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Additional file 2 The phylogenetic analysis of Populus and Arabidopsis VND/NST/SND proteins. (a) PhyML phylogenetic tree analysis. A total of 21 and 22 different loci from Populus and Arabidopsis, respectively, were identified as AtSND1 homologous proteins. The full-length amino acid sequences were subjected to PhyML. Potri005G018000, a receptor like protein, was used as an outgroup protein sequence in this phylogenic tree. The AtVND homolog cluster is highlighted by green color and these proteins are used for further analyses shown in Fig. 1. (b) Heatmap illustrating amino acid sequence similarity of 43 VND homologs from Populus and Arabidopsis.

Additional file 3. VND homologs in Populus and Arabidopsis. (a) Phylogenetic tree of Populus NAC domain-containing proteins and Arabidopsis NAC proteins known as the master switch transcription factors regulating secondary cell wall biosynthesis. Potri.015G127400 (PtrWND3A) shows high amino acid sequence similarity with AtVND4 and VND5. (b) The heatmap illustrating full-length amino acid sequence similarity between VND homologs in Populus and Arabidopsis. (c) Conserved domain in full-length amino acid sequence of proteins shown in panel (a) and (b). Note that NAC domain is the only conserved region among VND proteins in Populus and Arabidopsis.

Table S1. Percentage of the gene set in each individual species distributed into different ortholog clades. (DOCX 20 kb)

Additional file 5. Expression of PdWND3A in the Populus transgenic plants. Asterisk marks selected lines for further analysis presented in this study.

Additional file 4. The cDNA sequence alignment between Potri.012G126500 (PdWND3B) and Potri.015G127400 (PdWND3A). The missing sequence region between these two genes is highlighted with yellow color.

Additional file 2. Total lignin content and S/G ratio of P. deltoides wild-type (WT), vector control and PtGAUT12.1-OE lines. Values are mean ± SE, n = 25 for WT, n = 10–15 for vector control (V. Control-1-8) and PtGAUT12.1-OE lines (AB29.1–AB29.13). In bold and denoted with a star are transgenic values that are significantly different from WT and vector control lines at P

Figure S3. Positive selection region in chloroplast β-amylase (CT-BMY). (PDF 171 kb)

Table S4. Ortholog clades of C4 cycle genes & related transporters. (PDF 118 kb)

Table S6. List of Agave americana genes that have homologs in other three independent CAM lineages but not in 21 non-CAM plant species. (PDF 31 kb)

Table S9. List of Agave americana genes with Ka/Ks ratio greater than one in Agave-Arabidopsis and Agave-Oryza but not Agave-Zea pairs. (PDF 47 kb)

Table S8. Genes implicated in the CAM pathway undergoing positive selection as revealed from Ka/Ks ratio calculated from Agave-Arabidopsis, Agave-Oryza and Agave-Zea orthologous gene pairs. (PDF 91 kb)

Table S14. Intrinsically disordered protein region (IDPR) in protein sequences under positive selection. (PDF 33 kb)

Additional file 6. Glycosyl residue composition of (a) alcohol insoluble residue (AIR) and (b–h) wall fractions from stems of field-grown P. deltoides control and PtGAUT12.1-OE transgenic plants. Wall fractions were prepared by sequential extraction of AIR using increasingly harsh reagents: (b) 50 mM ammonium oxalate, (c) 50 mM Na2CO3, (d) 1 M KOH, (e) 4 M KOH, (f) 100 mM sodium chlorite (chlorite) and (g) 4 M KOH post-chlorite (4 M KOH PC). (h) The insoluble pellet remaining after all the extractions. Glycosyl residue composition was determined by GC–MS of trimetylsilyl (TMS) derivatives. Data are mean ± SE of three biological and two technical replicates, n = 5. *P

Figure S1. Overview charts of the gene ontology (GO) biological processes over-represented in individual co-expression modules. (PDF 464 kb)

Table S7. List of Agave americana genes with Ka/Ks ratio greater than one in Agave-Arabidopsis, Agave-Oryza and Agave-Zea pairs. (PDF 353 kb)

Table S1. Percentage of the gene set in each individual species distributed into different ortholog clades. (PDF 39 kb)

Additional file 3. Plant height and diameter of three-month-old P. deltoides wild-type (WT), vector control and PtGAUT12.1-OE lines. Values are mean ± SE, n = 25 for WT, n = 10–15 for vector control (V. Control-1-8) and PtGAUT12.1-OE lines (AB29.1–AB29.13). Transgenic values that are significantly different from WT and vector control lines are in bold and denoted with one star (P

Table S10. The positively selected sites of candidates with Ka/Ksâ >â 1 from Table S9. (PDF 304 kb)

Table S5. Distribution of co-expression modules in each individual ortholog clade in Agave americana. (PDF 90 kb)

Table S12. List of Agave americana genes with afternoon-to-night shift in expression pattern as compared with the orthologous genes in Arabidopsis. (PDF 69 kb)

Table S13. List of circadian clock genes in Agave americana and Arabidopsis thaliana. (PDF 18 kb)

Figure S2. The diel expression pattern of conserved CAM-specific genes in Agave. (PDF 111 kb)

Figure S6. Intrinsically disordered protein regions (IDPRs) and structural models of the circadian clock proteins in Agave americana. (PDF 153 kb)

Additional file 4. Correlation between the total GAUT12.1 transcript expression and plant growth in P. deltoides PtGAUT12.1-OE transgenic and control lines. a Height and b radial diameter of 3-month-old greenhouse-grown poplar plants are plotted against the transcript expression level. Blue diamonds—WT; black diamonds—vector controls; red diamonds—PtGAUT12.1-OE lines (please note that on the graphs the WT blue diamonds may be obscured by the vector control black diamonds). n = 25 for WT, n = 10–15 for vector control and PtGAUT12.1-OE lines.

Additional file 7. Mass of AIR, total material recovered in sequential AIR extracts and in the insoluble pellet from P. deltoides PtGAUT12.1-OE lines compared to controls. a Mass of AIR extracted per gram of ground dry stem tissues of PtGAUT12.1-OE and control lines. b–g Amount of material recovered in wall fractions extracted by b 50 mM ammonium oxalate, c 50 mM sodium carbonate, d 1 M KOH, e 4 M KOH, f 100 mM sodium chlorite, g 4 M KOH post chlorite (PC). h Total amount of material recovered in all wall fractions combined. i Amount of material remaining in the insoluble pellet, after all the extractions. Data in panels b through i are average mg extract per gram AIR ± SE, n = 4. Significance P values are expressed as *P

Table S3. Biological processes over-represented in ortholog clade C3:CAM:C4 in Agave americana. C3:CAM:C4 represents orthologs shared only by C3, CAM and C4 species. (PDF 17 kb)

Table S11. List of Agave americana genes with morning-to-night shift in expression pattern as compared with the orthologous genes in Arabidopsis. (PDF 70 kb)

Table S2. Biological processes over-represented (pâ

Figure S5. Phylogenetic trees and diel gene expression patterns of multi-gene ortholog groups (i.e., with a total of more than two Agave and Arabidopsis genes) listed Fig. 4b. (PDF 156 kb)

Additional file 5. Leaf phenotypes of 3-month-old, greenhouse-grown P. deltoides PtGAUT12.1-OE lines compared to controls. a Comparison of leaves (the sixth leaf from apex) from P. deltoides WT, vector control, and PtGAUT12.1-OE plants. b Length and c width of leaves of different developmental stages from 3-month-old plants. Every third leaf from the apex of ten plants was measured. d Developing (10th leaf from apex) and e fully expanded (20th leaf from apex) leaf areas measured from five plants. f The relative water content (RWC) of WT and PtGAUT12.1-OE lines. Error bars represent SE. *P

Alternative splicing events in MAPK pathway. (DOCX 19Â kb)

Additional file 2. PdDUF231A expression in OXPdDUF231A transgenic Populus. RT-PCR was performed using cDNA generated from total RNA isolated from petiole of mature leaves. PdUBCc was used as an internal control. Red asterisks indicate the two transgenic lines selected for subsequent analyses.

Additional file 3. The gene copy number of PdDUF231A in OXPdDUF231A transgenic plants.

Additional file 6. Protein motif analysis of PdDUF266A. a) Conserved motifs identified in the DUF266 domain. Four conserved regions were identified with 50% amino acid identity threshold. The predicted disordered region is marked in a red box in the diagram. b) Amino acid alignment of ATKMENK residues. ATKMENK fragment is marked in blue box. PdDUF266A is indicated by a black arrow. Sequence logo displays conserved amino acid sequence with over 50% similarity.

Additional file 8. RT-PCR analysis of PdDUF266A transcript in Populus transgenic lines.

Additional file 5. Amino acid alignment of DUF266 proteins from 10 different species by using MUSCLE. A total of 187 DUF266 proteins were identified from 10 plant species including moss (P. patens), lycophyte (S. moellendorffii), rice (O. sativa), corn (Z. mays), soybean (G. max), Amborella (A. thrichopoda), grape (V. vinifera), Eucalyptus (E. grandis), Populus (P. trichocarpa) and Arabidopsis (A. thaliana). Shown is amino acid sequence alignment of 169 DUF266 proteins excluding those with 500aa in length. The DUF266 domain is shown in blue box. Proteins with extra amino acid sequences or lacking N-terminal sequences are marked by arrows (Red arrows: with extra amino acids; blue arrows, lacking N-terminal sequences; green arrows, lacking C-terminal sequences within the DUF266 domain). Four corn DUF266 proteins having longer N-terminal sequences than others are indicated by purple arrows.

Additional file 2. Distribution of DUF266 proteins in 10 different species. Number denotes total number of DUF266 proteins identified from each species. Numbers in parenthesis indicated DUF266 proteins with 500 amino acids.

Alternative splicing events in MAPK pathway. (DOCX 19Â kb)

Correlation between JMJD6 expression and clinicopathologic characteristics of 88 melanoma patients by chi-square test. (DOCX 503Â kb)

Additional file 7. Potri.011G009500 expression in different tissues and organs. Normalized FPKM values for various tissues and organ were compiled from Populus Gene Atlas dataset integrated in the Phytozyme website ( https://phytozome.jgi.doe.gov ).

Correlation between JMJD6 expression and clinicopathologic characteristics of 88 melanoma patients by chi-square test. (DOCX 503Â kb)

The amino acid sequence alignment and conserved motifs of G-type PtLecRLKs. (A) Amino acid sequence alignment via CLUSTALW. To determine conserved amino acid regions, 50Â % sequence identity was used as a cutoff. Note that the amino acid sequence in the protein kinase domain is highly conserved in most G-type PtLecRLKs whereas the other domains such as S-locus glycoprotein, EGF, PAN, TM and signal peptide varied. Highly conserved motifs are boxed with labels a, b, c, d and e. The red arrows indicate truncated bulb lectin domains. The blue arrows indicate the truncated protein kinase domains. (B) The conserved motif sequences marked on panel A. Sequence logo was generated from the consensus amino acid sequence over 50Â % sequence identity. (PPTX 3107 kb)

Additional file 1: Figure S1. Alignment of cDNAs of AtHY2, AtTRY, AtLNG1, AtNPQ1, AtSEX1, AtMAX3 and AtGUN4.

Comparison of different types of PtLecRLKs. (A) Amino acid sequence alignment of randomly selected PtLecRLKs from each type by ClustalW. Amino acid identity is displayed by green color above the first row in the plot. Note that the protein kinase domains at the C-terminus are highly conserved whereas lectin domains at the N-terminus are very distinct. (B) Phylogenetic tree using neighbor joining method with 1000 bootstrapping of the randomly selected PtLecRLKs. Note that three types are separated clearly. The number on branch indicates bootstrapping value of each node formation. (C) The amino acid sequence identity of examined PtLecRLKs. (PPTX 477 kb)

Diagrams of protein domain architecture of lectin domain-containing proteins that lack the protein kinase domain in Populus. (PPTX 63 kb)

Additional file 2: Table S1. PCR primers used in this study.

The amino acid sequence alignment and conserved motifs of L-type PtLecRLKs. (A) Amino acid sequence alignment via CLUSTALW. Amino acid sequence identity of 50 % was used to determine cluster of protein domain and phylogenetic node of 50 L-type PtLecRLKs. The red arrows indicate truncated legume lectin domains. The blue arrows indicate the truncated protein kinase domains. (B) The conserved motifs marked in panel A. Sequence logo was generated from the consensus amino acid sequence over 50 % sequence identity. (PPTX 3150 kb)

Additional file 10. Glycosyl linkage analysis of the residual insoluble cell wall that remained after sequential extractions of AIR from P. deltoides WT, PdGAUT12.1-KD, and PtGAUT12.1-OE lines. Data are in mol percentages, provided by CCRC Analytical Services.

Additional file 10. Glycosyl linkage analysis of the residual insoluble cell wall that remained after sequential extractions of AIR from P. deltoides WT, PdGAUT12.1-KD, and PtGAUT12.1-OE lines. Data are in mol percentages, provided by CCRC Analytical Services.