SUPPLEMENTARY MATERIAL “Mutation of the inducible ARABIDOPSIS THALIANA CYTOCHROME P450 REDUCTASE 2 alters lignin composition and improves saccharification” Lisa Sundin, Ruben Vanholme, Jan Geerinck, Geert Goeminne, René Höfer, Hoon Kim, John Ralph and Wout Boerjan
Supplemental Figure S1. Phenolic profiling (A) PCA plot based on the 1067 peaks detected via UHPLC-MS. (B) Venn-diagrams with the number of significantly differential peaks in atr2-1 and atr2-2 as compared to wild-type (WT) samples.
Supplemental Figure S2. MS/MS spectra and the reasoning for the (tentative) structural identification of 33 metabolites of which the MS/MS spectra were not published before.
Compound 20: G 4-O-hexoside(8-O-4)S (+ formic acid)
Compound 20 eluted at 7.61 min with a calculated chemical formula of C28H37O15-, based on the theoretical m/z value (m/zth) 613.214 that best approximated the experimental m/z value (m/zexp) 613.212. A neutral loss of 208.054 Da could be assigned to a combined loss of dehydrated hexose and formic acid (C7H12O7, 208.059 Da). Based on the smaller fragments in the MS/MS spectrum, the aglycone with m/zexp 405.158 could be assigned to G(8-O-4)S (Morreel et al., 2004; 2010a; 2010b). Because compounds without acidic functionalities (e.g. carboxylic acid or phenolic function) tend to be detected as adducts with ions (e.g. formic acid) in the mobile phase, the hexose is most likely on the phenolic function of G(8-O-4)S and hence compound 20 was characterized as G 4-O-hexoside(8-O-4)S. (Note: formic acid was used to buffer the UHPLC mobile phase).
Compounds 21 and 22: G(red8-8)G hexoside
Compounds 21 and 22 eluted at 8.20 min and 9.04 min, respectively. The calculated chemical formula of C26H33O11- (m/zth 521.203) best approximated their m/zexp 521.203 and 521.202, respectively. Both MS/MS spectra had a base peak with m/zexp 329.136 that could be assigned to the result of a consecutive dehydrated hexose and formaldehyde loss (m/zth 329.139). In the MS/MS of compound 22, the aglycone was detected at low intensity (m/zexp 359.148). Since m/z 329.139 is the highest fragment in G(red8-8)G (lariciresinol, m/z 359.150, Morreel et al., 2003) compounds 21 and 22 were characterized as G(red8-8)G hexoside.
Compound 25: G(red8-5)G hexoside
Compound 25 eluting at 10.68 min had a calculated chemical formula of C26H33O11- (m/zth 521.203) matching the m/zexp 521.203. The MS/MS spectrum showed neutral losses of H2O and CH2O leading to m/zexp 503.192 and 491.191, respectively. In addition, the ions with m/zexp 341.130 and 329.133 can be assigned to neutral losses of H2O and CH2O, respectively, in combination with the neutral loss of a dehydrated hexose. Since m/zth 341.139 and 329.139 are the major fragments of G(red8-5)G (isodihydrodehydrodiconiferyl alcohol, IDDDC, Morreel et al., 2003), compound 25 was characterized as a hexosylated G(red8-5)G. More in particular, since the parent ion loses CH2O, which requires a free aliphatic alcohol on the 9-position of the A unit and a free phenolic function on the B unit, the hexose can not be linked on these positions. Thus, the hexose is most likely attached on the phenolic function of A unit or on the aliphatic function of the B-unit as in G 4-O-hexoside(red8-5)G or G(red8-5)G 8-O-hexoside.
Compounds 27 and 28: G(8-O-4)feruloyl hexose + 224 Da
Compounds 27 and 28 eluted at 11.68 min and 11.89 min, respectively. A chemical formula of C37H43O18- (m/zth 775.245) closely matched the measured m/zexp 775.246. An initial neutral loss of 224.068 Da gave rise to the m/z 551.178 second order ion with an estimated chemical formula of C26H31O13- (m/zth 551.177). About all other second order fragments could be assigned to G(8-O-4)ferulic acid hexoside (C26H31O13-, Matsuda et al., 2010). The fragments of 491.157, 461.142 and 431.134 are derived from ring-cleavage of the hexose and hint that the hexose esterified on the carboxylic function of the ferulic acid moiety. Because the structure of the neutral loss of 224.068 Da remained unresolved, these compounds were characterized as ‘G(8-O-4)feruloyl hexoside + 224 Da’.
Compounds 35 and 36: p-coumaroyl hexose + 224 Da
Compounds 35 and 36 eluted at 11.66 min (m/zexp 549.162) and 13.36 min (m/zexp 549.161), respectively. A chemical formula of C26H29O13- (m/zth 549.161) closely matched the measured m/zexp. In both cases, an initial neutral loss of about 224.069 Da gave rise to m/zexp 325.093 and 325.068 for 35 and 36, respectively, with estimated chemical formula of C15H17O8- (m/zth 325.291). About all other fragments further hinted the identity of the m/zexp 325.093 to be p-coumaroyl hexose (Vanholme et al., 2012). The MS/MS spectrum of 35 was contaminated with fragments originating from the m/z 551.178 fragment of G(8-O-4)feruloyl hexose + 224 da (compound 27), eluting at about the same retention time (data not shown). However, MS/MS of the co-eluting m/zexp 325.093 also indicated that this fragment could be assigned as p-coumaroyl hexose. Note the similarities with the spectra of compound 51 and 52. Because the structure of the neutral loss of 224.069 Da remained unresolved, these compounds were named ‘p-coumaroyl hexose + 224 Da’.
Compounds 39 and 40: p-coumaroyl shikimate
Compounds 39 and 40 eluted at 7.89 min and 9.49 min, respectively and had a calculated chemical formula of C16H15O7-, based on the m/zth 319.082 that approximated their m/zexp of 319.083 and 319.084, respectively. The MS/MS spectra of both compounds are nearly identical. Therefore, the structural elucidation of the compound 39 will be described in detail, the elucidation of the compound 40 can be deduced in a similar way. The neutral loss of 156.045 Da leading to a product ion at m/z 163.037 represents a molecule with C7H8O4 as chemical formula (with a theoretical mass of 156.042 Da), corresponding to dehydrated shikimic acid. The ions with m/z 173.035 and 155.031 can be assigned to deprotonated shikimic acid (m/zth 173.046) and deprotonated dehydrated shikimic acid (m/zth 155.035), respectively, and the ion with m/z 137.022 to a fragment of the latter after H2O loss (m/zth 137.024 Da). In addition, m/z 111.044 and 93.031 can be assigned to the loss of CO2 from m/z 155.031 and 137.022, respectively. The smaller product ion at m/z 145.027, 119.048 and 111.044 can be explained as the loss of H2O and CO2 and the combined loss of H2O and CO2 respectively, from the m/z 163.037 ion (Bandu et al., 2004). Therefore, the peak at m/z 163.037 can be assigned to p-coumarate. The ion with m/z 145.027 can be assigned to the p-coumaric acid-derived phenolate product ion. By comparing the mass, retention time and the MS/MS spectrum of compound 39 with those of an authentic standard compound, compound 39 was identified as trans-5-O-p-coumaroyl shikimate. Since the standard mixture had a second peak with m/z 335.077 at around 9.49 min, compound 40 is most likely the cis isomer of 5-O-p-coumaroyl shikimate.
Compounds 51 and 52: feruloyl hexose + 224 Da
Compounds 51 and 52 were integrated as m/z 217.051 (at 12.31 min) and m/z 355.104 (at 13.55 min), respectively. However, both appeared fragments of a bigger structures with m/zexp 579.173 and 597.170, respectively. In case of compound 51 the m/z 579.173 appeared not to be significant differential by the stringent criteria used and therefor did not end up in the list of differential peaks, while in case of compound 52 m/z 579.170 did not pass the criteria for integration in Transomics. Because both MS/MS spectra are very similar, only the spectrum of compound 51 will be discussed. A neutral loss of 224.071 Da resulted in an ion with m/zexp 355.102. All fragments further hinted the identity of the m/zexp 355.102 to be p-coumaroyl hexose (m/zth 355.103, Vanholme et al., 2012). Note the similarities with the spectra of compound 35 and 36. Because the structure of the neutral loss of 224.071 Da remained unresolved, these compounds were named ‘feruloyl hexose + 224 Da’.
Compound 60: benzoyl malate
Compound 60 eluted at 9.16 min. The m/zexp 237.041 suggested the chemical formula to be C11H9O6- (m/zth 237.040). The MS/MS showed a neutral loss of 116.017 Da (resulting in m/z 121.024) and an m/z 115.002 that could be the deprotonated neutral loss. In Arabidopsis phenolic metabolites, 116 Da often corresponds to dehydrated malate (e.g. sinapoyl malate and G(8-O-4)feruloyl malate; Mir Derikvand et al., 2008, Rohde et al., 2004). The m/z 77.034 could be the result of CO2 loss of m/z 121.024, hinting a carboxylic acid functionality after the loss of dehydrated malate. Taken together, compound 60 could be characterized as benzoyl malate.
Compound 61: p-hydroxybenzaldehyde
Compound 61 eluted at 5.44 min. The m/zexp 121.030 suggested the chemical formula to be C7H5O2- (m/zth 121.030). The retention time and exact mass of compound 61 fitted with those of p-hydroxybenzaldehyde injected as a standard.
Compound 63: p-hydroxybenzoic acid 4-O-hexoside
Compound 63 eluted at 1.85 min. The m/zexp 299.078 suggested the chemical formula to be C13H15O8- (m/zth 299.077). The MS/MS showed a loss dehydrated hexose (C6H10O5, 162.058 Da). The resulting second order ion with m/zexp 137.020 further lost fragments that could be assigned to H2O (resulting in m/z 132.002) and CO2 (resulting in m/z 93.030), hinting a carboxylic acid functionality. Therefore, the m/zexp 137.020 moiety was characterized as p-hydroxybenzoic acid (m/zth 137.024). In case the hexose was esterified to p-hydroxybenzoic acid, ions derived from ring-cleavage were expected (especially those of -60.021, -90.032 and -120.042 Da, Vanholme et al., 2012). Since these fragmentations are not observed the hexose is most likely not esterified, but attached at the phenolic function. Hence compound 63 is characterized as p-hydroxybenzoic acid 4-O-hexoside.
Compound 77: H(8-8)H 4-O-hexoside
Compound 77 eluted at 9.00 min. The parent ion with m/zexp 459.166 suggested the chemical formula to be C24H27O9- (m/zth 459.166). The MS/MS showed a loss of dehydrated hexose (C6H10O5, 162.059 Da). The mass of the aglycone m/z 297.108 closely matched the chemical formula of H(8-8)H (C18H17O4-, m/zth 297.113). The fragments of m/z 251.101 and 121.024 could be assigned to fragments of H(8-8)H: [M – H+ - HCOOH] and 2,5X-, respectively (Morreel et al., 2010a, 2010b). Therefore, compound 77 was characterized as H(8-8)H 4-O-hexoside.
Compound 78: H(8-8)G hexoside
Compound 78 eluted at 9.60 min. The parent ion with m/zexp 489.177 suggested the chemical formula to be C25H29O10- (m/zth 489.177). The MS/MS showed a loss of dehydrated hexose (C6H10O5, 162.061 Da). The mass of the aglycone m/z 327.116 closely matched the chemical formula of H(8-8)G (C19H19O5-, m/zth 327.124). The fragments of m/z 151.035 and 121.021 could be assigned to 2,5X- fragments of G and H moiety, respectively (Morreel et al., 2010a, 2010b). In addition, m/z 136.013 fits with the reported methyl radical loss of 2,5X- where X is the G unit (Morreel et al., 2010a, 2010b). Therefore, compound 78 was characterized as H(8-8)G hexoside.
Compound 79: H(8-8)G dihexoside
Compound 79 eluted at 6.59 min. The parent ion with m/zexp 651.230 suggested the chemical formula to be C31H39O15- (m/zth 651.229). The MS/MS showed twice a loss of dehydrated hexose (C6H10O5). The mass of the aglycone m/z 327.117 closely matched the chemical formula of H(8-8)G (C19H19O5-, m/zth 327.124). Further fragments further suggested the identity of the aglycone to be H(8-8)G, as discussed in the explanation of compound 78. In theory each of the phenolic functions of H(8-8)G could be coupled to a hexose, but such a molecule would not be easily charged, and would rather be detected as the formic acid adduct (see for instance compounds 20 and 80). Therefore, it is more likely that one of the phenol functions is substituted with a dihexose. In conclusion, compound 79 was characterized as H(8-8)G dihexoside.
Compound 80: H 4-O-hexoside(8-5)H (+ formic acid)
Compound 80 eluted at 9.16 min. The parent ion with m/zexp 505.171 suggested the chemical formula to be C25H29O11- (m/zth 505.172). The MS/MS showed a loss of 208.052 Da (resulting in m/zexp 297.119), which can be attributed to a combined dehydrated hexose and formic acid loss (C6H10O5 + CH2O2). The m/z of the resulting aglycone closely matched with the one of H(8-5)H (C18H17O4-, m/zth 297.113). In addition, further fragments with m/z 279.098 and 267.099 were reported before as fragments of H(8-5)H. Here we find an additional CH2O loss resulting in m/z 249.087 (derived from m/z 279.098) and m/z 237.085 (derived from m/z 267.099). The ion with m/z 325.091 can be assigned as the complex of m/z 279.098 with formic acid. Because the parent ion forms a complex with formic acid, the molecule most likely has no free phenolic function (that would be easily charged in the conditions used). Therefore the hexose must be linked at the phenolic moiety and compound 80 was thus characterized as H4-O-hexoside(8-5)H.
Compound 81: H 8-O-hexoside(8-5)H 8-O-hexoside
Compound 81 eluted at 7.50 min. The parent ion with m/zexp 621.219 suggested the chemical formula to be C30H37O14- (m/zth 621.219). The MS/MS showed a neutral loss of 162.062 Da (resulting in m/zexp 459.157), which can be attributed to the loss of a dehydrated hexose. Further fragments with m/z 441.151 and 429.157 can be assigned to a loss of H2O and CH2O, respectively, and hint the structure to bear a (8-5) functionality (Morreel et al., 2003, 2010a, 2010b). Fragments m/z 279.099 and 249.083 further hint a H(8-5)H aglycone structure (Morreel et al., 2010b, see also compound 80). In addition, m/z 173.062 and 161.033 have been reported before as fragments of H(8-5)H, as [M - H+ - H2O - 1,2A]- and [M - H+ - CH2O - 1,2A]-, respectively (Morreel et al., 2010b). The origin of m/z 179.047 is unclear, but might originate from the hexose moiety (m/zth 179.056). Thus, compound 81 exist out of a H(8-5)H aglycone linked to two hexose moieties. The fact that the compound is easily charged and does not elute as the formic acid adduct, hints that the phenolic unit is free. In addition, the fact the loss of a single hexose is sufficient to allow the typical (8-5) loss of H2O and CH2O, means that the 8 position of the A unit is linked with one hexose. The remaining hexose must thus be linked to the 8 position of the B-unit. Therefore, compound 81 is characterised as H 8-O-hexoside(8-8)H 8-O-hexoside.
Compound 82: H/G(8-5)G/H 4-O-hexoside (+ formic acid)
Compound 82 eluted at 8.53 min. The parent ion with m/zexp 535.181 suggested the chemical formula to be C26H31O12- (m/zth 535.182). The elucidation of MS/MS was similar as the one of compound 80. However, the intensity of the smaller fragments is insufficient to distinguish whether the A unit is H and the B unit is G or the other way around.