SUPPLEMENTARY MATERIAL

Table S1. List of primers used for qRT-PCR.

Table S2. Identification of N. tabacum putative clock genes using the corresponding Arabidopsis amino acids sequences as query in TBLASTN searches.

Table S3. Putative cis-acting regulatory elements (CARE) under circadian control and located in the promoter regions of the tobacco MXC-involved genes.

Table S4. Identification of Arabidopsis putative genes involved in MXC using homologs of tobacco genes as query in BLASTX searches. The BLASTX e-value and identity correspond to the local alignament between the tobacco nucleotide sequences and Arabidospsistranslated proteins.

Table S5. Putative cis-acting regulatory elements (CARE) under circadian control and located in the promoter regions of Arabidopsis MXC-related genes.

FigureS1. Schematic representation of time of growth, synchronization, phenol treatment and sampleharvesting in 2 and 3 week-old HRC. HRC were grown during 1 or 2 weeks under darkness condition and synchronized for at least 7 d by light/night cycles (12 h/12 h) in 2 or 3 week-old HRC, respectively. After this time, HRC were treated with phenol (100 mg/L) at ZT 3 during 24 h and then were harvested at different times (ZT 3, 7, 11, 15, 19, 23). White and black bars represent light and dark phases, respectively.

Figure S2. Analysis of clock gene expression: NtLHY (A), NtTOC1 (B), NtFKF1 (C), NtGI (D) and NtPRR9 (E)by q-RT PCR. N. tabacum hairy root cultures (HRC) were maintained in growth chamber during 2 weeks in solid MS mediumand synchronized by light/dark (12 h light/12 h dark) cycles at 24 ±2 °C for at least 7 d. Data are expressed as the mean of n=3 biological replicates ± SEM (standard error of the mean). White and black bars represent light and dark phases, respectively. Relative expression was normalized using Elongation factor 1 (NtEF1) as housekeeping gene.

Figure S3. Analysis of clock gene expression: NtLHY, NtTOC1 and NtFKF1by q-RT PCR during the subjective light phase. N. tabacum hairy root cultures (HRC) were maintained in growth chamber during 2 (A) and 3 (B) weeks in liquid MS medium under constant darkness (DD) conditions at 24 ±2 °C for at least 7 d.Data are expressed as the mean of n=3 biological replicates ± SEM (standard error of the mean). Black bar represents constant darkness condition. Relative expression was normalized using Elongation factor 1 (NtEF1) as housekeeping gene.

Table S1.

Gene / Forward Primer 5´ 3´ / Reverse Primer 5´ 3´ / PCR product size (bp)
N. tabacum clock genes
NtLHY / CACTCTTTTCAAGGAAGGTG / GTCGAAGGTGTTACAAGAGC / 241
NtTOC1 / ATCGTAGAACGGCAGCACTT / TCACAAACTGTCCCCTCACA / 129
NtFKF1 / ACAAGCCTACATGGAGAGAA / CCTCCAAGTCAATCGTGTAT / 165
NtPRR9 / GCTTATTTGTCACGCCACTTC / GCGGAGGAAGCAGATAATG / 104
NtGI
Putative MXC genes / CTCTCAGCACATCGGTTCTT / GCAGGACTGTGGATACCATTTA / 97
NtCYP71D21 / ACTTTGAGACTTCATCCACCG / TCTCTTCCCAATGCCCAAAC / 128
NtPXC8.1 / GTCGTGGAGTTCTGGAATCAG / CATTTTAACCATGGACCTGCC / 137
NtGsT / TCGAGAAGGAGCTGGATTTTG / GGATTGAGGGAAAGGTAAGGG / 85
NtGT / CGAGTGCTTGAAATGGCTTG / CAAGTTCGTGCAGTTGTGATG / 101
Reference gene
NtEF1 / TGAGATGCACCACGAAGCTC / CCAACATTGTCACCAGGAAGTG / 51

Table S2.

Gene Arabidopsis gene N. tabacumorthologgene N. tabacum name TBLASTN Identity (%) e-value

LHY AT1G01060.1LOC107775222 NtLHY-like 1E-50 33

TOC1 AT5G61380LOC107818975 NtTOC1-like 3E-161 49

FKF1 AT1G68050LOC107783380NtFKF1-like 0 77

PRR9 AT2G46790 LOC107780646 NtPRR9-like 4E-72 64

GI AT1G22770LOC107821341NtGI-like 0 69

Table S3.

Gene nameGene ID sequence position strand Gene description/putative phase of MXC

NtCYP71D21 107825018 CTAATTCAAC 4 -5-epiaristolochene 1.3-dihydroxylase-like/phase I

CTACAAGAAC 662 -

NTPXC8.1 107803841 CAACCCGATC 333 - Peroxidase N1-like/phase I

NtGsT 107795177 CTAAATGAACT 140 - Glutathione S-transferase PARB/phase II

CAAATTAATCA 1070 +

CTAATTTAAC 988 -

NtGT 107826982 CAATATTATC 923 -Scopoletinglucosyltransferase/phase II

Table S4.

Tobacco homolog gene / Arabidopsis gene names / Arabidopsis genes / BLASTX
E-value / Identity (%)
NtCYP71D21 / CYP71B26 / At3g26290 / 4E-57 / 53
NtPXC8.1 / ATPRX25 / At2G41480 / 4E-143 / 54
NtGsT / GST6 / At2G47730 / 1E-89 / 60
NtGT / UGT73B3 / At4G34131 / 0 / 56

Table S5.

Arabidopsis Arabidopsis gene sequence Binding site genomeBinding site

Gene name Start Enddescription

CYP71B26 At3g26290 aaaatatct 96370389637046Evening Element promoter motif

aaaatatct 9637154 9637162Evening Element promoter motif

ATPRX25At2G41480 aaaaatct 1730733917307346CCA1 binding site motif

aaaaatct 1730618017306187 CCA1 binding site motif

aaaatatct 17306658 17306666 Evening Element promoter motif

GST6 At2G47730 aaaaatct 1956345219563459CCA1 binding site motif aaaaatct 19564602 19564609 CCA1 binding site motif

UGT73B3 At4G34131 nd*

*No determine

Figure S1.

Figure S2.

Figure S3.