Supplement I. 1H and 31P-NMR spectra of cell wall standards
Standards of cell wall precursors were prepared at a theoretical concentration of 10mM and 10mM of methylphosphonic acid was added to each one as internal standard. Each standard was dried and resuspended in 600µL of D2O for 1H-NMR and 31P-NMR analyses. Spectra were acquired in a Varian 600MHz at 25°C using a 5 mm probe.1H NMR spectra were obtained at 599.82 MHz with a pulse of 11.5 µs (corresponding to an angle of 90°) using a relaxation time of 20s. 31P NMR spectra were obtained at 242.81 MHz with a pulse of 14.0 µs (corresponding to an angle of 90°) using a relaxation time of 45s.
(A)1H-NMR of Glc1P. The concentration of Glc1P was assessed by comparing the area of the 1H carried by the C1 of Glc1P to the area of the three 1H of the internal standard; (B)31P-NMR of Glc1P: The area of the 31P of Glc1P was compared to the area of the 31P of the methylphosphonic acid; (C)1H-NMR of UDP-GlcA; The areas of the 1H of the uracil group and the 1H of the glucuronic acid group are similar, showing that all the UDP is linked to the glucuronic acid; (D)31P-NMR of UDP-GlcA: The areas of the two 31P of UDP were compared to the area of the 31P of the methylphosphonic acid (E)1H-NMR of UDP-Xyl: There are several peaks between 5.0 and 5.8 ppm, revealing that UDP-Xyl is not pure. Furthermore, the area of the 1H of the uracil group is larger than one of the xylose group, which involves that there are other UDP-sugars and free UDP. The UDP-Xyl was quantified by comparing the area of the xylose group to the one of the internal standard.
Supplement II. 1H and 31P chemical shifts of cell wall standards
Standards of cell wall precursors were prepared and analysed as described in Supplement I. Chemical shifts (in ppm) for 1H and 31P-NMR are given below.Note that both 1H and 31P chemical shifts are sensitive to pH and other ion interactions.
Metabolite / 1H-NMR / 31P-NMRchemical group / H-shift (ppm) / P1-shift (ppm) / P2-shift (ppm)
ADP-Glc / H-adenine / 8.60 / -7.59 / -9.27
H-adenine / 8.37
H-ribose / 6.13
H-C1Glc / 5.53
Fru6P / nd / 4.85
Gal1P / H-C1Gal / 5.45 / 4.18
GDP-sugar / H-guanine / 8.70
H-ribose / 5.98
GDP-Fuc / H-C1Fuc / 5.39 / -7.47 / -9.22
GDP-Glc / H-C1Glc / 5.53 / -7.55 / -9.08
GDP-Man / H-C1Man / 5.44 / -7.98 / -10.28
Glc1P / H-C1Glc / 5.42 / 3.51
Glc6P / H-C1αGlc / 5.18 / 2.85
H-C1βGlc / 4.60
Man1P / H-C1Man / 5.33 / 2.18
Man6P / H-C1αMan / 5.11 / 2.9
H-C1βMan / 4.84
UDP-sugar / H-uracil / 7.88
UDP-Ara / H-C1Ara / 5.53 / nd
UDP-Gal / H-C1Gal / 5.47 / -8.90 / -10.83
UDP-GalA / H-C1GalA / 5.60 / -5.92 / -7.35
UDP-GalNAc / H-C1GalNAc / 5.47 / -9.24 / -10.83
-CH3 / 2.00
UDP-Glc / H-C1Glc / 5.53 / -9.14 / -10.78
UDP-GlcA / H-C1GlcA / 5.56 / -6.91 / -8.76
UDP-GlcNAc / H-C1GlcNAc / 5.44 / -9.29 / -10.98
-CH3 / 2.00
UDP-Xyl / H-C1Xyl / 5.48 / -8.53 / -10.21
Supplement III. Real concentration of standards of cell wall precursors
Standards of cell wall precursors were prepared at a theoretical concentration of 10mM, with the exception of UDP-GalA at 5mM. Real concentrations were determined by 1H-NMR and/or 31P-NMR after adding 10mM methylphosphonic acid as internal standard (as described in Supplement I).
Metabolite / Theoretical concentration (mM) / Real concentration (mM) / % differenceADP-Glc / 10.0 / 4.9 / 50.9
Fru6P / 9.9 / 8.1 / 18.1
Gal1P / 10.0 / 10.4 / 4.3
GDP-Fuc / 10.0 / 1.9 / 80.8
GDP-Glc / 10.0 / 9.0 / 10.2
GDP-Man / 10.0 / 8.3 / 17.5
Glc1P / 8.4 / 8.7 / 3.8
Glc6P / 10.5 / 11.2 / 6.4
Man1P / 10.0 / 4.6 / 54.1
Man6P / 10.5 / 11.5 / 9.4
UDP-Ara / 10.0 / 1.1 / 89.1
UDP-Gal / 10.0 / 4.3 / 56.6
UDP-GalA / 5.0 / 5.2 / 3.5
UDP-GalNAc / 10.0 / 4.7 / 52.9
UDP-Glc / 10.1 / 4.2 / 58.5
UDP-GlcA / 10.0 / 8.7 / 12.8
UDP-GlcNAc / 10.0 / 8.3 / 17.5
UDP-Xyl / 10.0 / 1.0 / 90.5
Supplement IV. LC-MS/MS analyses of Arabidopsis cell extracts
Arabidopsis T87 cell suspension culture were sampled as described in Material and Methods. Intracellular metabolites were extracted in boiling water, filtered and concentrated before being analyzed by LC-MS/MS using the separation and hardware described in the Material and Methods. (A)Hexose-Ps were monitored by LC-MS/MS at the transition 259/97. (B) NDP-sugars were analyzedby LC-MS/MS at different transitions: tr606/79 for UDP-GalNAc and UDP-GlcNAc, tr604/79 for GDP-Man and GDP-Glc, tr588/159 for ADP-Glc and GDP-Fuc, tr579/159 for UDP-GlcA and UDP-GalA, tr565/79 for UDP-Gal and UDP-Glc, tr549/79 for UDP-Rha, tr535/79 for UDP-Ara, UDP-Xyl and UDP-Api.
Supplement V. Extraction of the mass spectrum of glucose from UDP-Glc
In order to determine the mass spectrum of the sugar component (black bars) of UDP-Glc, the labeling of the free nucleotide (UMP; white bars) was used to correct themass spectrum of UDP-Glc (grey bars). The correction uses a subtraction algorithm that assumes the labeling in the nucleotide moiety is independently distributed relative to the labeling in the sugar component.