGD1::AtCCD4. NT means non-transgenic rice plant, cultivar Hwayoung. Two single-copy insertion lines of 1 (T-1) and 5 (T-5) confirmed by Taqman-PCR and Southern blot were in bold.

Fig. S2.Quantitative RT-PCR of three endogenous rice CCD genes (OsCCD7, 8a, and 8b) in two independent lines (T-1 and T-5) of AtCCD4-transgenic rice plants. The rice ubiquitin 5 gene (OsUBQ5) was used for normalization of the amounts of leaf RNA. NT means non-transgenic rice plants. Each value was the mean of three biological replicates.

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