Supplemental Material_1.Primers for quantitative RT-PCR of LtuCADhomologs. Poplar Tubulin A (TUA) primers served as the internal standard.

Homolog / Forward primer sequence (5’to 3’) / Reverse primer sequence (5’to 3’)
CAD1 / CGAACCATAGTCAACCGTCC / GGAGTATCTATCACGCAAAC
CAD2 / GCTTAGCATTCGTTTGTAGCAGC / TAGAAGGGATGAGTGAAGAGGC
CAD3 / GTTATACTGCCCAGAAGGATGGGT / GTGAGCATCTTAGCGCATGTTTCA
CAD4 / TCGGTGTATGAGCATCCGAAAGCA / ACTCTATCCATCCAGCATCTCCAG
CAD5 / ATGTTGCCCACCAAGTCATGTGTC / CATGGCCAAACTCTGAAAGCAGCA
CAD6 / TCTATGTGGGCCACCAAGTCATGT / TAGGTCCCACAAGGCGTATAACCA
CAD7 / ATCAGCATCAATGTGGCCCACCAA / TATAGGACCGTACACATGACTAGGCG
TUA / AGGTTCTGGTTTGGGGTCTT / TTGTCCAAAAGCACAGCAAC

Supplemental Material_2. Protein sequence alignment of CAD/SAD like enzymes. Sequences were from Liriodendron tulipifera ( Arabidopsis thaliana (AtCAD1: NP_177412; AtCAD2: NP_179765; AtCAD3: NP_179780; AtCAD4: NP_188576; AtCAD5: NP195149; AtCAD6: NP_195510; AtCAD7: NP195511; AtCAD8: NP_195512; AtCAD9: NP195643), Populus tremuloides (PtrSAD: AAK58693; PtCAD: AAF43140), Fragaria x ananassa (FxaCAD1: AAK28509), Eucalyptus gunnii (EuCAD2: P31655), and Pinus taeda (PinusCAD: P41637).The Rossmann fold domain is boxed by red dotted line. The catalytic Zn2+ binding sites are marked with blue dots. The structrural Zn2+ binding sites are marked with green dots. The NADP+/NADPH binding motifs are marked with yellow boxes. The key determinant residue site is marked with red star.

Supplemental Material_3. The predicted elements in the LtuCAD1 protein structure. C: coil; E: extended beta-sheet; H: alpha-helix.

Supplemental Material_4. The predicted 3D protein structure of LtuCAD1.

Supplemental Material_5.Cis-elements in LtuCAD1 promoter.

Name / Motif sequence / Position / Annotation
Abscisic acid responsive / DPBFCOREDCDC3 / ACACNNG / 1214, 2113 / DPBF-1 and 2 (Dc3 promoter-binding factor-1 and 2) core sequence, ABA responsive
DRE2COREZMRAB17 / ACCGAC / 1548 / "DRE" core in maize rab17 promoter, ABA responsive
MYB2CONSENSUSAT / YAACKG / 12, 1196, 1436 / MYB recognition site in Arabidopsis rd22, ABA responsive
MYCATRD22 / CACATG / 1747, 2114 / Binding site for MYC (rd22BP1), ABA, drought responsive
MYCCONSENSUSAT / CANNTG / 12, 805, 1196, 1263, 1341, 1423, 1436, 1747, 1758, 2114 / MYC recognition site in rd22 promoter, ABA responsive
PYRIMIDINEBOXHVEPB1 / TTTTTTCC / 1910 / "Pyrimidine box" in barley EPB-1 (cysteineproteinase) promoter, ABA responsive
WRKY71OS / TGAC / 210, 581, 809, 952, 1504, 1615, 1635, 1755, 1906 / "TGAC-containing W box" of Amy32b promoter, ABA responsive
Auxin responsive / ASF1MOTIFCAMV / TGACG / 1906 / "ASF-1 binding site" in CaMV 35S promoter, auxin or salicylic acid responsive, may be relevant to light regulation
SEBFCONSSTPR10A / YTGTCWC / 209 / Binding site of potato silencing element binding factor (SEBF) in PR-10a gene, auxin responsive
SURECOREATSULTR11 / GAGAC / 1332 / Sulfur-responsive element (SURE) in SULTR1 promoter, auxin responsive
Disease responsive / BIHD1OS / TGTCA / 660, 1345, 1641 / Binding site of OsBIHD1, defense/disease responsive
BOXLCOREDCPAL / ACCWWCC / 1933 / Box-L-like sequence, responsive to elicitor treatment
GT1GMSCAM4 / GAAAAA / 432 / "GT-1 motif" in soybean SCaM-4, pathogen responsive
PALBOXAPC / CCGTCC / 1487 / Box A of parsley phenylalanine ammonia-lyase (PAL) gene, elicitor, light responsive
PALBOXLPC / YCYYACCWACC / 1929 / Box L of parsley phenylalanine ammonia-lyase (PAL) gene, elicitor, light responsive
WBOXNTCHN48 / CTGACY / 1614 / "W box" in tobacco chitinase gene CHN48, elicitor responsive
Light responsive / EBOXBNNAPA / CANNTG / 12, 805, 1196, 1263, 1341, 1423, 1436, 1747, 1758, 2114 / E-box of napA storage-protein gene, light responsive
ASF1MOTIFCAMV / TGACG / 1906 / "ASF-1 binding site" in CaMV 35S promoter, auxin or salicylic acid responsive, may be relevant to light regulation
EVENINGAT / AAAATATCT / 1074 / "Evening element" in 31 cycling genes in Arabidopsis, circadian control of gene expression
GATABOX / GATA / 57, 1075, 1689, 1776, 2146 / "GATA box", light responsive
GT1CONSENSUS / GRWAAW / 66, 432, 604, 814, 1103, 1121 / Consensus GT-1 binding site, light responsive
GT1CORE / GGTTAA / 229 / GT-1 binding box II of rbcS, light responsive
INRNTPSADB / YTCANTYY / 1443, 2002 / "Inr (initiator)" elements in tobacco psaDb promoter, light responsive
PALBOXAPC / CCGTCC / 1487 / Box A of parsley phenylalanine ammonia-lyase (PAL) gene, elicitor, light responsive
PALBOXLPC / YCYYACCWACC / 1929 / Box L of parsley phenylalanine ammonia-lyase (PAL) gene, elicitor, light responsive
SORLIP1AT / GCCAC / 844 / "Sequences Over-Represented in Light-Induced Promoters" (SORIPs)
SORLIP2AT / GGGCC / 748, 1520 / "Sequences Over-Represented in Light-Induced Promoters" (SORIPs)
SV40COREENHAN / GTGGWWHG / 112 / "SV40 core enhancer", light responsive
TATABOX5 / TTATTT / 26, 674 / TATA box, light responsive
Salicylic acid responsive / ELRECOREPCRP1 / TTGACC / 223, 1268, 1391 / ElRE (Elicitor Responsive Element) core of parsley PR1 genes, elicitor and salicylic acid responsive
ASF1MOTIFCAMV / TGACG / 1906 / "ASF-1 binding site" in CaMV 35S promoter, auxin or salicylic acid responsive, may be relevant to light regulation
WBOXATNPR1 / TTGAC / 580, 1754 / "W-box" in Arabidopsis NPR1 promoter, salicylic acid responsive
Sugar responsive / CGACGOSAMY3 / CGACG / 1547 / "CGACG element" in rice Amy3D and Amy3E amylase, sugar responsive
CMSRE1IBSPOA / TGGACGG / 1486 / CSMRE-1 (Carbohydrate Metabolite Signal Responsive Element 1), sugar responsive
WBOXHVISO1 / TGACT / 809, 1615 / SUSIBA2 bind to W-box in barley iso1 (encoding isoamylase1) promoter, sugar responsive
Temperature responsive / CARGATCONSENSUS / CCWWWWWWGG / 1182 / "CArG consensus" sequence, involved in vernalization
LTRE1HVBLT49 / CCGAAA / 275 / "LTRE-1" (low-temperature-responsive element) in barley blt4.9 promoter
LTRECOREATCOR15 / CCGAC / 1548 / Core of low temperature responsive element (LTRE) of cor15a gene
Wound responsive / WBOXNTERF3 / TGACY / 809, 952, 1615, 1635 / "W box" in tobacco ERF3 promoter, wound responsive