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RESEARCH ARTICLE
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Integrative Dissection of Lignin Composition in Tartary
Buckwheat Seed Hulls for Enhanced Dehulling Efficiency
Wenqi Yang, Haiyang Duan, Ke Yu, Siyu Hou, Yifan Kang, Xiao Wang, Jiongyu Hao,
Longlong Liu, Yin Zhang, Laifu Luo, Yunjun Zhao, Junli Zhang, Chen Lan, Nan Wang,
Xuehai Zhang, Jihua Tang, Qiao Zhao,* Zhaoxia Sun,* and Xuebin Zhang*
The rigid hull encasing Tartary buckwheat seeds necessitates a laborious
dehulling process before flour milling, resulting in considerable nutrient loss.
Investigation of lignin composition is pivotal in understanding the structural
properties of tartary buckwheat seeds hulls, as lignin is key determinant of
rigidity in plant cell walls, thus directly impacting the dehulling process. Here,
the lignin composition of seed hulls from 274 Tartary buckwheat accessions is
analyzed, unveiling a unique lignin chemotype primarily consisting of G
lignin, a common feature in gymnosperms. Furthermore, the hardness of the
seed hull showed a strong negative correlation with the S lignin content.
Genome-wide detection of selective sweeps uncovered that genes governing
the biosynthesis of S lignin, specifically two caffeic acid O-methyltransferases
(COMTs) and one ferulate 5-hydroxylases, are selected during domestication.
This likely contributed to the increased S lignin content and decreased
hardness of seed hulls from more domesticated varieties. Genome-wide
association studies identified robust associations between FtCOMT1 and the
accumulation of S lignin in seed hull. Transgenic Arabidopsis comt1 plants
expressing FtCOMT1 successfully reinstated S lignin content, confirming its
conserved function across plant species. These findings provide valuable
metabolic and genetic insights for the potential redesign of Tartary buckwheat
seed hulls.
W. Yang, K. Yu, Y. Kang, X. Wang, J. Zhang, C. Lan, X. Zhang
State Key Laboratory of Crop Stress Adaptation and Improvement
Henan Joint International Laboratory for Crop Multi-Omics Research
School of Life Sciences
Henan University
Kaifeng , China
E-mail: xuebinzhang@henu.edu.cn
H. Duan, X. Zhang, J. Tang
National Key Laboratory of Wheat and Maize Crop Science
College of Agronomy
Henan Agricultural University
Zhengzhou , China
The ORCID identification number(s) for the author(s) of this article
can be found under https://doi.org/./advs.
© The Authors. Advanced Science published by Wiley-VCH GmbH.
This is an open access article under the terms of the Creative Commons
Attribution License, which permits use, distribution and reproduction in
any medium, provided the original work is properly cited.
DOI: 10.1002/advs.202400916
1. Introduction
Buckwheat is considered a functional food
due to its high content of aromatic com-
pounds, including flavonoids and pheno-
lic acids, which are well-documented for
their antioxidant, anticancer, and anti-
inflammatory activities.[1–4]Common buck-
wheat (Fagopyrum esculentum Moench)
and Tartary buckwheat (Fagopyrum tatar-
icum (L.) Gaertn.) are widely domesticated
and consumed around the world.[5]Tart a r y
buckwheat originated in southwest China
and is currently grown on marginal lands
in mountainous area and Himalayas, and
in several other countries and regions, in-
cluding Japan, Canada, and Europe.[6,7]It
has height tolerance to harsh climates and
acidic soils containing aluminum, which is
toxic to other crops.[8]Compared with com-
mon buckwheat, Tartary buckwheat, in par-
ticular, contains higher level of aromatic
compounds in its seeds, such as rutin, mak-
ing it of higher nutritional and medicinal
value.[9]However, the seeds of all Tartary
S. Hou, J. Hao, Y. Zhang, Z. Sun
College of Agriculture
Shanxi Agricultural University
Taigu , China
E-mail: zhaoxiasun@sxau.edu.cn
S. Hou, Z. Sun
Houji Lab of Shanxi Province
Taiyuan , China
L. Liu
Center for Agricultural Genetic Resources Research
Shanxi Agricultural University
Taiyuan , China
L. Luo, Y. Zhao
Key Laboratory of Plant Carbon Captureand CAS Center for Excellence in
Molecular Plant Sciences
Chinese Academy of Sciences
Shanghai , China
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buckwheat cultivars are encased in thick and rigid hulls, which
are difficult to be removed during the milling process.[10–12]
The current dehulling process, involving boiling, results in
low efficiency, broken groats, and nutrient loss, representing
a significant processing bottleneck in the Tartary buckwheat
industry.[12,13]
Lignin is one of the predominant cell wall components in the
seed hulls of both common and Tartary buckwheat.[10–12]Lignin
polymer crosslinks cell wall polysaccharides, providing strength,
rigidity, and hydrophobicity.[14,15]Recently, by employing solid-
state nuclear magnetic resonance (ssNMR), the nanoscale in-
teractions between lignin polymer and polysaccharides cellu-
lose, galactoglucomannan and xylan were unveiled,[16,17]which
could contribute the mechanical strengths of secondary cell
wall. In most plant tissues analyzed to date, the lignin poly-
mers are mainly synthesized from three monolignols, namely
p-coumaryl, coniferyl, and sinapyl alcohols, differing in their de-
gree of methoxylation.[18]Monolignols are synthesized in the cy-
tosol through the phenylpropanoid pathway and are exported
into the apoplastic space via diffusion driven by a concentration
gradient.[19]Once enter the apoplastic space, these monolignols
are integrated into the growing lignin polymer through radical
coupling to give rise to p-hydroxyphenyl (H), guaiacyl (G), and sy-
ringyl (S) lignin units, respectively.[14,15,20]Several cell-wall-bound
peroxidases, laccase and mitochondrial ascorbate peroxidase par-
ticipate in the oxidative lignin polymerization.[21,22]In addition,
the Casparian strip-located endodermal family of dirigent pro-
tein complex directs lignin polymerization on Casparian strip.[23]
In the polymerization process, lignin units are primarily inter-
linked via 𝛽-O-4 ether bonds. Interunit C–C linkages, including
𝛽-𝛽,𝛽-5, 5-5, are also present in the lignin polymer.[15,24]C─C
bonds, characterized by shorter bond lengths and higher dissoci-
ation energy than ether bonds, significantly influence the physic-
ochemical properties of the lignin polymer.[25]Different mono-
lignols have varying numbers of methoxy groups at the aromatic
ring, which could prevent the formation of interunit C–C link-
ages. Hence, the proportion ratio of these units determines the
diversity of interunit linkages, thereby influencing the physico-
chemical properties of the lignin polymer.[24,25]For example, a
higher proportion of S units would result in a lower proportion
of C–C linkages, making the lignin polymer less condensed and
more vulnerable to chemical or physical treatments.
Current knowledge of the lignin composition in buckwheat
seed hull is limited. In this study, by investigating the lignin com-
position of seed hull from 274 Tartary buckwheat accessions, we
unveiled a distinctive composition primarily consisting of G units
(over 75%), with relatively low levels of S and H units. Correla-
tion analysis indicated a robust negative correlation between the
S unit content in the lignin polymer and the hardness of the seed
N. Wang, Q. Zhao
Shenzhen Key Laboratory of Synthetic Genomics
Guangdong Provincial Key Laboratory of Synthetic Genomics
Key Laboratory of Quantitative Synthetic Biology
Shenzhen Institute of Synthetic Biology
Shenzhen Institute of Advanced Technology
Chinese Academy of Sciences
Shenzhen , China
E-mail: qiao.zhao@siat.ac.cn
hull. Genome-wide detection of selective sweeps and associa-
tion studies pinpointed that genes responsible for sinapyl alcohol
biosynthesis have undergone selection during modern breeding
or are associated with S unit proportion in seed hulls. Express-
ing FtCOMT1, one candidate gene that encodes a caffeic acid O-
methyltransferase, in Arabidopsis mutant comt1 successfully rein-
troduced S lignin units into the lignin polymer. These results ex-
panded our understanding of lignin composition and its impact
on the mechanical strength of buckwheat seed hulls, providing
valuable insights for further engineering desirable lignin traits
to facilitate the dehulling process.
2. Results
2.1. Lignin of Tartary Buckwheat Seed Hull is Primarily
Composed of G Units
In most dicotyledons, lignin polymer consists of G and S units,
and traces of H units can also be detected in dicotyledonous
angiosperm.[18]The exceptional toughness of Tartary buckwheat
seed hull makes us wonder whether it possesses a lignin compo-
sition that is different from other tissues or other dicots. Through
solution-state nuclear magnetic resonance (Solution NMR) spec-
troscopy, we analyzed the lignin composition of highly ligni-
fied stem tissue and seed hull of a Tartary buckwheat cultivar
(Heifeng No. 1). For simplicity, we define the total lignin units
as the sum of the H, G, and S units. The stem tissue exhibited a
regular lignin composition, in which S units represent 65.9% of
total lignin units, followed by 33.9% of G units and trace amount
of H units (Figure 1A). Interestingly, G units dominates the to-
tal lignin units (96.9%) in the seed hull, followed by 3.1% of H
units, while no S units were detected (Figure 1A). We then quan-
tified the abundance of each lignin unit in leaf, stem and see hull
of this Tartary buckwheat cultivar through thioacidolysis coupled
with gas chromatography-mass spectrometry (GC-MS). Charac-
teristic fragment ions of H, G, and S units, that are identical
to synthesized standards,[26]were detected in the thioacidolysis-
released products of all tested tissues (Figure 1B, Figure S1,Sup-
porting Information). In leaf, G units represent 72.2% of the
lignin units released by thioacidolysis, followed by 19.8% of S
units (Figure 2A). In stem, S units represent 75.3% of the lignin
units, followed by 24.5% of G unit (Figure 2A). In consistent with
the solution NMR results, lignin of seed hull was almost entirely
composed of G units (98.2%), with trace amount of H (0.1%) and
S (1.7%) units (Figure 2A). Significant differences of the ratio be-
tween S and G units (S/G ratio) were observed among tissues of
Tartary buckwheat, with the minimum of 0.016 in seed hull and
the maximum of 3.07 in stem (Figure 2B). Together, these results
demonstrate that G units are dominantly present, and S units are
depleted, in the lignin polymer of Tartary buckwheat seed hull.
2.2. S-Depleted Lignin Chemotype is Unique to Seed Hulls of
Tartary Buckwheat
The extremely low level of S units in Tartary buckwheat seed
hull is surprising, because G-dominant lignin chemotype is
commonly found in gymnosperms.[18]This S-depleted lignin
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Figure 1. Identification of H, G, and S lignin units in Tartary buckwheat cell wall. A) D NMR analysis of whole cell walls from Tartary buckwheat stem
and seed hull. Aromatic sub-regions of short range C–H correlation (HSQC) NMR spectra from cell wall gels of Tartary buckwheat stem and seed hull
are shown. Contour coloration matches that of lignin substructures displayed in each panel. G, Guaiacyl; S, Syringyl; H, p-Hydroxyphenyl; X, Cinnamyl
alcohol; FA, Ferulate; pCA, p-Coumarate. B) Selected ion chromatograms of three lignin monomers in the thioacidolysis-released products of indicated
Tartary buckwheat cell wall from GC-MS.
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Figure 2. S-depleted lignin chemotype is unique to seed hulls of Tartary buckwheat. A) Lignin composition of cell wall of indicated tissues from Tartary
buckwheat, Arabidopsis, and soybean. Error bars represent SD. B) S/G ratio of cell wall of indicated tissues from Tartary buckwheat, Arabidopsis,and
soybean. One-way ANOVA followed by Tukey’s honestly significant dierence test was used for statistical analysis (n =, p<.). All data points were
plotted to show the variation of data. Error bars represent SD. Letters indicate significant dierences. C) The cumulative expression of putative F5Hs
and COMTs in indicated tissues. One-way ANOVA followed by Tukey’s honestly significant dierence test was used for statistical analysis (n =, p<
.). All data points were plotted to show the variation of data. Error bars represent SD. Letters indicate significant dierences.
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Figure 3. Tartary buckwheat seed hull contain less S units and lower S/G ratio than that of common buckwheat. A) Photos of seeds of four Tartary
buckwheat varieties (Ft, Ft, Ft, and Ft) and two common buckwheat species (HHTQ and JQ). B) Lignin composition of seed hull cell wall
of indicated varieties. Error bars represent SD. C) S/G ratio of seed hull cell wall of indicated varieties. One-way ANOVA followed by Tukey’s honestly
significant dierence test was used for statistical analysis (n ≥, p<.). All data points were plotted to show the variation of data. Error bars represent
SD. Letters indicate significant dierences.
chemotype in seed hull prompted us to investigate whether it
is a common feature across different plant species. Seed hulls
are specialized structure developed from ovary wall to protect
the inner grains. Therefore, we analyzed the lignin composition
of the seed pods of Arabidopsis (Col-0 ecotype) and soybean
(W82 ecotype), two well-studied model dicotyledons, through
thioacidolysis coupled with GC-MS, as seed pods are as well de-
veloped from ovary wall. For comparison of lignin composition
in different tissues, we also took the leaf and stem tissues from
Arabidopsis and soybean for analysis. The results showed that
G and S units together account for the majority of lignin units
detected in all tested tissues from either Arabidopsis or soybean
(Figure 2A). Unlike the large variance exhibited by the propor-
tion of each lignin unit in different tissues of Tartary buckwheat,
the S/G ratio remains at a comparable level (around 0.4–0.6)
among different tissues in Arabidopsis and soybean (Figure 2B).
Collectively, these results suggest that the S-depleted lignin
chemotype in seed hull is unique to Tartary buckwheat.
To explore the genetic drivers influencing the variation of S
lignin unit proportion in different tissues, we performed tran-
scriptome analysis of the leaf, stem, and seed hulls (seed hulls
were harvested at three stages), and examined the expression
of genes that are specialized for the biosynthesis of sinapyl al-
cohol. Coniferaldehyde is the branching point for the biosyn-
thesis of coniferyl alcohol and sinapyl alcohol.[14,27]Ferulate 5-
hydroxylases (F5H) hydroxylate coniferaldehyde into 5-hydroxy-
coniferaldehyde, and then it is methylated by caffeic acid O-
methyltransferases (COMTs) into sinapaldehyde.[28,29]Transcrip-
tome analysis identified five copies of putative F5H and three
copies of putative COMT, and the cumulative expressions of both
F5Hs and COMTs are much higher in the stem tissue than other
tissues (Figure 2C). These results might explain the higher S unit
proportion found in the lignin of Tartary buckwheat stem tissues.
2.3. S Unit Content and S/G Ratio are Negatively Related to the
Hardness of Tartary Buckwheat Seed Hull
Despite the high nutritional value of Tartary buckwheat, its rigid
seed hull significantly restricts the dehulling process.[10,11]Be-
cause the proportion ratio of different lignin units could influ-
ence the physiochemical properties of the lignin polymer, we
speculated that the S-depleted lignin chemotype in Tartary buck-
wheat seed hull is related to its superior mechanical strength. It
is reported that the seed hull of common buckwheat is softer than
that of Tartary buckwheat.[30]To examine whether the lignin poly-
mer from common buckwheat seed hull would contain relatively
more S units, we analyzed the lignin composition of seed hulls
from randomly-selected four Tartary buckwheat varieties (Ft58,
Ft200, Ft268, and Ft271) and two common buckwheat varieties
(HHTQ and TQ, Figure 3A). As expected, the results showed that
although G units account for the majority of total lignin units in
the seed hulls of both buckwheat species (over 80%), the propor-
tion of S units, as well as the S/G ratio in common buckwheat
seed hull are significantly higher (Figure 3B), indicating that the
low proportion of S units of Tartary buckwheat seed hull might
contribute to its hardness.
We then performed population-level investigation on the
lignin composition (through thioacidolysis coupled with GC-MS)
and hardness of seed hulls from 274 Tartary buckwheat varieties.
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Figure 4. S unit content and S/G ratio are negatively related to the hardness of Tartary buckwheat seed hull. A) Population structure divided upon
breeding process and geographical distribution. B) Quantification of lignin monomers in the thioacidolysis-released products and hardness of seed
hull from Tartary buckwheat varieties. One-way ANOVA followed by Tukey’s honestly significant dierence test was used for statistical analysis (n=
, p<.). All data points were plotted to show the variation of data. Error bars represent SD. Letters indicate significant dierences. C) Pearson
correlation among variables in (B), the value and color of square represent the magnitude and the direction of the correlation. The probability values
(P-values) less than . are marked with **, indicating a strong statistical confidence.
These 274 varieties, collected from diverse habitats across con-
tinents, exhibit high phenotypic diversity (Figure 4A). Likewise,
the lignin composition and hardness of seed hulls from differ-
ent varieties exhibit large variation (Figure 4B). In a population
scale, the overall G units account for more than 75% of total
lignin units, and S units account for less than 13% (Figure 4B).
Pearson’s correlation analysis revealed an extremely strong cor-
relation between the content of G units and total lignin in seed
hull (Figure 4C), suggesting that variation of G unit content could
largely determine the variation of total lignin unit content. Inter-
estingly, the hardness of seed hull exhibited negative correlation
with S unit content and S/G ratio, but not with other traits in-
cluding G unit content (Figure 4C). In addition, the correlation
between S unit content exhibited stronger correlation with S/G
ratio than G unit content (Figure 4C), indicating that the content
of S unit and S/G ratio might be the key drivers of seed hull hard-
ness.
2.4. S Lignin Related Genes Undergone Selection During Tartary
Buckwheat Breeding
Domestication and modern breeding process could significantly
impact the genetic diversity of crops. As the tedious dehulling
process is unfavored in Tartary buckwheat industry, we specu-
lated that there might be a potential, if not dedicated, selection of
Tartary buckwheat varieties with lower hardness of seed hull. The
274 Tartary buckwheat varieties consist of six wild accessions,
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233 landraces, and 42 cultivars (Figure 4A), three groups that are
representing different stages of Tartary buckwheat domestication
and breeding. Hence, we examined whether differences in each
lignin unit and hardness of seed hull could be detected among
these groups. The results showed that the content of H and G
units, as well as total lignin units, are not different among differ-
ent groups (Figure 5A). On the contrary, we observed a signifi-
cantly higher S unit content, higher S/G ratio, and lower hard-
ness of seed hulls in widely domesticated cultivars of Tartary
buckwheat (Figure 5A), suggesting that the seed hulls of these
cultivars have become softer, as a result of an altered lignin com-
position with more S unit proportion, during the modern breed-
ing process.
F5H and COMT are the key enzymes for sinapyl alcohol
biosynthesis.[28,29]The potential selection for higher S lignin
units in Tartary buckwheat seed hull indicated that these genes
might be selected during modern breeding process. Therefore,
we performed genome-side scanning of selection sweeps by us-
ing available genetic polymorphism data of 274 Tartary buck-
wheat varieties. Tartary buckwheat has a 489.3 Mb genome.[7]
Analysis of FST values identified 3551 genomic regions that are
potentially selected by modern breeding process (FST >0.1)
(Table S3, Supporting Information). In these selective sweeps,
we observed that FtPinG0009454400, which encodes a predicted
O-methyltransferase (OMT) that catalyze the methylation of vari-
ous metabolites,[31–33]is consistently present in a highly selected
genomic region (Figure 5B).
Plant OMTs are categorized into two subgroups: caffeoyl-
CoA OMT (CCoAOMT) and COMT subgroups, according to the
substrate specificity.[33,34,35]In Arabidopsis,bothCCoAOMTand
COMT are involved in lignin biosynthesis. Because the extent
of sequence identity of OMTs shows a good correlation with
the structural classes of their methyl acceptor molecules,[31]we
performed phylogenetic analysis of FtPinG0009454400 and all
OMTs in Arabidopsis. The result showed that FtPinG0009454400
falls within the same clade together with AtCOMT1 (Figure S2,
Supporting Information). FtPinG0009454400 contains all five re-
gions that is conserved in COMT, and shares 66% and 66.8%
amino acid identity with AtCOMT1 and PtCOMT1, a COMT of
poplar,[36]respectively (Figure 4B), suggesting similar biochemi-
cal functions. Both AtCOMT1 and PtCOMT1 are responsible for
the biosynthesis of sinapyl alcohol.[28]Hence, we designated Ft-
PinG0009454400 as FtCOMT1. Together, these results suggest
that FtCOMT1, a gene likely related to S lignin unit content, had
undergone selection during Tartary buckwheat breeding.
We also found that FtCOMT2 (another putative COMT)was
selected in the comparison between wild species and more do-
mesticated varieties, and a putative F5H was selected in the com-
parison between wild species and cultivars (Figure 5B). Besides
genes dedicated to sinapyl alcohol biosynthesis, four other genes
involved in lignin biosynthesis, including Hydroxycinnamoyl-
CoA:shikimate/quinate hydroxycinnamoyl transferase (HCT) 6,
Cinnamyl alcohol dehydrogenase (CAD)1,Cinnamoyl-CoA reduc-
tase (CCR)4and CCoAOMT1, are present in the selective sweeps
identified in the comparison between wild species and more do-
mesticated varieties (Figure 5B). To further validate the identi-
fied selective sweeps, we performed Tajima’s D and nucleotide
diversity analysis, and focus on the genomic region containing
COMTs and F5Hs, due to their specific role for S lignin biosyn-
thesis. Both analyses suggested that these genes are located in ge-
nomic regions with reduced nucleotide diversity, at least in lan-
drace and cultivar (Figure S3A,B, Supporting Information). We
also zoomed into the genomic region containing each selected
gene, and found that FST indicate a selective sweep at the locus.
We then looked into the SNPs locating on each of the selected
genes, and for two out of three genes (FtCOMT1 and FtCOMT2),
we were able to find SNPs locating on the genes. Identically, the
frequencies of haplotype of both FtCOMT1 and FtCOMT2 varied
among three subpopulations, with landrace remaining in middle
consistently (Figure S3D, Supporting Information). These results
support the conclusion that these genes are selected by the tartary
buckwheat breeding. Based on these results, combing with the
lignin chemotype of seed hulls from Tartary buckwheat cultivars,
we propose that genes related to the biosynthesis of lignin, and
more specifically, S lignin units, have undergone selection dur-
ing the domestication and modern breeding process of Tartary
buckwheat.
2.5. FtCOMT1 is Associated with S/G Ratio in Tartary Buckwheat
Seed Hull
To identify the genetic basis controlling the accumulation of S
lignin unit in seed hull, we performed genome-wide associa-
tion studies (GWAS) by using six target traits including con-
tent of each unit, total lignin content, S/G ratio and hardness
(Figure 6A). In total, 3895 SNPs were identified to be associ-
ated with these traits, which defined 1078 non-redundant QTL
and 4716 candidate genes (Table S4, Supporting Information).
Among the candidate genes, FtCOMT1, a gene that was selected
during breeding of Tartary buckwheat, was found to be signifi-
cantly associated S/G ratio (Figure 6A,B), suggesting strong as-
sociation between FtCOMT1 and the accumulation of S lignin
units in seed hull.
COMT preferably methylate the 5-hydroxyl group of 5-
hydroxconiferaldehyde and 5-hydroxyconiferyl alcohol to
produce the precursors of S lignin unit.[37,38]Plants with
compromised activity of COMT show a significant decrease of
S units and an increased incorporation of 5-hydroxyguaiacyl
(5-OH-G) lignin units.[28,27]To investigate whether FtCOMT1
functions similarly with AtCOMT1, we created a transgenic Ara-
bidopsis line in Atcomt1 (a COMT-deficient mutant) background
by expressing FtCOMT1 under a 35S promoter (designated as
35Spro:FtCOMT1/Atcomt1, Figure S3, Supporting Information).
Analysis of the lignin composition in the highly lignified stem
tissue revealed no difference in either G or H lignin content
among Col-0, Atcomt1,and35Spro:FtCOMT1/Atcomt1, while S
lignin units are depleted and 5-OH-G units are highly enriched
(Figure 6C), which is consistent with previous reports.[28,39]
Heterologous expression of FtCOMT1 in Atcomt1 restored the
content of both S unit and 5-OH-G unit to the wild-type levels
(Figure 6C), suggesting a conserved function of COMT1 in both
Tartary buckwheat and Arabidopsis. Taken together, these results
suggest that FtCOMT1, a dedicated enzyme related to S lignin
units that had undergone selection during domestication and
breeding process of Tartary buckwheat, controls the S/G ratio
in the lignin polymer of seed hull, representing a promising
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Figure 5. Selection Elimination of lignin related genes in geographical distribution and breeding process. A) Lignin content and hardness of seed hull
from wild species, landraces and cultivars. Student’s T test was used for statistical analysis. All data points were plotted to show the variation of data.
Error bars represent SD. Asterisk indicate significant dierences. B) Genome-wide selection signals (FST value) of dierent Tartary buckwheat breeding
stages.
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Figure 6. FtCOMT is significantly associated with the S/G ratio in Tartary buckwheat seed hulls. A) Manhattan plot of six seed hull traits analyzed by
GWAS. Putative lignin biosynthesis related genes were identified within kb up- and down-stream of associated SNPs. B) Visualized result of GWAS for
S/G ratio of Tartary buckwheat seed hull lignin. The significant-associated SNP is highlighted in black. Sliding window represents for the kb up- and
down-stream region surrounding the indicated significant SNP. C) Quantification of lignin monomers in the thioacidolysis-released products of stems
from indicated genotypes. One-way ANOVA followed by Tukey’s honestly significant dierence test was used for statistical analysis (n ≥, p<.). All
data points were plotted to show the variation of data. Error bars represent SD. Letters indicate significant dierences.
breeding target for enhancing dehulling efficiency in Tartary
buckwheat industry.
3. Discussion
Plants have evolved highly differentiated structures to execute de-
sired functions. Secondary cell wall provides mechanical strength
for plant bodies. One of the key cell wall components, lignin, is a
complex phenolic polymer that varies in content and composition
among different plant tissues and affects the secondary wall me-
chanical properties. Previous studies have shown a correlation
between lignin absorbance and tensile stiffness in poplar trees
(Özparpucu et al., 2017) and the influence of lignin biosynthe-
sis on cell wall characteristics using specific mutants or small-
scale varieties.[24,15]However, the impact of lignin composition
on cell wall mechanical properties is still poorly understood. In
this study, we analyzed a large population of 274 Tartary buck-
wheat varieties with diverse lignin composition and seed hull
hardness. We found that Tartary buckwheat seed hull processes
a unique lignin chemotype that primarily consist of G lignin,
commonly found in gymnosperms (Figure 2). The lower S unit
content makes harder seed hulls in Tartary buckwheat compared
with common buckwheat (Figure 3), as indicated by the nega-
tive correlation between the hardness and the S lignin content
or S/G ratio of Tartary buckwheat seed hull (Figure 3). These ex-
perimental evidences collectively indicate a close relationship be-
tween lignin composition and cell wall mechanical properties.
During the polymerization process, lignin units are inter-
linked not only via 𝛽-O-4, but also through C–C linkage, includ-
ing 𝛽-𝛽,𝛽-5, and 5-5 bonds.[24]C─C bonds are, characterized by
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shorter bond lengths and higher dissociation energy than ether
bonds.[25]Therefore, the presence of more C─Cbondsleadsto,
the denser and more resistant lignin polymers. Recently, we iden-
tified that maize sporopollenin, which stand for the most in-
destructible biopolymer, primarily consists of H unit,[40]This
unique lignin composition not only protect the pollens against
heat waves and excess UV radiation during the growth season,[40]
but also likely provides the sporopollenin with superior durability
that could makes it persist in the sediments for over millions of
years.[40,41]This indicated that, denser lignin confers more rigid
property for cell wall. Compared with coniferyl alcohol, sinapyl
alcohol forms less C–C linkage, because the methoxy groups at
the aromatic ring can prevent the formation of 𝛽-5 and 5-5 C–C
linkages. Therefore, higher S unit content or S/G ratio likely re-
sults in looser structure that contributes to the reduction of seed
hull hardness.
Breeding of tartaty buckwheat is still in its infancy and is
mostly towards a higher content of nutrients such as rutin.
Through genome-wide analysis of selective sweeps, we showed
that genes that are responsible for higher S lignin units are con-
tinuously selected during the domestication and breeding pro-
cess of Tartary buckwheat, which likely explains the higher S/G
ratio and lower hardness of seed hulls of widely domesticated cul-
tivars. One of the selected genes, FtCOMT1, is also demonstrated
to be strongly associated with the S/G ration of seed hull. COMT
is an important enzyme in the phenylpropanoid metabolic path-
way that synchronously evolved from N-acetylserotonin methyl-
transferase (ASMT) with appearance of land plant by gene dupli-
cation and subsequent divergence.[42]Newly emergent COMTs
are more conserved the amino acid level but possess more open
conformation compared to ASMT, getting a new function on cat-
alyzing the biosynthesis of monolignols.[42]This development
directly contributed to plant colonization of land and the domi-
nance of vascular plants.[42]The crucial function of COMTs in in-
fluencing S unit content (and S/G ratio) in lignin polymer makes
it a promising target for engineering lignin composition of trans-
genic plants, to improve forage digestibility, pulping efficiency, or
utility in biofuel production.[24,43]Therefore, higher S/G ratio and
lower hardness of Tartary buckwheat seed hull would also bring
extra benefits. The high S/G ratio of lignin in Tartary buckwheat
stem indicate that the synthesis pathway of S unit is active in this
plant. Considering the expression pattern of COMTs and F5Hs,
a pathway that regulates the differential composition of S units
in different tissues by controlling the tissue-specific expression
of COMTs and F5Hs might exist in Tartary buckwheat. Exploring
and dissecting this pathway is of benefit in redesign of Tartary
buckwheat seed hulls.
Lodging and seed shattering are two other problems that affect
the yield and harvesting of Tartary buckwheat. Tartary buckwheat
plants have tall and brittle hollow stems, which make them prone
to lodging, bending, and pest attack. Lodging resistance affects
yield and mechanical harvesting significantly (Hagiwara et al.,
1999). Almost all buckwheat varieties have indeterminate inflo-
rescence, which results in a long flowering period and a broad
grain ripening period. Seeds of different maturity stages are
present at the same time during the ripening period (Funatsuki
et al., 2000). This makes it challenging to determine the opti-
mum harvesting time to avoid the shedding of early-mature seeds
and minimize the yield loss (Oba et al., 1998). Seed shattering
problem reduces the yield of buckwheat by about 40–50% when
harvested by machine compared to hand harvesting (Radics and
Mikóházi, 2010). The shattering problem is more severe in Tar-
tary buckwheat, because its pedicel has smaller diameter, break-
ing bending strength, and breaking tensile strength than those of
common buckwheat (Oba et al., 1998). Therefore, enhancing the
physical strength of pedicel is one of the effective ways to reduce
the yield loss during harvesting. Further exploration of the lignin
composition of specific tissues and the regulatory mechanism in
Tartary buckwheat has great potential for improving the variety
and increasing the yield. In summary, this work provides a theo-
retical basis for further improving the mechanical properties and
dehulling efficiency of Tartary buckwheat. Moreover, the genetic
insights on the lignin composition of tartaty buckwheat offered
by this work could largely facilitate the targeted breeding process.
4. Experimental Section
Plant Materials and Growth Conditions:All buckwheat materials were
grown during the cropping season (March to September of and )
on the experimental farm of the College of Agronomy, Shanxi Agricultural
University, Taigu, Shanxi, China (°′N, °′E). For Arabidopsis,Col-
ecotype, and T-DNA insertion mutants Atcomt1 (Salk_) were pre-
served in the lab. The detailed growth condition was the same as described
previously.[]For soybean, William ecotype was preserved in the lab,
and planted in greenhouse with a temperature setting as °C.
A total of Tartary buckwheat accessions, including varieties
from Himalayan region ( germplasms), Yun-Gui Plateau region (
germplasms), Loess Plateau region ( germplasms), and Southern
Plain region ( germplasms) of China, and varieties from United
States, Nepal, Bhutan, Japan, and Russia, were used for correlation analy-
sis and GWAS in this study (Table S, Supporting Information). The hulls
of mature seeds were collected for lignin composition and hardness anal-
ysis.
Preparation of Cell Wall Residues:All tissues were collected after buck-
wheat seeds were fully matured. The preparation of cell wall residues and
thioacidolysis was performed as described previously.[]Briefly, the fine-
milled tissue powders were extracted with % ethanol at °C for three
times each for h. The residues were extracted with chloroform/methanol
(:, v/v) and then washed by acetone, and each for three times. The
residues were dried overnight and de-starched by amylase and pullu-
lanase. After briefly washing with water and acetone, the residues were
dried overnight, resulting in de-starched extractive-free cell wall residues
(CWRs). The CWRs were enzymatically hydrolyzed at °Cfordaysusing
a mixture of Macerozym R and Cellulase Onozuka R (Yakult, Nishi-
nomiya, Japan) (% of each in . sodium acetate buer, pH .). The
residues were pelleted and washed with water and acetone. The over-night
dried residues were considered as cellulolytic enzyme lignin (CEL).
2D NMR Spectroscopy:Transfer mg ball-milled CEL into a mm
NMR tube. Add μL of DMSO-d/pyridine-d (:; v/v) into the NMR
tube, and treated with ultrasonic bath for – h until the sample be-
comes apparently homogeneous. NMR spectra were acquired on Agilent
MHz spectrometer equipped with a mm H-(C/N) C Enh
Cryo-Probe. The parameters of adiabatic Nuclear Magnetic Resonance
Spectroscopy (HSQC) NMR experiments (“gNhsqc”) were consistent with
the reference.[]MestReNova was used for NMR data analysis and pro-
cessing. The central DMSO solvent peak was used as an internal reference
(𝛿H/𝛿C . ppm/. ppm). The typical matched Gaussian apodization
in F, squared cosine-bell and one level of linear prediction ( coe-
cients) in F to obtain the HSQC plots. The signals were assigned ac-
cording to the previous literature.[,]For quantification analysis of lignin
aromatic units, C-H/C-H or C′-H′/C′-H′correlations form S/,
S′/ and H/ and C-H correlations form G were integrated and the
S/, S′/ and H/ signals were logically halved. The values of relative
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signal intensities in Figure were expressed on /(S/+S′/) +G +
/ H/ = basis.
Thioacidolysis of CWRs: mg CWRs was incubated with mL reac-
tion solution (dioxane: boron trifluoride diethyl etherate: ethanethiol =
::, v/v/v) at °C for h. Gently mix the samples every hour,
and in the last hour, mix the samples every min. After the reac-
tion mixture was cooled, its pH was adjusted to – by . NaHCO
and then extracted with mL dichloromethane. The dichloromethane
layer was filtered through anhydrous sodium sulfate into a -mL Ep-
pendorf tube, and the solvent was evaporated. The resulting residues
were re-dissolved in μL pyridine, and derivatized using μL N,O-
bis(trimethylsilyl)trifluoroacetamide (BSTFA) at room temperature for h.
Aliquots of this solution were analyzed by GC/MS.
Gas Chromatography–Mass Spectrometry:An Agilent A gas chro-
matography coupled to a B mass spectrometer was used for detec-
tion of lignin monomers presented the thioacidolysis lysate. Separation
was achieved on an Agilent HP-MS column ( mm ×. mm, . μm
film thickness). The following temperature gradient was used with a -min
solvent delay: Initial hold at °Cformin;a°C per minute ramp to
a °C and hold for min. The flow rate was set at . mL per minute.
Peaks were identified by characteristic mass spectrum ions of , ,
and m z−for H, G and S units, respectively.[]The calculation of
lignin monomer quantify is based on the equation as follows.
C=
AS
AIS ×WIS ×K
CWR ()
In this equation, C refers to the concentration (μmol g−), ASrefers
to the peak area of lignin unit, AIS refers to the peak area of internal
standard, WIS refers to the amount (μmol) of the internal standard, K
refers to the response factors of lignin units versus internal standard,
CWR refers to the mass (g) of cell wall residues. In our protocol, . mg
,′-ethylenebisphenol was added into mL dichloromethane as internal
standard. Thus, WIS is ., K of H, G, and S units are ., ., .,
respectively.[]
Hardness of Tartary Buckwheat Seed Hull:The seed hull was peeled
from mature seeds. Hardness of seed hull were measured using a Grain
Hardness Tester (WDGAGE, Model: GW-).
RNA Sequence Analysis:Leaf, stem, and seed hulls at three develop-
mental stages (S: grain filling stage, S: grain green mature stage, S:
grain mature stage) were used for RNA isolation and each stage has three
replicates. The total RNA of samples was extracted according to the man-
ual instructions in the RNeasy plant mini kit (Qiagen, Germany) kit, and
then the quality was examined. Complementary DNA (cDNA) libraries
were constructed by the Beijing Biomax Biotechnology Co., Ltd, then se-
quenced by the Illumina high-throughput sequencing platform. Trimmo-
matic (version) was used to perform quality control on the raw sequence
data.[]HISAT was adopted to compare the clean data to the reference
genome,[,]whereas StringTie was applied to compare and assemble
the reads.[]FPKM (fragments per kilobase of transcript per million frag-
ments mapped) value was used for the quantification of gene expression.
Genome-Wide Detection of Selective Sweeps:Genome-wide detection
of selective sweeps was performed by using the FST value as an indica-
tor. Population dierentiation analyses were conducted between dierent
subgroups by using vcftools software.[]For analysis of FST (Fixation in-
dex), the sliding window was set to kb, and the step size was set to
kb. The FST values ranged from to , with a higher value indicating
a greater degree of dierentiation and a higher level of selection for the
QTL/gene(s).
Genome-Wide Association Analyses:The genotype dataset of lines
of Tartary buckwheat used in this study includes SNPs (.
SNPs) with a minimum allele frequency (MAF) of ≥.. Integrating geno-
type and phenotype data, the PCA+K model — a Mixed Linear Model
(MLM) that corrects for both principal component analysis (PCA) and rel-
ative kinship (K) — was employed utilizing TASSEL . software for con-
ducting a genome-wide association study (GWAS).[,]The MLM model
was represented by the equation y =X𝛼+Z𝛽+Wμ+e, where y represents
trait values. The components include X𝛼(principal component, acting as
a covariate) and Z𝛽(SNPs or marker eects) as fixed eects, Wμ(rela-
tive kinship matrix) as the random eect, and e as the residual error.[]
To avoid the occurrence of type I and type II errors (false positives and
false negatives), a stringent threshold (/) was utilized to assess
the presence of a significant association between the SNP and target trait.
As reported in previous studies, a QTL was defined as a total interval of
kb, encompassing kb upstream and downstream of the significant
SNP.[]The confidence interval of significant QTL was compared with the
Pinku reference genome[](http://www.mbkbase.org/Pinku) to identify
genes potentially involved in regulating Tartary buckwheat hull. For visu-
alization, the “CMplot” package was utilized in R to generate Manhattan
plots, and used the “LDheatmap” package for LD plots.
Phylogenetic Analysis:The protein sequences of OMT family in Ara-
bidopsis (ATG, ATG, ATG, ATG, ATG,
ATG, ATG, ATG, TG, ATG,
ATG, ATG, ATG, ATG, ATG,
ATG, ATG, ATG, ATG, ATG,
ATG, ATG, ATG) were retrieved from TAIR. The
protein sequence of FtPinG were extracted from the reference
genome for Pinku. The Maximum-Likelihood phylogenetic tree (boot-
strap value set as ) was constructed based on the protein sequences
of these homologs by using MEGA X software.[]
Transformation of Plants:For the complementation of Atcomt1 with
FtCOMT, the coding sequence of FtCOMT1 was cloned into the entry
vector pCR using NovoRec plus One step PCR Cloning Kit (NovoPro-
tein), and then subcloned into the destination vector pMDC vector. The
construct was transformed into Agraobacterium tumefaciens GV, and
then transformed into Arabidopsis plants using floral dip method.[]For
screening transgenic lines, positive transformants were screened using
hygromycin (Roche).
Statistical Analysis of Phenotypic Traits:Statistical analysis was per-
formed by using GraphPad Prism software. For each figure, the sta-
tistical method was specified in the figure legend. Pearson correlation
among all traits were calculated R function stats::corr.test and visualized
by R package corrplot (https://cran.r-project.org/web/packages/corrplot/
citation.html).
Supporting Information
Supporting Information is available from the Wiley Online Library or from
the author.
Acknowledgements
The authors would like to thank the sta members of the Nuclear Magnetic
Resonance System at the National Facility for Protein Science in Shanghai
(NFPS), Shanghai Advanced Research Institute, Chinese Academy of Sci-
ences, China for providing technical support and assistance in data col-
lection and analysis. The authors would also like to thank members in
Xuebin Zhang’s lab and Zhaoxia Sun’s lab for their assistance in exper-
iments and helpful discussion. This work was supported by grants from
the National Natural Science Foundation of China (No. to Xue-
bin Zhang and No. to Zhaoxia Sun); Natural Science Founda-
tion of Henan Province ( to Wenqi Yang); the grand science
and technology special project in Shanxi Province ( to
Zhaoxia Sun); National guides local science and technology development
fund projects (YDZJSXA to Zhaoxia Sun); China Agriculture Re-
search System of MOF and MARA(CARS--A- to Zhaoxia Sun); The spe-
cial fund for Science and Technology Innovation Teams of Shanxi Province
( to Zhaoxia Sun). Guangdong Provincial Key Laboratory
of Synthetic Genomics (B to Qiao Zhao).
Conflict of Interest
The authors declare no conflict of interest.
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Author Contributions
W.Y., H.D., K.Y., and S.H. contributed equally to this work. X.B.Z. and Z.X.S.
conceived and supervised the project. X.B.Z., Z.X.S., W.Q.Y., H.Y.D., K.Y.
and S.Y.H. designed the experiments. W.Q.Y., H.Y.D., Y.F.K., and X.W. per-
formed most of the experiments and analyzed the data. S.Y.H., J.Y.H., and
L.L.L. performed the transcriptome analysis. L.F.L. and Y.J.Z. performed
NMR spectroscopy. Y.Z., J.L.Z., and C.L. assisted in experiments including
sample preparation and GC-MS. X.H.Z. and J.H.T helped in experimental
design. Q.Z. and N.W. helped in manuscript writing. W.Q.Y., H.Y.D., K.Y.,
and S.Y.H. prepared graphs, and wrote the manuscript with input from all
co-authors. All authors read and approved the manuscript.
Data Availability Statement
The data that support the findings of this study are available from the cor-
responding author upon reasonable request.
Keywords
COMT, GWAS, lignin, seed hull, tartary buckwheat
Received: January ,
Revised: March ,
Published online: March ,
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