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Differential expression and functional analysis of two short-chain alcohol dehydrogenases/reductases in Hedychium coronarium

Hua Chen
Hua Chen
, 
Yuechong Yue
Yuechong Yue
, 
Rangcai Yu
Rangcai Yu
 and 
Yanping Fan
Yanping Fan
   | May 18, 2020
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Article Category: Research Article
Published Online: May 18, 2020
Page range: 97 - 114
Received: Jan 30, 2020
Accepted: Apr 14, 2020
DOI: https://doi.org/10.2478/fhort-2020-0010
Keywords
© 2020 Hua Chen et al., published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

Figure 1

Photographs of H. coronarium flowers in different developmental stages. −40 = 40 h pre-anthesis; −32 = 32 h pre-anthesis; −24 = 24 h pre-anthesis; −16 = 16 h pre-anthesis; −8 = 8 h pre-anthesis; 0 = 0 h anthesis; 8 = 8 h post-anthesis.
Photographs of H. coronarium flowers in different developmental stages. −40 = 40 h pre-anthesis; −32 = 32 h pre-anthesis; −24 = 24 h pre-anthesis; −16 = 16 h pre-anthesis; −8 = 8 h pre-anthesis; 0 = 0 h anthesis; 8 = 8 h post-anthesis.

Figure 2

Alignment of the deduced amino acid sequences of HcADH2, HcADH3 and alcohol dehydrogenases/reductases from various plant species. RWR85228 = Cinnamomum micranthum SDR; HcADH = Hedychium coronarium SDR (KF358245); BAK09296 = Zingiber zerumbet SDR; RWR94702 = Cinnamomum micranthum secoisolariciresinol dehydrogenase. Grey boxes = identical residues; black boxes = similar residues; black triangle = the aspartate at position 52 of HcADH2 and position 67 of HcADH3. SDR, short-chain dehydrogenase/reductase.
Alignment of the deduced amino acid sequences of HcADH2, HcADH3 and alcohol dehydrogenases/reductases from various plant species. RWR85228 = Cinnamomum micranthum SDR; HcADH = Hedychium coronarium SDR (KF358245); BAK09296 = Zingiber zerumbet SDR; RWR94702 = Cinnamomum micranthum secoisolariciresinol dehydrogenase. Grey boxes = identical residues; black boxes = similar residues; black triangle = the aspartate at position 52 of HcADH2 and position 67 of HcADH3. SDR, short-chain dehydrogenase/reductase.

Figure 3

Phylogenetic tree of HcADH2 and HcADH3 with known alcohol dehydrogenase/reductase from other species. Aa = Artemisia annua; Ao = Asparagus officinalis; At = Arabidopsis thaliana; Cm = Cinnamomum micranthum; Chl = Chasmanthium latifolium; Cl = Citrullus lanatus; Cr = Catharanthus roseus; Hc = Hedychium coronarium; Le = Lycopersicon esculentum; Ma = Musa acuminate; Mc = Macleaya cordata; Mp = Mentha x piperita; Nt = Nicotiana tomentosiformis; Ol = Orthoclada laxa; Os = Oryza sativa; Pa = Parasponia andersonii; Pb = Papaver bracteatum; Pd = Phoenix dactylifera; Pm = Persicaria minor; Ps = Paeonia suffruticosa; Rc = Ricinus communis; St = Solanum tuberosum; Td = Tripsacum dactyloides; Zz = Zingiber zerumbet. Scale bars = 20% sequence differences.
Phylogenetic tree of HcADH2 and HcADH3 with known alcohol dehydrogenase/reductase from other species. Aa = Artemisia annua; Ao = Asparagus officinalis; At = Arabidopsis thaliana; Cm = Cinnamomum micranthum; Chl = Chasmanthium latifolium; Cl = Citrullus lanatus; Cr = Catharanthus roseus; Hc = Hedychium coronarium; Le = Lycopersicon esculentum; Ma = Musa acuminate; Mc = Macleaya cordata; Mp = Mentha x piperita; Nt = Nicotiana tomentosiformis; Ol = Orthoclada laxa; Os = Oryza sativa; Pa = Parasponia andersonii; Pb = Papaver bracteatum; Pd = Phoenix dactylifera; Pm = Persicaria minor; Ps = Paeonia suffruticosa; Rc = Ricinus communis; St = Solanum tuberosum; Td = Tripsacum dactyloides; Zz = Zingiber zerumbet. Scale bars = 20% sequence differences.

Figure 4

The expression analysis of HcADH2 and HcADH3 in different tissues of H. coronarium. (A) The HcADH2 relative expression analysis in different tissues using qRT-PCR. (B) The HcADH3 relative expression analysis in different tissues using qRT-PCR. (C) The HcADH3 relative expression analysis in different parts of flower using qRT-PCR. OL = outer abellum; Se = petal; LP = laterral petal; Pe = pedicel; An = anter; Fi = filament; SS = style and stigma. Numbers = ±SD (n = 3). Asterisks = statistically significant differences compared with the ones with the highest expression level (*p < 0.05; **p < 0.01).
The expression analysis of HcADH2 and HcADH3 in different tissues of H. coronarium. (A) The HcADH2 relative expression analysis in different tissues using qRT-PCR. (B) The HcADH3 relative expression analysis in different tissues using qRT-PCR. (C) The HcADH3 relative expression analysis in different parts of flower using qRT-PCR. OL = outer abellum; Se = petal; LP = laterral petal; Pe = pedicel; An = anter; Fi = filament; SS = style and stigma. Numbers = ±SD (n = 3). Asterisks = statistically significant differences compared with the ones with the highest expression level (*p < 0.05; **p < 0.01).

Figure 5

The expression analysis of HcADH2 (A) and HcADH3 (B) in different species of Hedychium. 1 = H. gardnerianum; 2 = H. forrestii; 3 = H. coronarium; 4 = H. coccineum Buch.–Ham. Numbers = ±SD (n = 3). Asterisks = statistically significant differences compared with the ones with the highest expression level (*p < 0.05; **p < 0.01).
The expression analysis of HcADH2 (A) and HcADH3 (B) in different species of Hedychium. 1 = H. gardnerianum; 2 = H. forrestii; 3 = H. coronarium; 4 = H. coccineum Buch.–Ham. Numbers = ±SD (n = 3). Asterisks = statistically significant differences compared with the ones with the highest expression level (*p < 0.05; **p < 0.01).

Figure 6

The expression analysis of HcADH3 in different developmental stages of H. coronarium flower. −40 = 40 h pre-anthesis; −32 = 32 h pre-anthesis; −24 = 24 h pre-anthesis; −16 = 16 h pre-anthesis; −8 = 8 h pre-anthesis; 0 = anthesis (0 h); 8 = 8 h post-anthesis. Numbers = ±SD (n = 3). Asterisks = statistically significant differences compared with the ones with the highest expression level (*p < 0.05; **p < 0.01).
The expression analysis of HcADH3 in different developmental stages of H. coronarium flower. −40 = 40 h pre-anthesis; −32 = 32 h pre-anthesis; −24 = 24 h pre-anthesis; −16 = 16 h pre-anthesis; −8 = 8 h pre-anthesis; 0 = anthesis (0 h); 8 = 8 h post-anthesis. Numbers = ±SD (n = 3). Asterisks = statistically significant differences compared with the ones with the highest expression level (*p < 0.05; **p < 0.01).

Figure 7

Expression analysis of HcADH2 and HcADH3 mRNA levels in response to external environmental stimuli. (A) HcADH2 expression at 0–36 h after wounding. (B) HcADH2 expression analysis of MeJA treatments and natural leaves (control) at different times. (C) HcADH3 expression at 0–36 h after wounding. (D) HcADH3 expression analysis of MeJA treatments and natural leaves (control) at different times. Numbers = ±SD (n = 3). Asterisks = statistically significant differences compared with the ones with the highest expression level (*p < 0.05; **p < 0.01).
Expression analysis of HcADH2 and HcADH3 mRNA levels in response to external environmental stimuli. (A) HcADH2 expression at 0–36 h after wounding. (B) HcADH2 expression analysis of MeJA treatments and natural leaves (control) at different times. (C) HcADH3 expression at 0–36 h after wounding. (D) HcADH3 expression analysis of MeJA treatments and natural leaves (control) at different times. Numbers = ±SD (n = 3). Asterisks = statistically significant differences compared with the ones with the highest expression level (*p < 0.05; **p < 0.01).

Figure 8

Analyses of products generated by the recombinant HcADH3 enzyme with geraniol as substrate. (A) Total ion chromatogram of the products obtained from the empty vector pET30a. (B) Total ion chromatogram of products obtained from pET30a-HcADH3 with NAD (peak 1, geraniol; peak 2, citral). (C) Mass spectrum of peak 2 (citral). (D) Mass spectrum of authentic citral in the NIST08 library.
Analyses of products generated by the recombinant HcADH3 enzyme with geraniol as substrate. (A) Total ion chromatogram of the products obtained from the empty vector pET30a. (B) Total ion chromatogram of products obtained from pET30a-HcADH3 with NAD (peak 1, geraniol; peak 2, citral). (C) Mass spectrum of peak 2 (citral). (D) Mass spectrum of authentic citral in the NIST08 library.

Figure S1

The RT-PCR amplification products of HcADH2 and HcADH3. M = DNA DL 2000 Marker; 1 = HcADH2 PCR product; 2 = HcADH3 PCR product.
The RT-PCR amplification products of HcADH2 and HcADH3. M = DNA DL 2000 Marker; 1 = HcADH2 PCR product; 2 = HcADH3 PCR product.

Figure S2

Full-length sequence of HcADH2 and HcADH3 genes. Nucleotide sequences with green colour = the primer sequences for full cDNA synthesis. Nucleotide sequences with red colour = initiator codon (ATG), terminators (TGA).
Full-length sequence of HcADH2 and HcADH3 genes. Nucleotide sequences with green colour = the primer sequences for full cDNA synthesis. Nucleotide sequences with red colour = initiator codon (ATG), terminators (TGA).

Figure S3

Relative terpenes volatile aroma contents of Hedychium coronarium during flower development. Numbers = ± SD (n = 3). Asterisks = statistically significant differences compared with the ones with the highest expression level (*p < 0.05; **p < 0.01).
Relative terpenes volatile aroma contents of Hedychium coronarium during flower development. Numbers = ± SD (n = 3). Asterisks = statistically significant differences compared with the ones with the highest expression level (*p < 0.05; **p < 0.01).

Primer sequences used in quantitative real-time PCR assays and their characteristics

Gene namePrimer (forward/reverse) 5′→3′Amplicon Tm (°C)Amplicon length (bp)Amplication efficiency (%)R2
GAPDHTAACATCATTCCCAGCAGCACTGGTGGATCTCACTGTCAGGCTC83.2013694.80.998
HcADH2TGCTACGTCAGCGGACAGAACGGACCGTCACCGACATAAGG90.7112879.40.991
HcADH3GTCAGTGGTGGCAGGGATCAGAGAATAGGCGAGCGG85.712499.70.988

Amino acid residues sequences of comp45280.c0 and comp41433.c0

NameAmino acid residue sequences
comp45280_c0MAADGSNGNAVPASPSRRLEGKVAFITGGAAGSGEATARLFVLHGAKVVIGDVRDEFGRAAASSIGGEDVITYVHCDVSKEADVERAVDLAVAKYGRLDIVFSNAAVLDECRGVAVAEADDFDRVMAVNVRGVFLGTKHAARAMMAAGVRGSIINNGSVATVVAGVASHAYVASKHAVLGLTRSAAAELGQHGIRVNCVSPFAYATSLACDFIHMDQKQIEQFIGAVSNLKGAVLRADDVARAAVYLASDESCYVSGQNIIIDGGFTAVNHAFGLFKN
comp41433_c0MLGMALRVKRGLAIRTGIRTQQQQFSTHPTPARLAGKVAIITGAASGVGRATAAEFIHHGAQVVLADIQHELGKSVAAELGPGATFVPCDVTQEPQVAAVVDLAVAKHGRLDIMYNNAGICGPMTFAVTDVDLTEFDRVMAVNVRSVVAGIKHAARVMIPRRAGSILCTASITGFVGGLAPLAYSLSKAAVAAAVRLSAAELSKHGIRVNCISPASLPTPFGIKAIREILPDLEEQRAVEMIELSSAELAGTKCEVEDVAKAATFLASDEAKYISGHNLMVDGGFTTSKRLNFSPE

Sequences description of accession numbers in GenBank on the phylogenetic tree

Accession numbersName
AAB57737.1Short-chain alcohol dehydrogenase
AAM10204.1Tropinone reductase
AAU20370.1Secoisolaticiresinol dehydrogenase
AB018559.1Short-chain alcohol dehydrogenase
ACZ34296.1Short-chain alcohol dehydrogenase/reductase
AJO70763.1Alcohol dehydrogenase
AK103462.1Short-chain alcohol dehydrogenase/reductase
BAG96093.1Short-chain alcohol dehydrogenase/reductase
BAG99023.1Short-chain alcohol dehydrogenase/reductase
BAK09296.1Short-chain alcohol dehydrogenase/reductase
DQ384222.1Short-chain dehydrogenase/reductase
DQ384263.1Short-chain dehydrogenase/reductase
EEF41657.1Short chain alcohol dehydrogenase
EF184229.1Salutaridine reductase
HQ283448.1Dihydroflavonol 4-reductase
KF358245Short-chain dehydrogenase/reductase
KM515811.1Short-chain dehydrogenase/reductase
KM515812.1Short-chain dehydrogenase/reductase
NP_001233856.1Short-chain dehydrogenase/reductase
OVA14417.1Short-chain dehydrogenase/reductase
PON43718.1Short-chain dehydrogenase/reductase
PWA82834.1Glucose/ribitol dehydrogenase
RWR74505.1Short-chain dehydrogenase/reductase
RWR85228.1Short-chain dehydrogenase/reductase
RWR94702.1Secoisolaticiresinol dehydrogenase
XP_006366369.1Zerumbone synthase
XP_008811702.1Momilactone A synthase
XP_009404353.1Secoisolariciresinol dehydrogenase
XP_009587781.1Short-chain dehydrogenase/reductase
XP_015645551.1Momilactone A synthase
XP_018675432.1Secoisolariciresinol dehydrogenase
XP_020246099.1Momilactone A synthase

Primer sequences of HcADH2 and HcADH3 used in RT-PCR assays

MethodsForward primerReverse primerPCR product size (bp)
RT PCRHcADH2TCTTCCACTCAGATGGCGGCTTCAGTTCTTGAAGAGGCCAAA849
HcADH3TTGACAGAAACCATGCTCGGAATTCACTCTGGTGAAAAATTTAAGCG903
PET-30a:HcADH2 vectorCGCGGATCCATGGCGGCTGACGGCCCAAGCTTTCAGTTCTTGAAGAGGCCAAAG
PET-30a:HcADH3 vectorCGCGGATCCATGCTCGGAATGCCCAAGCTTTCACTCTGGTGAAAAAT

Substrate of enzyme catalysed reaction

SubstrateNumber of carbonStructure
GeraniolC10
FarnesolC15
l-LinaloolC10
CamphorC10
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