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A sucrose ferulate cycle linchpin for feruloylation of arabinoxylans in plant commelinids

Nature Plants volume 10, pages 1389–1399 (2024) Cite this article

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Abstract

A transformation in plant cell wall evolution marked the emergence of grasses, grains and related species that now cover much of the globe. Their tough, less digestible cell walls arose from a new pattern of cross-linking between arabinoxylan polymers with distinctive ferulic acid residues. Despite extensive study, the biochemical mechanism of ferulic acid incorporation into cell walls remains unknown. Here we show that ferulic acid is transferred to arabinoxylans via an unexpected sucrose derivative, 3,6-O-diferuloyl sucrose (2-feruloyl-O-α-d-glucopyranosyl-(1′→2)-3,6-O-feruloyl-β-d-fructofuranoside), formed by a sucrose ferulate cycle. Sucrose gains ferulate units through sequential transfers from feruloyl-CoA, initially at the O-3 position of sucrose catalysed by a family of BAHD-type sucrose ferulic acid transferases (SFT1 to SFT4 in maize), then at the O-6 position by a feruloyl sucrose feruloyl transferase (FSFT), which creates 3,6-O-diferuloyl sucrose. An FSFT-deficient mutant of maize, disorganized wall 1 (dow1), sharply decreases cell wall arabinoxylan ferulic acid content, causes accumulation of 3-O-feruloyl sucrose (α-d-glucopyranosyl-(1′→2)-3-O-feruloyl-β-d-fructofuranoside) and leads to the abortion of embryos with defective cell walls. In vivo, isotope-labelled ferulic acid residues are transferred from 3,6-O-diferuloyl sucrose onto cell wall arabinoxylans. This previously unrecognized sucrose ferulate cycle resolves a long-standing mystery surrounding the evolution of the distinctive cell wall characteristics of cereal grains, biofuel crops and related commelinid species; identifies an unexpected role for sucrose as a ferulate group carrier in cell wall biosynthesis; and reveals a new paradigm for modifying cell wall polymers through ferulic acid incorporation.

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Fig. 1: The maize dow1 mutant has defective cell walls.
The alternative text for this image may have been generated using AI.
Fig. 2: Conversion of Peak 1 to Peak 2 by transfer of FA from Fer-CoA catalysed by recombinant MBP–DOW1, and in vitro conversion of Peak 2 to Peak 3 mediated by a cell membrane extract from wild-type kernels.
The alternative text for this image may have been generated using AI.
Fig. 3: Recombinant BAHD transferases MBP–SFT1, MBP–SFT2, MBP–SFT3 and MBP–SFT4 catalyse the synthesis of 3-O-Fer sucrose (Peak 1) from Fer-CoA and sucrose.
The alternative text for this image may have been generated using AI.
Fig. 4: Incorporation of FA from sucrose 3,6-O-diFer sucrose (Peak 2) and intermediates into cell wall arabinoxylans.
The alternative text for this image may have been generated using AI.
Fig. 5: A proposed sucrose ferulate cycle required for ferulate incorporation into arabinoxylans of grass-type plant cell walls.
The alternative text for this image may have been generated using AI.

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Acknowledgements

We thank T. Beuerle for the gift of recombinant plasmid 4CL/pCRT7 for the prokaryotic expression of 4Cl and S. Cao and M. Hou for assistance in RT–qPCR, confocal and DNA blotting experiments. This research was supported by the National Natural Science Foundation of China (project no. 32230075, B.-C.T.).

Author information

Authors and Affiliations

  1. Key Laboratory of Plant Development and Environmental Adaptation Biology, Ministry of Education, School of Life Science, Shandong University, Qingdao, China

    Dalin Yang, Hui Liu & Bao-Cai Tan

  2. Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences, Shenzhen, China

    Xiaojie Li

  3. Guangdong Provincial Key Laboratory of Plant Molecular Breeding, College of Agriculture, South China Agricultural University, Guangzhou, China

    Yafeng Zhang

  4. State Key Laboratory of Microbial Technology, Shandong University, Qingdao, China

    Xingwang Zhang, Mingyu Liu & Shengying Li

  5. School of Life Sciences, Qilu Normal University, Jinan, China

    Huanhuan Yang

  6. Hoirticultural Sciences Department, University of Florida, Gainesville, FL, USA

    Karen E. Koch & Donald R. McCarty

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Contributions

D.Y. and B.-C.T. conceived and designed the experiments. D.Y. performed most of the experiments. X.L., Y.Z. and H.Y. cloned Dow1. X.Z., M.L. and S.L. analysed the NMR data, and X.Z. contributed to experimental discussions. H.L. performed the prokaryotic expression of SFT1 to SFT4. D.Y. and B.-C.T. analysed the data and wrote the paper. K.E.K. and D.R.M. revised the paper.

Corresponding author

Correspondence to Bao-Cai Tan.

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Extended data

Extended Data Fig. 1 Genetic verification of Dow1 and proteins prokaryotic expression.

a, b, 18 DAP ears segregating for the dow1-1 (a) and dow1-2 (b) mutation, respectively. c, dow1-1/dow1-2 ear. d, dow1-2/dow1-1 ear; Bar=2 cm in (a), (b), (c), and (d). e, RT-PCR analysis of Dow1 expression in the dow1 mutants. Total RNA was isolated from the dow1 mutants and WT embryos at 15 DAP, reverse-transcribed, and used as templates in the RT-PCR analysis. f, Southern blot analysis identified a 2.8 kb Spe I fragment carrying a Mu8 insertion linked to the Dow1 mutation. DNAs from a segregating F2 population of dow1-1 were digested with Spe I, blotted, and hybridized with a Mu8-specific probe. N, non-segregating (WT); S, segregating (heterozygous). g, Specificity of DOW1 antibody. T: total proteins from 7-day-old seedlings; WT: total proteins from 15 DAP WT embryos; dow1-1 and dow1-2: total proteins from 15 DAP dow1-1 and dow1-2 embryos. For each sample, 60 μg total protein was loaded. h, MBP-DOW1, MBP-SFT1, MBP-SFT2, MBP-SFT3, MBP-SFT4, and MBP prokaryotic expression. For each protein, 2 μg protein was loaded. MBP-DOW1: 90 kD; MBP-SFT1: 91 kD; MBP-SFT2: 91 kD; MBP-SFT3: 91 kD; MBP-SFT4: 90; MBP: 44 kD. Experiments were repeated independently with similar results at least 3 times (e-h).

Source data

Extended Data Fig. 2 Dow1 encodes a BAHD acyltransferase protein localized in the cytosol and membrane.

a, Gene structure of Dow1 and locations of the Mu insertions in two independent alleles. Exons are closed boxes, and introns are lines. b, Co-transfecting the maize protoplasts, Arabidopsis protoplasts, and tobacco leaves with GFP-fused DOW1 and RFP-fused BiP, GFP-fused DOW1 and RFP-fused BiP, RFP-fused DOW1 and GFP-fused BiP. Scale bar=5 μm for maize protoplasts, 5 μm for Arabidopsis protoplasts, and 20 μm for tobacco leaves. BiP-RFP was used to indicate endoplasmic reticulum (ER). BF, bright field. c, Immunoblot analysis of total proteins and nucleus proteins. DOW1 does not accumulate in the nucleus. Anti-H3 was used to indicate the nucleus. T: total proteins from 3-day-old seedlings; N: nuclear protein. d, Immunoblot analysis of cytoplasm non-membranous proteins and cell total membranous proteins. Cyt: Cytoplasm non-membranous proteins; CM1: cell total membranous proteins; CM2: 5 times the concentration of CM1; W: supernatant of washing cell total membranous proteins; Anti-BiP was used to indicate ER. Abs, antibodies. e, Immunoblot analysis of the fractions obtained from maize seedlings by a sucrose density gradient centrifugation. The distribution pattern of DOW1 is identical to that of BiP, which is an ER marker, indicating that DOW1 is localized to ER. Anti-BiP and anti-PIP1s antibodies were used to indicate ER and plasma membrane. Abs, antibodies. f, RT-qPCR analysis of the Dow1 transcript levels in major maize tissues. Error bars=mean ± SD (n = 3 replicates of assays with independent samples). Total RNA was isolated from all tissues, reverse-transcribed, and used as templates. Kernels are indicated as DAP (days after pollination). Experiments were repeated independently with similar results at least 3 times (b-e).

Source data

Extended Data Fig. 3 Preparations of 6-[2H3]-P2, 6-[2H3]-P3 and 3-[2H3]-P2.

a, Preparation of 6-[2H3]-P2. b, Preparation of 6-[2H3]-P3. c, Preparation of 3-[2H3]-P2. [2H3]-FA: [2H3]-ferulic acid, [2H3]-Fer-CoA: [2H3]-feruloyl-CoA. d, HPLC analysis of 21.58 uM [2H3]-FA (trace 1), 21.58 uM 6-[2H3]-P2 (trace 2), and 21.58 uM 3-[2H3]-P2 (trace 3). e, HPLC analysis of 18.69 uM [2H3]-FA (trace 1) and 18.69 uM 6-[2H3]-P3 (trace 2). These three labeled compounds were used for the subsequent labeling experiment. f, HPLC-HR-MS spectra analysis of 6-[2H3]-P2, 3-[2H3]-P2, and 6-[2H3]-P3. For 6-[2H3]-P2, 3-[2H3]-P2 and 6-[2H3]-P3, m/z 696.2215, 696.2288, 520.1832 corresponding to [M-H]- respectively. For unlabelled Peak 2 and Peak 3, the observed m/z of [M-H]- are 693.2003 and 517.1568, respectively.

Extended Data Fig. 4 HPLC-HR-MS analysis of cell wall esterified FA from 3-[2H3]-P2, 6-[2H3]-P2 and [2H3]-FA treatment groups and control.

a, EIC of cell wall esterified FA (193.0495, C10H9O4, [M-H]-) from control (1), and 13.9 μM 3-[2H3]-P2 (2), 13.9 μM 6-[2H3]-P2 (3), 13.9 μM [2H3]-FA (4) and 27.8 μM [2H3]-FA (5) treatment groups. b, EIC of cell-wall esterified [2H3]-FA (196.0684, C10H6D3O4, [M-H]-) from traces unlabeled controls (trace 1), and seedlings fed with 13.9 μM 3-[2H3]-P2 (2), 13.9 μM 6-[2H3]-P2 (3), 13.9 μM [2H3]-FA (4), or 27.8 μM [2H3]-FA (5). The ion chromatogram marked red is selected to show the observed mass spectra of FA from the control and the treatment groups (c). c, Mass spectra of FA (Calcd. of C10H9O4 is 193.0495, [M-H]-) and [2H3]-FA (Calcd. of C10H6D3O4 is 196.0684, [M-H]-) from the control and 13.9 μM 3-[2H3]-P2, 13.9 μM 6-[2H3]-P2, 13.9 μM [2H3]-FA and 27.8 μM [2H3]-FA treatment groups.

Extended Data Fig. 5 HPLC-HR-MS analysis of cell wall esterified non-decarboxylated diFA from 3-[2H3]-P2, 6-[2H3]-P2, [2H3]-FA treatment groups and control.

a, EIC of non-decarboxylated di-FA (385.0918, C20H17O8, [M-H]-). a-k refers to the EIC of non-decarboxylated di-FA. b, EIC of single FA-labeled non-decarboxylated diFA ([2H3]-non-decarboxylated diFA) (388.1106, C20H14D3O8, [M-H]-). a–j: EIC from treatment groups compared to control. c, EIC of double FA-labeled non-decarboxylated diFA ([2H6]-non-decarboxylated diFA) (391.1295, C20H11D6O8, [M-H]-). a–j: EIC from treatment groups compared to control. 1: control; 2 to 5: 13.9 μM 3-[2H3]-P2, 13.9 μM 6-[2H3]-P2, 13.9 μM [2H3]-FA and 27.8 μM [2H3]-FA treatment groups. Ion chromatogram marked red is selected to show the observed mass spectra of non-decarboxylated di-FA, [2H3]-non-decarboxylated diFA, and [2H6]-non-decarboxylated diFA from control and the treatment groups (d). d, Mass spectra of non-decarboxylated diFA (Calcd. of C20H17O8 is 385.0918, [M-H]-), [2H3]-non-decarboxylated diFA (Calcd. of C20H14D3O8 is 388.1106, [M-H]-), [2H6]-non-decarboxylated diFA (Calcd. of C20H11D6O8 is 391.1295, [M-H]-) from control and 13.9 μM 3-[2H3]-P2, 13.9 μM 6-[2H3]-P2, 13.9 μM [2H3]-FA, 27.8 μM [2H3]-FA treatment groups.

Extended Data Fig. 6 HPLC-HR-MS analysis of cell wall esterified decarboxylated diFA from 3-[2H3]-P2, 6-[2H3]-P2, [2H3]-FA treatment groups and control.

a, EIC of decarboxylated diFA (341.1020, C19H17O6, [M-H]-). a-h: refer to EIC of decarboxylated diFA. b, EIC of single FA-labeled decarboxylated diFA ([2H3]-decarboxylated diFA) (344.1208, C19H14D3O6, [M-H]-). c, EIC of double FA-labeled decarboxylated diFA ([2H6]-decarboxylated diFA) (347.1396, C19H11D6O6, [M-H]-). 1: control; 2 to 5: 13.9 μM 3-[2H3]-P2, 13.9 μM 6-[2H3]-P2, 13.9 μM [2H3]-FA and 27.8 μM [2H3]-FA treatment groups. a-h: EIC from treatment groups compared to control. Ion chromatogram marked red is selected to show the observed mass spectra of [2H3]-decarboxylated diFA and [2H6]-decarboxylated diFA from the control and the treatment groups (d). d, Mass spectra of decarboxylated diFA (Calcd. of C19H17O6 is 341.1020, [M-H]-), [2H3]-decarboxylated diFA (Calcd. of C19H14D3O6 is 344.1028, [M-H]-), [2H6]-decarboxylated diFA (Calcd. of C19H11D6O6 is 347.1396, [M-H]-) from control and 13.9 μM 3-[2H3]-P2, 13.9 μM 6-[2H3]-P2, 13.9 μM [2H3]-FA and 27.8 μM [2H3]-FA treatment groups.

Extended Data Fig. 7 HPLC-HR-MS analysis of cell wall esterified FA from 6-[2H3]-P3, and [2H3]-FA treatment groups and control.

a, EIC of cell wall esterified FA from control (1), 6.95 μM 6-[2H3]-P3 (2), and 6.95 μM [2H3]-FA (3) treatment groups. c: trans-FA; d: cis-FA. The ion chromatogram marked red is selected to show the observed mass spectra of FA from the control and the treatment groups (c). b, The EIC of cell-wall esterified [2H3]-FA from control seedlings (1) and 6.95 μM 6-[2H3]-P3 (2), 6.95 μM [2H3]-FA (3) treatments. The ion chromatogram marked red shows the observed mass spectra of [2H3]-FA fed seedlings (c). c: trans-FA; d: cis-FA. c, Mass spectra of FA (Calcd. of C10H9O4 is 193.0495, [M-H]-), and [2H3]-FA (Calcd. of C10H6D3O4 is 196.0684, [M-H]-) from control and 6.95 μM 6-[2H3]-P3, 6.95 μM [2H3]-FA treatment groups.

Extended Data Fig. 8 HPLC-HR-MS analysis of cell wall esterified non-decarboxylated diFA from 6-[2H3]-P3, [2H3]-FA treatment groups and control.

a, EIC of non-decarboxylated diFA (385.0918, C20H17O8, [M-H]-). a-k refers to non-decarboxylated diFA. b, EIC of [2H3]-non-decarboxylated diFA (388.1106, C20H14D3O8, [M-H]-). a-i: EIC from treatment groups compared to control. 1: control; 2: 6.95 μM 6-[2H3]-P3 treatment group; 3: 6.95 μM [2H3]-FA treatment group. Ion chromatogram marked red is selected to show the observed mass spectra of non-decarboxylated diFA and [2H3]-non-decarboxylated diFA from the control and the treatment groups (c). c, Mass spectra of non-decarboxylated diFA (Calcd. of C20H17O8 is 385.0918, [M-H]-), and [2H3]-non-decarboxylated diFA (Calcd. of C20H14D3O8 is 388.1106, [M-H]-) from control and 6.95 μM 6-[2H3]-P3, 6.95 μM [2H3]-FA treatment groups.

Extended Data Fig. 9 HPLC-HR-MS analysis of cell wall esterified decarboxylated diFA from 6-[2H3]-P3, [2H3]-FA treatment groups and control.

a, EIC of decarboxylated diFA (341.1020, C19H17O6, [M-H]-). a-h refers to decarboxylated diFA. b, EIC of [2H3]-decarboxylated diFA (344.1208, C19H14D3O6, [M-H]-). a-h: EIC from treatment groups compared to control. 1: control; 2: 6.95 μM 6-[2H3]-P3 treatment group; 3: 6.95 μM [2H3]-FA treatment group. Ion chromatogram marked red is selected to show the observed mass spectra of decarboxylated diFA and [2H3]-decarboxylated diFA from the control and the treatment groups (c). c, Mass spectra of decarboxylated diFA (Calcd. of C19H17O6 is 341.1020, [M-H]-), and [2H3]-decarboxylated diFA (Calcd. of C19H14D3O6 is 344.1028, [M-H]-) from control and 6.95 μM 6-[2H3]-P3, 6.95 μM [2H3]-FA treatment groups.

Extended Data Fig. 10 HPLC-MS analysis of root cell wall hydroxycinnamate (HCA) conjugates from the control and 6-[2H3]-P3, [2H3]-FA treatment groups by the 50 mM TFA mild acidolysis method.

a, EIC of cell wall FA-Ara (325.0918, C15H18O8, [M-H]-) from control (1) and 6.95 μM 6-[2H3]-P3 (2), 6.95 μM [2H3]-FA (3) treatment groups. b, EIC of [2H3]-FA-Ara (328.1106, C15H15D3O8, [M-H]-) from control (1) and 6.95 μM 6-[2H3]-P3 (2), 6.95 μM [2H3]-FA (3) treatment groups. c, Mass spectra of [2H3]-FA-Ara (328.1106, C15H15D3O8, [M-H]-) from control and 6.95 μM 6-[2H3]-P3, 6.95 μM [2H3]-FA treatment groups. d, EIC of cell wall Ara-FA-FA-Ara (649.1763, C30H34O16, [M-H]-) from the control (1) and 6.95 μM 6-[2H3]-P3 (2), 6.95 μM [2H3]-FA (3) treatment groups. e, EIC of Ara-[2H3]-FA-FA-Ara (652.1905, C30H31D3O16, [M-H]-) from the control (1) and 6.95 μM 6-[2H3]-P3 (2), 6.95 μM [2H3]-FA (3) treatment groups. f, Mass spectra of Ara-[2H3]-FA-FA-Ara (652.1905, C30H31D3O16, [M-H]-) from the control (1) and 6.95 μM 6-[2H3]-P3 (2), 6.95 μM [2H3]-FA (3) treatment groups.

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Yang, D., Liu, H., Li, X. et al. A sucrose ferulate cycle linchpin for feruloylation of arabinoxylans in plant commelinids. Nat. Plants 10, 1389–1399 (2024). https://doi.org/10.1038/s41477-024-01781-1

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