Available via license: CC BY-NC-ND 4.0
Content may be subject to copyright.
VAMP726 from maize and Arabidopsis confers
pollen resistance to heat and UV radiation by
influencing lignin content of sporopollenin
Wenqi Yang
1,6
, Dongdong Yao
1,6
, Haiyang Duan
2,6
, Junli Zhang
1,2
, Yaling Cai
1
, Chen Lan
1
,
Bing Zhao
1
, Yong Mei
1
, Yan Zheng
1
, Erbing Yang
4
, Xiaoduo Lu
5
, Xuehai Zhang
2
, Jihua Tang
2,3
,
Ke Yu
1,
*and Xuebin Zhang
1,
*
1
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 475004, China
2
National Key Laboratory of Wheat and Maize Crop Science, College of Agronomy, Henan Agricultural University, Zhengzhou 450002, China
3
The Shennong Laboratory, Zhengzhou 450002, China
4
College of Chemistry, Zhengzhou University, Zhengzhou 450001, China
5
National Engineering Laboratory of Crop Stress Resistance, School of Life Science, Anhui Agricultural University, Hefei 230036, China
6
These authors contributed equally to this article.
*Correspondence: Xuebin Zhang (xuebinzhang@henu.edu.cn), Ke Yu (keyu@henu.edu.cn)
https://doi.org/10.1016/j.xplc.2023.100682
ABSTRACT
Sporopollenin in the pollen cell wall protects male gametophytes from stresses. Phenylpropanoid deriva-
tives, including guaiacyl (G) lignin units, are known to be structural components of sporopollenin, but the
exact composition of sporopollenin remains to be fully resolved. We analyzed the phenylpropanoid deriv-
atives in sporopollenin from maize and Arabidopsis by thioacidolysis coupled with nuclear magnetic reso-
nance (NMR) and gas chromatography–mass spectrometry (GC–MS). The NMR and GC–MS results
confirmed the presence of p-hydroxyphenyl (H), G, and syringyl (S) lignin units in sporopollenin from maize
and Arabidopsis. Strikingly, H units account for the majority of lignin monomers in sporopollenin from these
species. We next performed a genome-wide association study to explore the genetic basis of maize sporo-
pollenin composition and identified a vesicle-associated membrane protein (ZmVAMP726) that is strongly
associated with lignin monomer composition of maize sporopollenin. Genetic manipulation of VAMP726
affected not only lignin monomer composition in sporopollenin but also pollen resistance to heat and UV
radiation in maize and Arabidopsis, indicating that VAMP726 is functionally conserved in monocot and dicot
plants. Our work provides new insight into the lignin monomers that serve as structural components of
sporopollenin and characterizes VAMP726, which affects sporopollenin composition and stress resistance
in pollen.
Key words: pollen cell wall, sporopollenin, lignin monomers, heat stress, UV radiation
Yang W., Yao D., Duan H., Zhang J., Cai Y., Lan C., Zhao B., Mei Y., Zheng Y., Yang E., Lu X., Zhang X., Tang
J., Yu K., and Zhang X. (2023). VAMP726 from maize and Arabidopsis confers pollen resistance to heat and UV
radiation by influencing lignin content of sporopollenin. Plant Comm. 4, 100682.
INTRODUCTION
Pollen viability is vital to the yield of many cereal crops. The
increasing frequency of extreme climate events severely affects
pollen viability and leads to significant yield losses, posing great
challenges to food security (Stapleton, 1992;Dolferus et al.,
2011;Wheeler and von Braun, 2013;Chaturvedi et al., 2021).
Plants have evolved the pollen cell wall to protect male
gametophytes from unfavorable environmental conditions
(Edlund et al., 2004). The exine is the outermost layer of the
pollen cell wall; it consists mainly of sporopollenin, a biopolymer
that is considered to be the toughest biomaterial in nature
(Ariizumi and Toriyama, 2011). The intine is the inner cell wall
Published by the Plant Communications Shanghai Editorial Office in
association with Cell Press, an imprint of Elsevier Inc., on behalf of CSPB and
CEMPS, CAS.
Plant Communications 4, 100682, November 13 2023 ª2023 The Author(s).
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
1
Plant Communications
Research article
llll
layer deposited between the plasma membrane and the exine,
and it has the typical composition of a primary cell wall (Jiang
et al., 2013). For decades, the exact chemical composition
of sporopollenin has remained elusive. Use of thioacidolysis
coupled with solid-state nuclear magnetic resonance (NMR)
analysis recently revealed that sporopollenin consists mainly of
crosslinked long-chain polyvinyl alcohol units and coumaroylated
C16 aliphatic units (Li et al., 2019). Phenylpropanoid derivatives,
including naringenin, p-hydroxybenzoate (p-BA), p-coumarate
(p-CA), ferulate (FA), and guaiacyl (G) lignin units, are also present
in sporopollenin (Li et al., 2019;Xue et al., 2020).
Phenylpropanoids comprise a wide variety of secondary metab-
olites that originate from phenylalanine (Fraser and Chapple,
2011). These aromatic compounds are essential for plant
growth, development, and adaptive responses to environmental
changes (Dong and Lin, 2021). For example, phenylpropanoid
derivatives in sporopollenin can confer pollen resistance to
ultraviolet (UV) radiation (Xue et al., 2020). Lignin is an
important polymeric phenylpropanoid derivative that is mainly
deposited in the secondary cell wall to provide mechanical
strength and hydrophobicity (Bonawitz and Chapple, 2010); it
also provides other benefits, such as resistance to biotic and
abiotic stresses (Cesarino, 2019). Formation of the lignin
polymer requires three major monolignols, p-coumaryl alcohol,
coniferyl alcohol, and sinapyl alcohol. Once these monolignols
enter the apoplastic space, they are activated by cell-wall-
bound oxidation systems, initiating radical coupling to form
lignin polymers (Bonawitz and Chapple, 2010). Accordingly,
lignin polymers typically consist of the three most abundant
monomers, p-hydroxyphenyl (H), G, and syringyl (S) lignin units,
which are derived from p-coumaryl alcohol, coniferyl
alcohol, and sinapyl alcohol, respectively (Bonawitz and
Chapple, 2010). Different plant species have distinct lignin
monomer compositions, and lignin also exhibits tissue-specific
differences in composition within the same plant (Campbell and
Sederoff, 1996). Interestingly, only G units were detected in
sporopollenin of Cryptomeria (gymnosperm) and Lilium
(angiosperm), and no lignin units were detected in
sporopollenin of two seedless plants, Ophioglossum (fern) and
Lycopodium (moss) (Xue et al., 2020), indicating that
monolignol biosynthesis may have been involved in the
environmental adaptation of early vascular plants.
Sporopollenin precursors are synthesized in the tapetum, a spe-
cific cell layer that surrounds the developing pollen grains (Wang
et al., 2018). The absence of S units in sporopollenin of tested
plant species is likely due to the low expression of genes
encoding enzymes required for sinapyl alcohol biosynthesis in
the tapetum, such as caffeic acid O-methyltransferase (COMT)
and ferulic acid 5-hydroxylase (F5H) (Bonawitz and Chapple,
2010;Xue et al., 2020). Once synthesized, these precursors are
translocated from the tapetal cells to the locules between the
tapetum and immature pollen grains, then incorporated into
primexine (the surface of the developing pollen grains) to form
the exine (Shi et al., 2015). Transport proteins, such as ATP-
binding-cassette transporters and lipid-transfer proteins, have
been proposed to mediate delivery of sporopollenin precursors
across the plasma membrane (Zhang et al., 2010;Choi et al.,
2011,2014;Huang et al., 2013;Quilichini et al., 2014).
Secretory vesicles derived from the endoplasmic reticulum-
trans-Golgi network might also be involved in the secretion of
sporopollenin precursors (Ichino and Yazaki, 2022).
In this study, we analyzed the phenylpropanoid derivatives in
sporopollenin from Zea mays (hereafter maize) and Arabidopsis
thaliana (hereafter Arabidopsis). We found that H, G, and S lignin
units are structural components of maize and Arabidopsis sporo-
pollenin, and H units is much more abundant than the other two
monomers. We confirmed that the monolignol biosynthetic
pathway is required for pollen resistance to heat and UV
stresses and identified vesicle-associated membrane protein
726 (VAMP726), which positively influences lignin monomer
composition in sporopollenin, through a genome-wide associa-
tion study (GWAS). Genetic manipulation of VAMP726 expres-
sion strongly affected pollen resistance to heat and UV radiation
in both maize and Arabidopsis. Finally, we showed that overex-
pression of VAMP726 resulted in enrichment of proteins related
to lignification in the apoplastic space. This work reveals a meta-
bolic strategy deployed by plants to enhance pollen stress
resistance.
RESULTS
H units comprise the majority of lignin monomers in
sporopollenin from maize and Arabidopsis
Several phenylpropanoid derivatives, such as p-BA, p-CA, FA,
naringenin, and G lignin units, have been detected in thioacidol-
ysis lysates of sporopollenin from vascular plants (Li et al.,
2019;Xue et al., 2020). The exclusive presence of G lignin
units, but not other major lignin monomers, in sporopollenin
attracted our attention. Gymnosperm lignin is composed mainly
of G units, whereas angiosperms mostly produce G- and S-
type lignin (Ralph et al., 2019). The absence of S and H lignin
units in sporopollenin of Cryptomeria (gymnosperm) and Lilium
(dicot) (Xue et al., 2020) caused us to wonder whether ‘‘G-
lignin-only’’ sporopollenin is a universal phenomenon across
the plant kingdom. To test this possibility, we collected pollen
grains from field-grown maize, one of the most important
monocot cereal crops, and prepared sporopollenin (Figure 1A).
We then analyzed the presence of lignin monomers and other
phenylpropanoid derivatives in the thioacidolysis lysate of
sporopollenin by solution-state two-dimensional (2D)-NMR
spectroscopy. The thioacidolysis products of maize
sporopollenin yielded typical NMR signal patterns of H and G
lignin units, suggesting that, in addition to G units, H units are
also present in maize sporopollenin (Figure 1B). G and S lignin
units comprise the vast majority of lignin monomers in most
lignified tissues of maize (Kim et al., 2017;Del Rio et al., 2018;
Sun et al., 2018). To compare lignin monomer
composition between maize sporopollenin and other maize
tissues, we analyzed the lignin monomer composition of a
mixture of tissues (stem, leaf, and leaf sheath) by NMR. The
thioacidolysis products of these maize tissues exhibited a
typical lignin monomer composition consisting of H, G, and S
units (Figure 1B). Because p-BA and p-CA can yield signature
features of H units, they might interfere with the detection of H
lignin units derived from p-coumaryl alcohol, resulting in an
exaggerated level of this specific monomer (Ralph et al., 2019).
To rule out the possibility that thioacidolysis breaks down p-BA
or p-CA, we looked for the NMR signal patterns of these
2Plant Communications 4, 100682, November 13 2023 ª2023 The Author(s).
Plant Communications VAMP726 confers pollen resistance to heat and UV
Figure 1. H, G, and S lignin units are structural components of maize sporopollenin.
(A) Electron micrographs of maize pollen grains (top), the fine powder after ball milling (middle), and the destarched extractive-free cell-wall residues of
maize pollen (bottom). Scale bar, 500 mm.
(B) 2D-NMR spectra of thioacidolysis-released products of maize sporopollenin and cell walls extracted from a mixture of lignified maize tissues (stem,
leaf, and leaf sheath), showing the aromatic signals of H, G, and S lignin units, as well as tricin, p-BA, p-CA, and FA.
(C) Selected ion chromatograms and MS
2
spectra of the indicated lignin monomers in thioacidolysis-released products of maize sporopollenin or maize
stems from GC–MS. The structures of characteristic base peak fragment ions are shown. Results obtained from the maize inbred line MN are used here
for representation.
Plant Communications 4, 100682, November 13 2023 ª2023 The Author(s). 3
VAMP726 confers pollen resistance to heat and UV Plant Communications
phenolics in the thioacidolysis-released fractions of maize sporo-
pollenin and cell-wall residues from other tissues. The results
confirmed the presence of p-CA and FA in maize sporopollenin,
and all three phenolics were present in other maize tissues
(Figure 1B), suggesting that H units detected in sporopollenin
by NMR are not derived from p-BA or p-CA. Tricin is a
flavonoid that is incorporated as a lignin monomer into the
lignin polymers of monocot plants (Lan et al., 2015). We also
identified the presence of tricin in the NMR spectra of mixed
maize tissues but not of sporopollenin (Figure 1B).
To validate the NMR results, we used gas chromatography–mass
spectrometry (GC–MS) to determine the presence of each
lignin monomer in maize sporopollenin. In this case, we per-
formed a population-wide investigation of lignin monomer
composition in sporopollenin from 117 field-grown maize inbred
lines (Supplemental Dataset 1). In all of these lines, we detected
the characteristic chromatograms and mass fragment ions of
H, G, and S units in sporopollenin (Figure 1C), which were
mostly identical to those of synthesized standards (Yue et al.,
2012) and to lignin monomers detected in the xylem (maize
stem, Figure 1C). We wondered whether pollen grains of a
dicotyledonous plant, e.g., Arabidopsis, would be encased by
sporopollenin with a similar lignin monomer composition, as
dicots typically contain few H lignin units (Ralph et al., 2019).
We therefore analyzed the lignin monomer composition of
Arabidopsis sporopollenin by thioacidolysis coupled with GC–
MS. The results showed that Arabidopsis sporopollenin also
contained H, G, and S lignin units (Supplemental Figure 1).
Moreover, we were able to detect the presence of p-BA, p-CA,
and FA in maize and Arabidopsis sporopollenin through GC–MS
(Supplemental Figure 2). Although we did not detect the NMR
signals of S units in sporopollenin, this may be because the
abundance of S units in the sample prepared for NMR analysis
was too low to be captured. Nonetheless, combining the results
from NMR and GC–MS, we conclude that H, G, and S lignin
units are structural components of maize and Arabidopsis
sporopollenin.
The presence of p-BA, p-CA, and FA, as well as three major lignin
monomers, in sporopollenin prompted us to investigate how they
integrate into sporopollenin. Thioacidolysis is routinely used in
the analysis of lignin monomer composition of the plant cell
wall; it mainly breaks ether bonds and ester bonds between
different lignin monomers (Eudes et al., 2012;Ralph et al.,
2019). We wondered whether these phenylpropanoid
derivatives could be attached to the aliphatic units of
sporopollenin through ester bonds. Hence, we used methanol
or NaOH treatment during preparation of maize sporopollenin
to release soluble phenolics and wall-bound phenolics (through
ester bonds) from the pollen cell wall, then subjected the remain-
ing residues to thioacidolysis (Supplemental Figure 3A). We found
that p-CA was present in the wall-bound phenolics but not in the
soluble fractions (Supplemental Figure 3B). FA and p-BA were
detected in both wall-bound and soluble phenolics
(Supplemental Figure 3B). These results confirmed that these
phenolics could be attached to metabolite units (lignin
monomers or aliphatic units) in maize sporopollenin through
ester bonds. In addition, we did not detect any signal of lignin
monomers in these washing fractions (Supplemental Figure 3B),
and we could still detect all three major lignin monomers in the
thioacidolysis-released products (Supplemental Figure 3C),
suggesting that these lignin monomers were not attached to
metabolite units of maize sporopollenin through ester bonds.
These data indicate that the lignin monomers detected in the
thioacidolysis-released fractions of sporopollenin likely form a
lignin polymer or crosslink with some other metabolite units of
sporopollenin through ether bonds. More sophisticated analytical
methods will be required to dissect the exact linkage types.
We quantified the absolute abundance of each lignin unit in sporo-
pollenin from each maize inbred line on the basis of peak areas in
the GC–MS chromatograms. For simplicity, we defined the total
content of lignin monomers as the sum of the abundance of H,
G, and S units. We found that, at a population scale, the overall
H units accounted for more than 70% of lignin monomers in maize
sporopollenin, and no difference was observed between the abun-
dance of G and S units (Figure 2A and 2B). This is surprising,
because H units usually represent a minor constituent (less than
30%) of total lignin monomers in most lignified plant tissues
(Ralph et al., 2019). In Arabidopsis sporopollenin, the abundance
of H units was highest (accounting for more than half of total
lignin monomers) and that of S units was lowest (Supplemental
Figure 4). To ensure the reliability of the analytical methods, we
analyzed the lignin monomer composition of other tissues from
maize cultivar B73, including the leaf, stem, tassel (male flowers
were removed), glume, and leaf sheath. We detected an
extremely high amount of H units in B73 sporopollenin,
significantly higher than that of G and S units (Figure 2C). In
other tissues of B73, lignin consisted mainly of G and S units,
and H units accounted for less than 2% of total lignin monomers
(Figure 2D), consistent with previous reports (Kim et al., 2017;
Del Rio et al., 2018;Sun et al., 2018). We also noticed that the
total yield of lignin monomers from other tissues was at least 20
times that from sporopollenin (Figure 2C and 2D). Collectively,
these results confirmed that H, G, and S lignin units are present
in sporopollenin of maize and Arabidopsis and that H units are
the dominant lignin monomers in sporopollenin.
The monolignol biosynthetic pathway is required for
lignin monomer accumulation in sporopollenin and
pollen stress resistance in Arabidopsis
The maize population we used for analysis of lignin monomer
composition could be divided into two groups on the basis of
geographic origin (Supplemental Dataset 1). Interestingly, the
total content of lignin monomers in sporopollenin was
significantly higher in maize inbred lines from tropical/subtropical
areas than in those from temperate areas (Figure 3A), suggesting
that lignin monomers in sporopollenin may be important for maize
adaptation to local environmental conditions. The UV-absorbing
property of phenylpropanoid derivatives could protect plants
from damage caused by excess exposure to solar radiation
(Dixon and Paiva, 1995;Qian et al., 2015). In Arabidopsis,
mutations in key enzymes of monolignol biosynthesis, such as
cinnamate-4-hydroxylase (C4H) and cinnamoyl-coenzyme A
(CoA) reductase (CCR), resulted in defective sporopollenin and hy-
persensitivity of pollen to UV radiation (Xue et al., 2020). In the field,
maize pollen grains are often exposed to high temperatures and
high doses of UV during summer periods, both of which could
markedly affect pollen viability. We wondered whether lignin
monomer composition of maize sporopollenin is associated with
4Plant Communications 4, 100682, November 13 2023 ª2023 The Author(s).
Plant Communications VAMP726 confers pollen resistance to heat and UV
resistance of pollen to heat. Therefore, we harvested pollen grains
of selected maize inbred lines that contained the highest or lowest
levels of total lignin monomers in sporopollenin (Supplemental
Dataset 2) and examined their pollen viability under heat
treatment using triphenyl tetrazolium chloride (TTC) staining
(Alexander, 1969). The results showed that maize inbred lines that
contained more lignin monomers in sporopollenin overall
produced pollen grains that were better able to cope with heat
stress (Figure 3BandSupplemental Figure 5), suggesting that
lignin monomers in sporopollenin may contribute to pollen heat
resistance.
We speculated that deficiency in monolignol biosynthesis would
affect lignin monomer content in sporopollenin and lead to
impaired pollen resistance to stresses, including heat. To test
this possibility, we took advantage of well-studied Arabidopsis
mutants (ccr1,comt, and ccomt) that are defective in monolignol
biosynthesis. CCR, COMT, and caffeoyl-CoA 3-O-methyltrans-
ferase (which was mutated in ccomt) all catalyze critical steps
in monolignol biosynthesis (Bonawitz and Chapple, 2010), and
mutations in these genes significantly alter lignin monomer
composition in Arabidopsis (Do et al., 2007;Rinaldi et al., 2016).
We used tt4, in which chalcone synthase (CHS) is mutated, as
a control, because lack of CHS would block the biosynthesis of
flavonoids but not of lignin (Li et al., 2010). We first examined
whether pollen grains from these mutants were more prone to
heat stress. We used germination rate as an alternative metric
to assess pollen viability because the TTC staining protocol did
not work well for Arabidopsis pollen. The results showed that
ccr1,comt, and ccomt pollen had significantly lower resistance
to heat (Figure 3C). The tt4 mutant also exhibited moderately
reduced heat resistance, but to a much lesser extent
(Figure 3C). After exposure to high temperature for 30 min,
pollen resistance of tt4 did not differ significantly from that of
Columbia-0 (Col-0) and was only moderately reduced when the
treatment was extended to 60 min (Figure 3C).
We next investigated whether lignin monomer content in sporo-
pollenin of these mutants was altered as predicted. The results
showed that abundance of H units in sporopollenin was not
significantly different in ccr1, but it was significantly lower in
ccomt than in Col-0 (Supplemental Figure 6). The abundance of
G and S units in sporopollenin of ccr1 and ccomt did not
change significantly (Supplemental Figure 6). However, the
abundance of total lignin monomers in sporopollenin was
significantly lower in ccr1 and ccomt than in Col-
0(Supplemental Figure 6). These results suggest that mutations
in monolignol biosynthesis negatively affect the lignin monomer
Figure 2. H units comprise the majority of lignin monomers in maize sporopollenin.
(A) Quantification of lignin monomers in thioacidolysis-released products of sporopollenin from 117 maize inbred lines. One-way ANOVA followed by
Tukey’s honestly significant difference test was used for statistical analysis (n= 117, P< 0.05). Error bars represent SD. Letters indicate significant
differences.
(B) Descriptive statistics for lignin monomer composition in sporopollenin of 117 maize inbred lines.
(C) Quantification of lignin monomers in thioacidolysis-released products of sporopollenin from B73. One-way ANOVA followed by Tukey’s honestly
significant difference test was used for statistical analysis (n=4,P< 0.05). Error bars represent SD. Letters indicate significant differences.
(D) Quantification of lignin monomers in thioacidolysis-released products of leaf, stem, tassel, glume, and leaf sheath tissues of B73. Two-way ANOVA
followed by Tukey’s honestly significant difference test was used for statistical analysis (n=4,P< 0.05). Error bars represent SD. Letters indicate
significant differences in the abundance of individual monomers within each tissue.
Plant Communications 4, 100682, November 13 2023 ª2023 The Author(s). 5
VAMP726 confers pollen resistance to heat and UV Plant Communications
content of Arabidopsis sporopollenin. We also analyzed lignin
monomer composition in sporopollenin of tt4 and found no
significant differences in the abundance of individual units or
total lignin monomers compared with Col-0 (Supplemental
Figure 6), consistent with its stronger pollen heat resistance
compared with the other mutants (Figure 3C). On the basis of
our data and previous observations of reduced pollen
resistance to UV radiation in c4h and ccr1 (Xue et al., 2020), we
concluded that the monolignol biosynthetic pathway is required
for lignin monomer accumulation in sporopollenin and pollen
resistance to heat and UV radiation in Arabidopsis.
Natural variation in ZmVAMP726 is associated with
lignin monomer composition in maize sporopollenin
The essential role of monolignol biosynthesis in pollen stress
resistance prompted us to investigate the genetic basis for lignin
monomer composition in maize sporopollenin. The genetic diver-
sity of 117 maize inbred lines enabled us to perform a GWAS us-
ing the total content of lignin monomers in sporopollenin as the
target trait. Using a mixed linear model, we detected two adjacent
SNPs, chr9.S_153426472 and chr9.S_153426479, that ex-
ceeded the significance threshold for association (Figures 4A
and 7A; Supplemental Dataset 3). We performed an additional
GWAS using the abundance of H units in sporopollenin as the
target trait in order to explore the potential mechanism by
which maize deposits this prominent lignin monomer in
sporopollenin. Interestingly, the two GWASs revealed identical
significant SNPs (Supplemental Figure 7B and Supplemental
Dataset 3), suggesting that the variation in total content of lignin
monomers in maize sporopollenin is likely driven by variation in
the abundance of H units. We designated the 60-kb (±30-kb)
genomic region surrounding each SNP as a quantitative trait
locus (QTL). The two QTLs almost completely overlapped and
explained over 30% of the phenotypic variation (Supplemental
Dataset 3). Six candidate genes were present in this
region (Supplemental Dataset 3), and we examined their
developmental expression patterns using the Maize eFP
Browser (Hoopes et al., 2019). Finally, GRMZM2G075588 was
selected for further analysis, as it was the only gene specifically
expressed in anthers (Supplemental Figure 8).
GRMZM2G075588 is predicted to encode a VAMP. VAMPs
belong to a subgroup of soluble N-ethylmaleimide-sensitive fac-
tor attachment protein receptors (SNAREs), which mainly
mediate vesicle trafficking of proteins or metabolites via mem-
brane fusion (Sanderfoot et al., 2000). The protein sequence
encoded by GRMZM2G075588 shares 74.1% amino acid
identity with VAMP726 in Arabidopsis (Figure 4B), and we
therefore designated it ZmVAMP726. Expression analysis by
quantitative real-time PCR confirmed that GRMZM2G075588
was expressed mainly in tassels (Figure 4C), suggesting a
Figure 3. Monolignol biosynthesis is essential for pollen resistance to stresses in Arabidopsis.
(A) Comparison of total lignin monomers in thioacidolysis-released products of sporopollenin from maize inbred lines originating from tropical/subtropical
and temperate areas. Student’s t-test was used for statistical analysis (tropical/subtropical, n= 65; temperate, n= 52; P< 0.05). Error bars represent SD.
Asterisk indicates a significant difference.
(B) Heatmap showing the percentage of defective pollen in inbred lines with the highest and lowest levels of total lignin monomers in sporopolle nin before
and after heat treatment. Five biological replicates (shown in five rows) were analyzed for each inbred line.
(C) Pollen viability of Arabidopsis before and after heat treatment. Fresh pollen grains were exposed to 37C for the indicated times and then tested for
in vitro germination. Scale bar represents 200 mm. Two-way ANOVA followed by Tukey’s honestly significant difference test was used for statistical
analysis (n= 10, P< 0.05). Error bars represent SD. Letters indicate significant differences in germination rate at each time point.
6Plant Communications 4, 100682, November 13 2023 ª2023 The Author(s).
Plant Communications VAMP726 confers pollen resistance to heat and UV
potential role during pollen development. To validate the effect of
ZmVAMP726 on lignin monomer composition in sporopollenin,
we obtained two maize mutants (in the B73 background)
carrying null mutations in ZmVAMP726. One of the mutants
(named Zmvamp726-1 and carrying a premature stop codon in
the first exon) exhibited severe defects in pollen production
owing to anther indehiscence (Supplemental Figure 9A). We
therefore used the remaining mutant, which carried a premature
stop codon in the third exon (named Zmvamp726-2, and here-
after Zmvamp726,Supplemental Figure 9A and 9B) for further
analysis. We analyzed the ultrastructure of the pollen cell wall in
wild-type maize B73 and Zmvamp726 using scanning electron
microscopy and transmission electron microscopy. The results
revealed no visible changes in the surface or cell wall of pollen
grains (Supplemental Figure 9C). However, the total content of
lignin monomers and the abundance of H units in sporopollenin
were significantly lower in Zmvamp726 than in the B73 wild
type, although levels of G and S lignin units were not altered
(Figure 4D). Collectively, these results demonstrate that natural
variation in ZmVAMP726 is associated with lignin monomer
composition in maize sporopollenin.
VAMP726 is involved in pollen resistance to heat and UV
radiation in maize and Arabidopsis
The decreased content of total lignin monomers in sporopollenin
of Zmvamp726 suggested that its pollen grains may be hypersen-
sitive to stresses such as heat and UV radiation. TTC staining re-
vealed that a rather large number of pollen grains produced by
Zmvamp726 were nonviable in the absence of stress treatment
Figure 4. Natural variation in ZmVAMP726 is associated with lignin monomer composition in maize sporopollenin.
(A) GWAS results for total lignin monomer content in thioacidolysis-released products of maize sporopollenin. The lead SNP is highlighted in red. The
window (middle) represents the 60-kb genomic region surrounding the most significant SNP. The LD heatmap (bottom) shows pairwise R
2
values be-
tween polymorphisms in the 60-kb region centered on the most significant SNP.
(B) Protein sequence alignment of AtVAMP726 and GRMZM2G075588. Identical residues are highlighted in gray, and similar residues are highlighted in
yellow and cyan.
(C) Developmental expression pattern of ZmVAMP726. Relative expression was calculated using the 2
DDCt
method. Zm00001d015327 (UBQ) was used
as the reference. Error bars represent SD (n= 3).
(D) Quantification of H, G, and S units and total lignin monomers in thioacidolysis-released products of sporopollenin from B73 and Zmvamp726.
Student’s t-test was used for statistical analysis (n= 10, P< 0.05). Error bars represent SD. Asterisks indicate significant differences. n.s., not significant.
Plant Communications 4, 100682, November 13 2023 ª2023 The Author(s). 7
VAMP726 confers pollen resistance to heat and UV Plant Communications
(Figure 5A). The proportion of defective pollen grains increased
with prolonged stress treatment in both B73 and Zmvamp726
(Figure 5A). However, heat treatment resulted in a significantly
higher rate of defective pollen grains in Zmvamp726 than in
B73 (Figure 5A). The percentage of defective pollen grains
produced by Zmvamp726 was 75% at 60 min after treatment,
whereas this percentage was 30% in B73 (Figure 5A).
Likewise, most pollen grains of Zmvamp726 (>95%) completely
lost viability after exposure to UV for 60 min, whereas more
than half of the pollen grains from B73 remained viable
(Figure 5B). These results suggest that ZmVAMP726 promotes
pollen resistance to heat and UV radiation.
Phylogenetic analysis of VAMP7 family proteins revealed that
ZmVAMP726 falls within the same clade as AtVAMP721,
AtVAMP722, AtVAMP725, and AtVAMP726, as well as several
other VAMP7s from Glycine max,Solanum lycopersicum,Triti-
cum aestivum,Oryza sativa, and Setaria viridis (Supplemental
Figure 10A), suggesting the functional conservation of
VAMP726 across different plant species. A gene expression
map of Arabidopsis development and b-glucuronidase (GUS)
staining of an AtVAMP726
pro
:GUS reporter line showed that At-
VAMP726 is mainly expressed in pollen (Supplemental
Figure 11), indicating that, like ZmVAMP726,AtVAMP726 may
also be associated with pollen development. A previous report
found that an AtVAMP726-GFP fusion protein was localized
mainly on the plasma membrane and was likely associated with
vesicle trafficking (Uemura et al., 2004). We co-expressed
AtVAMP726 fused to mCherry and ZmVAMP726 fused to GFP
in Nicotiana benthamiana. The fluorescent signals of the two
fusion proteins clearly overlapped (Supplemental Figure 10B).
The similar spatiotemporal expression patterns of ZmVAMP726
and AtVAMP726 suggest that they may have similar functions.
We obtained an Arabidopsis T-DNA insertion mutant (At-
vamp726-tdna) and two null-mutant alleles created via the
CRISPR–Cas9 system (Atvamp726-gr1 and Atvamp726-
gr2)(Supplemental Figure 12A). In addition, we created
aVAMP726-overexpressing line in the Col-0 background
(35S
pro
:AtVAMP726,Supplemental Figure 12C). No obvious
differences in reproductive growth were observed between
these materials and Col-0 (Supplemental Figure 12D). However,
pollen grains of the mutants were generally smaller than those
of Col-0 and 35S
pro
:AtVAMP726 and showed an increased
incidence of shape distortion (Supplemental Figure 12E). In the
absence of stress, pollen germination rate was slightly but not
significantly reduced in Atvamp726-tdna compared with Col-
0 and 35S
pro
:AtVAMP726, but pollen germination was more
strongly reduced in Atvamp726-gr1 and Atvamp726-gr2
(Figure 6A). After heat treatment for 30 min and 60 min, pollen
grains of all three mutants exhibited a significantly reduced
germination rate, and the declines were much steeper for
Figure 5. ZmVAMP726 is involved in maize pollen resistance to heat and UV radiation.
(A) Pollen viability of B73 and Zmvamp726 after heat treatment. Fresh pollen grains were exposed to 37C for the indicated times and then stained with
TTC. Student’s t-test was used for statistical analysis (n=5,P< 0.05). Error bars represent SD. Asterisks indicate significant differences. Scale bar,
500 mm.
(B) Pollen viability of B73 and Zmvamp726 after UV radiation treatment. Fresh pollen grains were exposed to UV for the indicated times and then stained
with TTC. Student’s t-test was used for statistical analysis (n=5,P< 0.05). Error bars represent SD. Asterisks indicate significant differences. Scale bar,
500 mm.
8Plant Communications 4, 100682, November 13 2023 ª2023 The Author(s).
Plant Communications VAMP726 confers pollen resistance to heat and UV
Figure 6. Both AtVAMP726 and ZmVAMP726 are involved in resistance of Arabidopsis pollen to heat and UV radiation.
(A) AtVAMP726 is involved in resistance of Arabidopsis pollen to heat and UV radiation. Fresh pollen grains of the indicated Arabidopsis genotypes were
exposed to 37C or UV radiation for the indicated times and then tested for in vitro germination. Two-way ANOVA followed by Tukey’s honestly significant
(legend continued on next page)
Plant Communications 4, 100682, November 13 2023 ª2023 The Author(s). 9
VAMP726 confers pollen resistance to heat and UV Plant Communications
Atvamp726-gr1 and Atvamp726-gr2 (Figure 6A). Notably, pollen
germination was less affected by heat treatment in
35S
pro
:AtVAMP726 than in Col-0 (Figure 6A). Prolonged heat
treatment (to 90 min) resulted in similar germination rates (less
than 10%) of Col-0 and the three mutants, whereas over 20%
of the 35S
pro
:AtVAMP726 pollen grains remained viable
(Figure 6A). Similar patterns in germination rate were observed
when pollen grains were exposed to UV radiation (Figure 6A).
We next examined the lignin monomer composition in sporopol-
lenin from the mutants and overexpression line. The abundance
of H units and the total content of lignin monomers were signifi-
cantly higher in sporopollenin of 35S
pro:AtVAMP726
than in that of
Col-0 and the two null mutants, but there were no significant dif-
ferences in the abundance of G and S lignin units (Supplemental
Figure 12F). However, in sporopollenin of Atvamp726-gr1 and At-
vamp726-gr2, we did detect a slight but not significant reduction
in the abundance of each lignin monomer or the total content of
lignin monomers (Supplemental Figure 12F). This might be
attributed to the overall low level of lignin monomer
accumulation in Arabidopsis sporopollenin. Nonetheless, these
results suggest that AtVAMP726 mediates pollen resistance to
heat and UV radiation, and genetic enhancement of AtVAMP726
expression improved pollen resistance to these stresses, likely by
influencing lignin monomer composition in sporopollenin.
We next wondered whether monocot and dicot VAMP726s
could compensate for one another in the promotion of
pollen stress resistance. We created transgenic Arabidopsis
plants expressing AtVAMP726 or ZmVAMP726 under a 35S
promoter in the Atvamp726-gr1 background (35S
pro
:
AtVAMP726/Atvamp726-gr1 and 35S
pro
:ZmVAMP726/At-
vamp726-gr1,Supplemental Figure 13) and examined
their pollen viability under heat and UV treatment. Under
both stress conditions, constitutive expression of either
AtVAMP726 or ZmVAMP726 in Atvamp726-gr1 resulted in
pollen grains that were more resistant to heat and UV treatment
compared with Col-0 and Atvamp726-gr1 (Figure 6B).
Although the pollen resistance of 35S
pro
:AtVAMP726/
Atvamp726-gr1 and 35S
pro
:ZmVAMP726/Atvamp726-gr1 was
slightly lower than that of 35S
pro
:AtVAMP726 (Col-0 back-
ground, Figure 6B), there was no difference in pollen viability
between these two complemented lines, suggesting that
heterologous expression of ZmVAMP726 can fully restore
pollen resistance of Atvamp726-gr1. Together, these results
demonstrate the functional conservation of VAMP726 in maize
and Arabidopsis.
Overexpression of VAMP726 led to enrichment of
peroxidases involved in lignification in the apoplastic
space
We speculated that VAMP726 was a key determinant of lignin
monomer composition in sporopollenin. As a protein involved in
vesicle trafficking, VAMP726 likely mediates the transport of
either metabolite units of sporopollenin or proteins involved in
sporopollenin development from the cytosol to the apoplast.
However, the cargo of VAMP726 may not necessarily be mono-
lignols, because sporopollenin also contains long-chain polyvinyl
alcohol units and coumaroylated aliphatic units (Li et al., 2019).
Owing to technical limitations, we were not able to quantify
these metabolite units in sporopollenin from maize or
Arabidopsis. Nevertheless, the available facilities enabled us to
determine which kinds of aliphatic units were present in
thioacidolysis-released fractions of maize and Arabidopsis
sporopollenin. We identified three fatty acids and three
glycerides in the thioacidolysis-released products of maize
sporopollenin (Supplemental Figure 14A). Semi-quantification re-
vealed no differences in their abundance between sporopollenin
from B73 and Zmvamp726 (Supplemental Figure 14B).
Similarly, there was no significant difference in the abundance
of two fatty acids and two glycerides detected in the
thioacidolysis-released products of sporopollenin from Col-0,
Atvamp726 mutants, and 35S
pro
:AtVAMP726 (Supplemental
Figure 14C). These results suggest that at least the abundance
of aliphatic units released from sporopollenin by thioacidolysis
is not associated with VAMP726.
Another member of the VAMP72 subgroup, VAMP721, has been
shown to mediate translocation of cell-wall-modifying enzymes
from the cytosol to the apoplastic space (Uemura et al., 2019).
We speculated that VAMP726 may also transport proteins
involved in the development of sporopollenin. To explore this
possibility, we analyzed the proteome of apoplastic washing fluid
(AWF) from mature leaves of Arabidopsis plants overexpressing
VAMP726 or an empty vector (Supplemental Datasets 4 and 5).
To reduce costs, we did not use the Atvamp726 mutant
because of the low expression of VAMP726 in its mature leaf
tissues (Supplemental Figure 11). Proteomics analysis revealed
that the abundance of 761 proteins in the apoplastic space was
significantly altered by overexpression of VAMP726
(Supplemental Dataset 6). Gene ontology (GO) enrichment
analysis of these differentially accumulated proteins revealed
significant enrichment of a number of biological processes,
including ‘‘peroxidase activity’’ (Figure 7A and Supplemental
Dataset 7), suggesting that overexpression of VAMP726 may
significantly affect this process. Eight peroxidases (PRXs) and
three ascorbate peroxidases (APXs) were associated with this
GO term (Supplemental Dataset 8). PRXs and laccases are two
major classes of oxidative enzymes that catalyze the radical
coupling of monolignols to form lignin (Tobimatsu and Schuetz,
2019). A recent study also showed that a mitochondrial APX is
responsible for autonomous lignification at the earliest stage of
xylem development (Zhang et al., 2022). Therefore, proteins
associated with this GO term may be involved in the lignin
polymerization process. Because PRXs and APXs include many
members and not all of them are likely to be involved in lignin
polymerization, we focused on the 11 proteins associated with
this GO term and looked for published evidence supporting
their potential role in lignification. PRX4 and PRX25 have
difference test was used for statistical analysis (n=5,P< 0.05). Error bars represent SD. Letters indicate significant differences in germination rate at each
time point. Scale bar, 200 mm.
(B) ZmVAMP726 restored pollen stress resistance in Atvamp726. Fresh pollen grains were exposed to 37C or UV radiation for the indicated times and
then tested for in vitro germination. Two-way ANOVA followed by Tukey’s honestly significant difference test was used for statistical analysis (n=5,
P< 0.05). Error bars represent SD. Letters indicate significant differences in germination rate at each time point. Scale bar, 200 mm.
10 Plant Communications 4, 100682, November 13 2023 ª2023 The Author(s).
Plant Communications VAMP726 confers pollen resistance to heat and UV
previously been shown to participate in lignin polymerization
of Arabidopsis (Shigeto et al., 2013,2014,2015;Fernandez-
Perez et al., 2015). Likewise, PRX49 and PRX53 have been
proposed to function in lignin polymerization (Ostergaard et al.,
2000;Herrero et al., 2013;Shigeto et al., 2014), although
more experimental evidence is needed. These four PRXs were
all among the proteins enriched in AWF of the VAMP726-
overexpressing line (Figure 7B), suggesting that VAMP726
potentially mediates accumulation of these PRXs in the apoplas-
tic space. No other PRXs annotated with this GO term have
been reported or predicted to function in lignin polymerization
in Arabidopsis. By contrast, the abundance of three APXs
in the apoplastic space was negatively affected by
overexpression of VAMP726 (Figure 7B). APX1, also known as
C3H, is involved in the early steps of monolignol biosynthesis in
Arabidopsis (Barros et al., 2019). There is no evidence to
support a role for APX1 in lignin polymerization in the apoplast.
The other two APXs have not been reported or predicted to
function in lignin-related processes in Arabidopsis. Together,
these results suggest that VAMP726 is associated with the
enrichment of PRXs involved in lignin polymerization in the apo-
plastic space.
These enriched PRXs might speed up the lignification process,
resulting in more rapid consumption of monolignols in the apoplas-
Figure 7. Overexpression of AtVAMP726 al-
ters the proteome of the apoplastic space.
(A) GO enrichment analysis of differentially accu-
mulated proteins in apoplastic washing fluids ob-
tained from Arabidopsis plants overexpressing
VAMP726. The top ten most significantly enriched
GO terms are listed.
(B) Relative abundance of proteins associated with
the GO term ‘‘peroxidase activity.’’ The protein
abundances were normalized by Zscore and
plotted. Proteins were organized according to hi-
erarchical clustering. Three biological replicates per
genotype are represented by three columns. Color
scale represents the Z-score value.
(C) Quantification of p-coumaryl alcohol in apo-
plastic washing fluids obtained from Arabidopsis
plants. Student’s t-test was used for statistical
analysis (n=3,P< 0.05). Error bars represent SD.
Asterisks indicate a significant difference.
tic space. We therefore attempted to quantify
the concentration of monolignols in the AWF.
We were able to detect the presence of p-cou-
maryl alcohol in the AWF but were unable to
detect the other two monolignols (Figure 7C).
The results showed that the abundance of
p-coumaryl alcohol in the AWF was
significantly lower in 35S
pro
:AtVAMP726
(Figure 7C), which is consistent with our
speculation and with the increased lignin
monomers detected in sporopollenin of
35S
pro
:AtVAMP726 (Supplemental Figure
12F). Although the results presented above
do not fully clarify the mechanism by which
VAMP726 influences lignin monomer content
in sporopollenin, they do suggest the possibil-
ity that VAMP726 may mediate translocation of enzymes involved
in lignin polymerization to the apoplastic space.
DISCUSSION
The tough nature of sporopollenin makes it extremely resistant to
physical and chemical degradation (Grienenberger and Quilichini,
2021). This property has hindered the elucidation of its chemical
composition for decades. Two recent reports confirmed that
aliphatic and phenolic compounds are structural components
of sporopollenin in vascular plants (Li et al., 2019;Xue et al.,
2020). Here, we provide compelling evidence that H, G, and S
lignin units are also present in sporopollenin of maize and
Arabidopsis. Interestingly, only G units were found previously in
sporopollenin of Cryptomeria (gymnosperm) and Lilium (dicot)
(Xue et al., 2020). This could be explained by the fact that the
lignin monomer composition is species specific (Campbell and
Sederoff, 1996). Nonetheless, our results and previous reports
strongly support the notion that lignin monomers are structural
components of plant sporopollenin.
Formation of the lignin polymer is considered to be a major event
that enabled vascular plants to colonize terrestrial environments
(Weng and Chapple, 2010). The lignin polymer can crosslink
with hemicelluloses to form a specialized structure that permits
Plant Communications 4, 100682, November 13 2023 ª2023 The Author(s). 11
VAMP726 confers pollen resistance to heat and UV Plant Communications
plants to transport water and stand upright (Terrett and Dupree,
2019). In addition, it is essential for helping plants to cope with
diverse environmental stresses (Cesarino, 2019). For example,
lignin can be deposited at infection sites of incompatible
pathogens to form a physical barrier, thereby restricting their
proliferation (Lee et al., 2019). Accordingly, disruption of
monolignol biosynthesis in Arabidopsis drastically impairs
disease resistance (Quentin et al., 2009;Tronchet et al., 2010).
Likewise, monolignol biosynthesis is also indispensable for
pollen resistance to excessive UV radiation (Xue et al., 2020).
Here, we showed that deficiency in monolignol biosynthesis
also impairs pollen resistance to heat stress, highlighting the
essential role of monolignol biosynthesis in determining the
outcome of plant–environment interactions. Although we were
able to identify and quantify lignin monomers in sporopollenin, it
remains unclear how they are incorporated into the main polymer
structure. Thioacidolysis mainly breaks b-O-4 ether bonds be-
tween lignin monomers and ester bonds. Our data suggest that
lignin monomers are not attached to other metabolite units of
sporopollenin through ester bonds, indicating that they may
form a lignin polymer in sporopollenin. However, they may also
be linked to other metabolite units of sporopollenin through ether
bonds. Novel analytical methods, such as solid-state NMR or
energy-resolved mass spectrometry (Li et al., 2019;Dong et al.,
2022), should be used to dissect the exact linkage patterns of
lignin monomers with each other and with other sporopollenin
constituents.
G and S lignin units are the dominant lignin monomers in the stem
tissues of most plant species analyzed to date. H units usually
comprise the minority of total lignin monomers in plants, with
some exceptions in which the monolignol biosynthetic pathway
was artificially modified (Franke et al., 2002;Li et al., 2010).
Lignin monomer composition also exhibits tissue specificity.
Lignin of water-conducting tracheids and vessels is particularly
enriched in G units, whereas the proportion of S units increases
significantly in lignin of fiber cells (Meyer et al., 1998;
Nakashima et al., 2008). In Japanese cypress (Chamaecyparis
obtusa, a gymnosperm), the proportion of H units is
significantly higher in compression wood (22%) than in normal
wood (1%) (Hiraide et al., 2021). Here, we found that H lignin
comprised the majority of lignin monomers in sporopollenin of
maize and Arabidopsis. This unique feature of sporopollenin is
intriguing, as other parts of the plant rarely deposit such a
high proportion of H units in the cell wall (which contains mainly
G and S units). The composition of lignin monomers defines the
physical and chemical characteristics of the lignin polymer.
During lignin polymerization, the three lignin monomers are
mostly interlinked via C–C and C–O linkages, and C–C linkages
require higher dissociation energy than C–O linkages (Dong
et al., 2019). Methoxy groups on the aromatic rings of lignin
units can prevent the formation of interunit C–C linkages
(Anderson et al., 2019). Therefore, the ratio of different lignin
monomers can have a significant effect on the texture of the
lignin polymer, and specific lignin chemotypes may be
associated with specific functions (Renault et al., 2019). An
increased proportion of H units would result in a highly
condensed lignin structure, which is often associated with
stress, as their relatively short biosynthetic pathway could
provide readily accessible monolignols for rapid lignification to
cope with stresses (Cesarino, 2019). If indeed the lignin
monomers in sporopollenin form an integral polymer, it likely
contributes to the superior durability of sporopollenin. In fact,
sporopollenin can persist over millions of years, and the
presence of relatively intact fossil sporopollenin in sediments
provided the earliest evidence of land plants (Wellman et al.,
2003). Previous work and our results demonstrate the key role
of monolignol biosynthesis in pollen stress resistance (Xue
et al., 2020). We therefore speculate that the unique enrichment
of H units in sporopollenin might be an evolutionary adaptation
that enables plants to cope with increased stresses, such as
higher temperature and UV exposure, associated with
colonization of the terrestrial environment. In this case, plants
deposit more H units in sporopollenin to form a more
condensed lignin polymer that protects genetic material from
environmental stress.
We found that VAMP726 was positively involved in lignin mono-
mer composition in sporopollenin and pollen resistance to heat
and UV radiation. VAMPs, also known as R-type SNAREs, typi-
cally localize to membrane vesicles and drive membrane fusion
with other organelles (Fujimoto et al., 2020). We made initial
attempts to identify the cargo of VAMP726, and the results
showed that overexpression of VAMP726 resulted in enrichment
of PRXs involved in lignin polymerization, supporting a possible
role for VAMP726 in mediating the translocation of proteins
involved in lignification. However, we also need to consider the
possibility that VAMP726 is directly involved in transporting
monolignols, as evidenced by its key role in the formation of au-
tophagosomes in Arabidopsis (He et al., 2022). It was recently
shown to deliver monolignols from the cytosol to the cell wall to
facilitate rapid lignification upon pathogen attack (Jeon et al.,
2023). Nonetheless, although previous reports have shown that
transporter-based systems might mediate the translocation of
monolignols (Miao and Liu, 2010;Alejandro et al., 2012), it
remains debatable whether this mechanism is indispensable in
planta (Perkins et al., 2019). A recent study demonstrated that
monolignols could diffuse across the plasma membrane when a
concentration gradient was present, and this process was
unaffected by inhibition of transporter activity (Perkins et al.,
2022). Therefore, the involvement of VAMP726 in monolignol
transport might be conditional. VAMP726 is one of eight mem-
bers of the VAMP72 subgroup (Zhang et al., 2015). Previous
reports have demonstrated that two members of this group,
VAMP721 and VAMP722, can form a SNARE complex with
plasma-membrane-localized Qa-SNAREs to transport immune
proteins, cell-wall-modification enzymes, secondary metabo-
lites, and cell-wall components to the extracellular space (Kwon
et al., 2008;Uemura et al., 2019;Yun et al., 2022). Therefore, it
is likely that VAMP726 also interacts with other SNAREs to
mediate translocation of metabolites or proteins required for
accumulation of lignin monomers in sporopollenin. More compre-
hensive investigations are required to address the exact role
of VAMP726 in the incorporation of lignin monomers into
sporopollenin.
METHODS
Plant materials
The association mapping panel of maize materials was kindly
provided by Dr. Jianbing Yan (Huazhong Agricultural
12 Plant Communications 4, 100682, November 13 2023 ª2023 The Author(s).
Plant Communications VAMP726 confers pollen resistance to heat and UV
University) and consisted of 415 inbred lines (Yang et al., 2010).
Among these, we were able to harvest sufficient pollen for
sporopollenin analysis from 117 inbred lines. Hence, data
obtained from 117 inbred lines were used for GWASs.
Zmvamp726-1 and Zmvamp726-2 mutants carrying premature
stop codons were obtained from an ethyl methanesulfonate
mutant library of B73 (Lu et al., 2018). The premature stop
codon in Zmvamp726-1 is located in the first exon, and the pre-
mature stop codon in Zmvamp726-2 is located in the third
exon. Because Zmvamp726-1 showed anther indehiscence,
only Zmvamp726-2 was backcrossed with B73, and its progeny
were self-crossed for two generations (BC
1
F
2
). This BC
1F
2
gener-
ation of Zmvamp726-2 was used in subsequent work. For Arabi-
dopsis, the Col-0 ecotype was preserved in the lab. T-DNA inser-
tion mutants tt4-3 (CS66119), ccr1 (GK-622C01), comt
(Salk_135290), ccomt (Salk_055103), and vamp726
(Salk_082690) were obtained from Nottingham Arabidopsis
Stock Center (Scholl et al., 2000). For simplicity, these mutants
are mentioned without specific allele names in the main text. A
detailed description of growth conditions is included in
supplemental information.
Thioacidolysis of cell-wall residues
Pollen grains or other maize tissues were collected as described
in supplemental information. Cell-wall residues were prepared
according to a previously described protocol (Xue et al., 2020)
with slight modifications. Thioacidolysis of cell-wall residues
was performed as described previously (Foster et al., 2010).
Detailed descriptions of these procedures are included in
supplemental information.
Solution-state two-dimensional nuclear magnetic
resonance
Approximately 2 g of cell-wall residue was treated with 300 ml of
solution containing 1% Macerozyme R10 and 1% Cellulase Ono-
zuka R10 (w/v, dissolved in 0.1 M sodium acetate buffer [pH 4.5])
at 37C for 16 h. The resulting materials were washed with water
and acetone three times and then subjected to thioacidolysis. For
solution-state 2D-NMR spectroscopy, the thioacidolysis lysates
of cell-wall residues were dissolved in 0.55 ml of deuterated
DMSO-d6 (99.9%, Sigma). A detailed description of this proced-
ure is included in supplemental information.
Gas chromatography–mass spectrometry
For GC–MS, at least 2 mg of cell-wall residue was required for thi-
oacidolysis, and the thioacidolysis lysate was then derivatized as
described previously (Foster et al., 2010). A detailed description
of this procedure is included in supplemental information.
Assessment of pollen viability
For heat treatment, fresh pollen grains of maize were placed in
a37
C incubator for the indicated times. For UV radiation, the
pollen grains were placed under a UV lamp (6 W, 25 cm distance)
for the indicated times. Arabidopsis pollen grains were spread
onto solid basic medium before the stresses were applied. Pollen
viability was then examined immediately. TTC staining was used
to determine maize pollen viability (Alexander, 1969), and an
in vitro germination assay was used to determine Arabidopsis
pollen viability. Detailed descriptions of these procedures are
included in supplemental information.
Genome-wide association study
The MLM model implemented in TASSEL 3.0 was used for GWAS
(Bradbury et al., 2007;Li et al., 2013). Outliers were removed
before the phenotype data were imported into TASSEL. The sug-
gested Pvalue of 2.04 310
6
(1/En) was used as the genome-
wide threshold for significant SNP–trait associations, as is
common in plant GWASs. The previously estimated decay dis-
tance of linkage disequilibrium (LD) in this association mapping
panel (30 kb, R
2
= 0.1) was used to define a 60-kb
QTL interval, i.e., the 30 kb upstream and downstream of each
SNP. The B73 reference genome (RefGen_v2) was used for anno-
tation of candidate genes. Other details of the GWASs are
included in supplemental information.
Proteome analysis of apoplastic washing fluid
AWF of Arabidopsis plants was obtained using the infiltration
method as described previously (Chen et al., 2022). Proteins
were extracted as described in supplemental information. The
protein pellets were dissolved in 8 M urea, and the protein
concentrations were determined using a detergent-compatible
Bradford Protein Assay Kit (Beyotime Biotechnology). Label-
free proteome analysis was performed as described previously
(Yu et al., 2022).
Owing to space limitations, detailed descriptions of liquid chro-
matography coupled with triple-quadrupole mass spectrometry,
scanning electron microscopy, collection of washing fractions
during sporopollenin preparation, quantitative real-time PCR,
GUS staining, phylogenetic analysis, and plant transformation
are included in supplemental information.
DATA AND CODE AVAILABILITY
The data that support the findings of this study are available from
the corresponding author upon reasonable request.
SUPPLEMENTAL INFORMATION
Supplemental information is available at Plant Communications Online.
FUNDING
This work was supported by grants from the National Natural Science
Foundation of China (31970323 and 32170269 to Xuebin Zhang;
32171980 to Xuehai Zhang), the Henan Key Scientific Research Programs
to Universities and Colleges (22ZX006 to Xuebin Zhang), the Open Project
Funding of the State Key Laboratory of Crop Stress Adaptation and
Improvement (2021KF07 to Xuehai Zhang), the Open Project Funding of
the State Key Laboratory of Wheat and Maize Crops Science
(30501194 to K.Y.), the Henan Overseas Expertise Introduction Center
for Discipline Innovation (CXJD2020004), and the Program for Innovative
Research Team (in Science and Technology) of the University of Henan
Province (21IRTSTHN019).
AUTHOR CONTRIBUTIONS
Xuebin Zhang conceived and supervised the project. Xuebin Zhang, K.Y.,
W.Y., D.Y., and H.D. designed the experiments. W.Y., D.Y., and H.D. per-
formed most of the experiments and analyzed the data. J.Z. established
methods for detection of monolignols. Y.C. performed analysis of sporo-
pollenin derived from Arabidopsis mutants. J.Z., Y.C., C.L., B.Z., and Y.M.
assisted in experiments, including collection of maize materials in the field,
Plant Communications 4, 100682, November 13 2023 ª2023 The Author(s). 13
VAMP726 confers pollen resistance to heat and UV Plant Communications
sample preparation, and GC–MS. Y.Z. performed initial sporopollenin
analysis of maize inbred lines. E.Y. performed NMR spectroscopy. X.L.,
Xuehai Zhang, and J.T. provided materials and helped with experimental
design and data analysis. W.Y., K.Y., and Xuebin Zhang prepared
graphs and wrote the manuscript with input from all co-authors. All au-
thors read and approved the manuscript.
ACKNOWLEDGMENTS
We thank all members of Xuebin Zhang’s lab at Henan University for col-
lecting maize materials in the field. We thank Drs. Jianbing Yan (Huazhong
Agricultural University) for providing maize inbred lines; Yunjun Zhao
(Shanghai Institute of Plant Physiology and Ecology) and Xiaowei Han
(Huazhong Agricultural University) for critical reading of the manuscript
and helpful suggestions; and Xiao Wang (Henan University) for proteome
analysis. We thank Weipeng Wang for help with the phylogenetic analysis.
No conflict of interest is declared.
Received: June 20, 2023
Revised: August 28, 2023
Accepted: August 31, 2023
Published: September 9, 2023
REFERENCES
Alejandro, S., Lee, Y., Tohge, T., Sudre, D., Osorio, S., Park, J.,
Bovet, L., Lee, Y., Geldner, N., Fernie, A.R., and Martinoia, E.
(2012). AtABCG29 is a monolignol transporter involved in lignin
biosynthesis. Curr. Biol. 22:1207–1212. https://doi.org/10.1016/j.
cub.2012.04.064.
Alexander, M.P. (1969). Differential staining of aborted and nonaborted
pollen. Stain Technol. 44:117–122. https://doi.org/10.3109/105202
96909063335.
Anderson, E.M., Stone, M.L., Katahira, R., Reed, M., Muchero, W.,
Ramirez, K.J., Beckham, G.T., and Roma
´n-Leshkov, Y. (2019).
Differences in S/G ratio in natural poplar variants do not predict
catalytic depolymerization monomer yields. Nat. Commun. 10:2033.
https://doi.org/10.1038/s41467-019-09986-1.
Ariizumi, T., and Toriyama, K. (2011). Genetic regulation of sporopollenin
synthesis and pollen exine development. Annu. Rev. Plant Biol.
62:437–460. https://doi.org/10.1146/annurev-arplant-042809-112312.
Barros, J., Escamilla-Trevino, L., Song, L., Rao, X., Serrani-Yarce,
J.C., Palacios, M.D., Engle, N., Choudhury, F.K., Tschaplinski,
T.J., Venables, B.J., et al. (2019). 4-Coumarate 3-hydroxylase in the
lignin biosynthesis pathway is a cytosolic ascorbate peroxidase. Nat.
Commun. 10:1994. https://doi.org/10.1038/s41467-019-10082-7.
Bonawitz, N.D., and Chapple, C. (2010). The genetics of lignin
biosynthesis: connecting genotype to phenotype. Annu. Rev. Genet.
44:337–363. https://doi.org/10.1146/annurev-genet-102209-163508.
Bradbury, P.J., Zhang, Z., Kroon, D.E., Casstevens, T.M., Ramdoss,
Y., and Buckler, E.S. (2007). TASSEL: software for association
mapping of complex traits in diverse samples. Bioinformatics
23:2633–2635. https://doi.org/10.1093/bioinformatics/btm308.
Campbell, M.M., and Sederoff, R.R. (1996). Variation in lignin content
and composition (mechanisms of control and implications for the
genetic improvement of plants). Plant Physiol. 110:3–13. https://doi.
org/10.1104/pp.110.1.3.
Cesarino, I. (2019). Structural features and regulation of lignin deposited
upon biotic and abiotic stresses. Curr. Opin. Biotechnol. 56:209–214.
https://doi.org/10.1016/j.copbio.2018.12.012.
Chaturvedi, P., Wiese, A.J., Ghatak, A., Za
´veska
´Dra
´bkova
´, L.,
Weckwerth, W., and Honys, D. (2021). Heat stress response
mechanisms in pollen development. New Phytol. 231:571–585.
https://doi.org/10.1111/nph.17380.
Chen, A., He, B., and Jin, H. (2022). Isolation of extracellular vesicles from
Arabidopsis. Curr. Protoc. 2:e352. https://doi.org/10.1002/cpz1.352.
Choi, H., Jin, J.Y., Choi, S., Hwang, J.U., Kim, Y.Y., Suh, M.C., and Lee,
Y. (2011). An ABCG/WBC-type ABC transporter is essential for
transport of sporopollenin precursors for exine formation in
developing pollen. Plant J. 65:181–193. https://doi.org/10.1111/j.
1365-313X.2010.04412.x.
Choi, H., Ohyama, K., Kim, Y.Y., Jin, J.Y., Lee, S.B., Yamaoka, Y.,
Muranaka, T., Suh, M.C., Fujioka, S., and Lee, Y. (2014). The role
of Arabidopsis ABCG9 and ABCG31 ATP binding cassette
transporters in pollen fitness and the deposition of steryl glycosides
on the pollen coat. Plant Cell 26:310–324. https://doi.org/10.1105/
tpc.113.118935.
Del Rı
´o, J.C., Rencoret, J., Gutie
´rrez, A., Kim, H., and Ralph, J. (2018).
Structural characterization of lignin from maize (Zea mays L.) fibers:
evidence for diferuloylputrescine incorporated into the lignin polymer
in maize kernels. J. Agric. Food Chem. 66:4402–4413. https://doi.
org/10.1021/acs.jafc.8b00880.
Dixon, R.A., and Paiva, N.L. (1995). Stress-Induced Phenylpropanoid
Metabolism. Plant Cell 7:1085–1097. https://doi.org/10.1105/tpc.7.
7.1085.
Do, C.T., Pollet, B., The
´venin, J., Sibout, R., Denoue, D., Barrie
`re, Y.,
Lapierre, C., and Jouanin, L. (2007). Both caffeoyl Coenzyme A 3-
O-methyltransferase 1 and caffeic acid O-methyltransferase 1 are
involved in redundant functions for lignin, flavonoids and sinapoyl
malate biosynthesis in Arabidopsis. Planta 226:1117–1129. https://
doi.org/10.1007/s00425-007-0558-3.
Dolferus, R., Ji, X., and Richards, R.A. (2011). Abiotic stress and control
of grain number in cereals. Plant Sci. 181:331–341. https://doi.org/10.
1016/j.plantsci.2011.05.015.
Dong, L., Lin, L., Han, X., Si, X., Liu, X., Guo, Y., Lu, F., Rudi
c, S.,
Parker, S.F., Yang, S., and Wang, Y. (2019). Breaking the limit of
lignin monomer production via cleavage of interunit carbon–carbon
linkages. Chem 5:1521–1536. https://doi.org/10.1016/j.chempr.2019.
03.007.
Dong, N.Q., and Lin, H.X. (2021). Contribution of phenylpropanoid
metabolism to plant development and plant-environment
interactions. J. Integr. Plant Biol. 63:180–209. https://doi.org/10.
1111/jipb.13054.
Dong, X., Mayes, H.B., Morreel, K., Katahira, R., Li, Y., Ralph, J., Black,
B.A., and Beckham, G.T. (2022). Energy-Resolved Mass
Spectrometry as a Tool for Identification of Lignin Depolymerization
Products. ChemSusChem. https://doi.org/10.1002/cssc.202201441.
Edlund, A.F., Swanson, R., and Preuss, D. (2004). Pollen and stigma
structure and function: the role of diversity in pollination. Plant Cell
Suppl. 16:S84–S97. https://doi.org/10.1105/tpc.015800.
Eudes, A., George, A., Mukerjee, P., Kim, J.S., Pollet, B., Benke, P.I.,
Yang, F., Mitra, P., Sun, L., Cetinkol, O.P., et al. (2012).
Biosynthesis and incorporation of side-chain-truncated lignin
monomers to reduce lignin polymerization and enhance
saccharification. Plant Biotechnol. J. 10:609–620. https://doi.org/10.
1111/j.1467-7652.2012.00692.x.
Ferna
´ndez-Pe
´rez, F., Vivar, T., Pomar, F., Pedren
˜o, M.A., and Novo-
Uzal, E. (2015). Peroxidase 4 is involved in syringyl lignin formation in
Arabidopsis thaliana. J. Plant Physiol. 175:86–94. https://doi.org/10.
1016/j.jplph.2014.11.006.
Foster, C.E., Martin, T.M., and Pauly, M. (2010). Comprehensive
compositional analysis of plant cell walls (Lignocellulosic biomass)
part I: lignin. J. Vis. Exp. https://doi.org/10.3791/1745.
Franke, R., Hemm, M.R., Denault, J.W., Ruegger, M.O., Humphreys,
J.M., and Chapple, C. (2002). Changes in secondary metabolism
and deposition of an unusual lignin in the ref8 mutant of Arabidopsis.
Plant J. 30:47–59. https://doi.org/10.1046/j.1365-313x.2002.01267.x.
14 Plant Communications 4, 100682, November 13 2023 ª2023 The Author(s).
Plant Communications VAMP726 confers pollen resistance to heat and UV
Fraser, C.M., and Chapple, C. (2011). The phenylpropanoid pathway in
Arabidopsis.Arabidopsis Book 9, e0152. https://doi.org/10.1199/
tab.0152.
Fujimoto, M., Ebine, K., Nishimura, K., Tsutsumi, N., and Ueda, T.
(2020). Longin R-SNARE is retrieved from the plasma membrane by
ANTH domain-containing proteins in Arabidopsis. Proc. Natl. Acad.
Sci. USA 117:25150–25158. https://doi.org/10.1073/pnas.2011152117.
Grienenberger, E., and Quilichini, T.D. (2021). The toughest material in
the plant kingdom: an update on sporopollenin. Front. Plant Sci. 12,
703864. https://doi.org/10.3389/fpls.2021.703864.
He, Y., Gao, J., Luo, M., Gao, C., Lin, Y., Wong, H.Y., Cui, Y., Zhuang,
X., and Jiang, L. (2022). VAMP724 and VAMP726 are involved in
autophagosome formation in Arabidopsis thaliana. Autophagy
19:1406–1423. https://doi.org/10.1080/15548627.2022.2127240.
Herrero, J., Esteban-Carrasco, A., and Zapata, J.M. (2013). Looking for
Arabidopsis thaliana peroxidases involved in lignin biosynthesis. Plant
Physiol. Biochem. 67:77–86. https://doi.org/10.1016/j.plaphy.2013.
02.019.
Hiraide, H., Tobimatsu, Y., Yoshinaga, A., Lam, P.Y., Kobayashi, M.,
Matsushita, Y., Fukushima, K., and Takabe, K. (2021). Localised
laccase activity modulates distribution of lignin polymers in
gymnosperm compression wood. New Phytol. 230:2186–2199.
https://doi.org/10.1111/nph.17264.
Hoopes, G.M., Hamilton, J.P., Wood, J.C., Esteban, E., Pasha, A.,
Vaillancourt, B., Provart, N.J., and Buell, C.R. (2019). An updated
gene atlas for maize reveals organ-specific and stress-induced
genes. Plant J. 97:1154–1167. https://doi.org/10.1111/tpj.14184.
Huang, M.D., Chen, T.L.L., and Huang, A.H.C. (2013). Abundant type III
lipid transfer proteins in Arabidopsis tapetum are secreted to the locule
and become a constituent of the pollen exine. Plant Physiol. 163:1218–
1229. https://doi.org/10.1104/pp.113.225706.
Ichino, T., and Yazaki, K. (2022). Modes of secretion of plant lipophilic
metabolites via ABCG transporter-dependent transport and vesicle-
mediated trafficking. Curr. Opin. Plant Biol. 66, 102184. https://doi.
org/10.1016/j.pbi.2022.102184.
Jeon, H.S., Jang, E., Kim, J., Kim, S.H., Lee, M.H., Nam, M.H.,
Tobimatsu, Y., and Park, O.K. (2023). Pathogen-induced autophagy
regulates monolignol transport and lignin formation in plant immunity.
Autophagy 19:597–615. https://doi.org/10.1080/15548627.2022.
2085496.
Jiang, J., Zhang, Z., and Cao, J. (2013). Pollen wall development: the
associated enzymes and metabolic pathways. Plant Biol.
15:249–263. https://doi.org/10.1111/j.1438-8677.2012.00706.x.
Kim, H., Padmakshan, D., Li, Y., Rencoret, J., Hatfield, R.D., and
Ralph, J. (2017). Characterization and elimination of undesirable
protein residues in plant cell wall materials for enhancing lignin
analysis by solution-state nuclear magnetic resonance spectroscopy.
Biomacromolecules 18:4184–4195. https://doi.org/10.1021/acs.
biomac.7b01223.
Kwon, C., Neu, C., Pajonk, S., Yun, H.S., Lipka, U., Humphry, M., Bau,
S., Straus, M., Kwaaitaal, M., Rampelt, H., et al. (2008). Co-option of
a default secretory pathway for plant immune responses. Nature
451:835–840. https://doi.org/10.1038/nature06545.
Lan, W., Lu, F., Regner, M., Zhu, Y., Rencoret, J., Ralph, S.A., Zakai,
U.I., Morreel, K., Boerjan, W., and Ralph, J. (2015). Tricin, a
flavonoid monomer in monocot lignification. Plant Physiol. 167:1284–
1295. https://doi.org/10.1104/pp.114.253757.
Lee, M.H., Jeon, H.S., Kim, S.H., Chung, J.H., Roppolo, D., Lee, H.J.,
Cho, H.J., Tobimatsu, Y., Ralph, J., and Park, O.K. (2019). Lignin-
based barrier restricts pathogens to the infection site and confers
resistance in plants. EMBO J. 38, e101948. https://doi.org/10.15252/
embj.2019101948.
Li, F.S., Phyo, P., Jacobowitz, J., Hong, M., and Weng, J.K. (2019). The
molecular structure of plant sporopollenin. Nat. Plants 5:41–46. https://
doi.org/10.1038/s41477-018-0330-7.
Li, H., Peng, Z., Yang, X., Wang, W., Fu, J., Wang, J., Han, Y., Chai, Y.,
Guo, T., Yang, N., et al. (2013). Genome-wide association study
dissects the genetic architecture of oil biosynthesis in maize kernels.
Nat. Genet. 45:43–50. https://doi.org/10.1038/ng.2484.
Li, X., Bonawitz, N.D., Weng, J.K., and Chapple, C. (2010). The growth
reduction associated with repressed lignin biosynthesis in Arabidopsis
thaliana is independent of flavonoids. Plant Cell 22:1620–1632. https://
doi.org/10.1105/tpc.110.074161.
Lu, X., Liu, J., Ren, W., Yang, Q., Chai, Z., Chen, R., Wang, L., Zhao, J.,
Lang, Z., Wang, H., et al. (2018). Gene-indexed mutations in maize.
Mol. Plant 11:496–504. https://doi.org/10.1016/j.molp.2017.11.013.
Meyer, K., Shirley, A.M., Cusumano, J.C., Bell-Lelong, D.A., and
Chapple, C. (1998). Lignin monomer composition is determined by
the expression of a cytochrome P450-dependent monooxygenase in
Arabidopsis. Proc. Natl. Acad. Sci. USA 95:6619–6623. https://doi.
org/10.1073/pnas.95.12.6619.
Miao, Y.C., and Liu, C.J. (2010). ATP-binding cassette-like transporters
are involved in the transport of lignin precursors across plasma and
vacuolar membranes. Proc. Natl. Acad. Sci. USA 107:22728–22733.
https://doi.org/10.1073/pnas.1007747108.
Nakashima, J., Chen, F., Jackson, L., Shadle, G., and Dixon, R.A.
(2008). Multi-site genetic modification of monolignol biosynthesis in
alfalfa (Medicago sativa): effects on lignin composition in specific cell
types. New Phytol. 179:738–750. https://doi.org/10.1111/j.1469-
8137.2008.02502.x.
Ostergaard, L., Teilum, K., Mirza, O., Mattsson, O., Petersen, M.,
Welinder, K.G., Mundy, J., Gajhede, M., and Henriksen, A. (2000).
Arabidopsis ATP A2 peroxidase. Expression and high-resolution
structure of a plant peroxidase with implications for lignification. Plant
Mol. Biol. 44:231–243. https://doi.org/10.1023/a:1006442618860.
Perkins, M., Smith, R.A., and Samuels, L. (2019). The transport of
monomers during lignification in plants: anything goes but how?
Curr. Opin. Biotechnol. 56:69–74. https://doi.org/10.1016/j.copbio.
2018.09.011.
Perkins, M.L., Schuetz, M., Unda, F., Chen, K.T., Bally, M.B., Kulkarni,
J.A., Yan, Y., Pico, J., Castellarin, S.D., Mansfield, S.D., and
Samuels, A.L. (2022). Monolignol export by diffusion down a
polymerization-induced concentration gradient. Plant Cell 34:2080–
2095. https://doi.org/10.1093/plcell/koac051.
Qian, Y., Qiu, X., and Zhu, S. (2015). Lignin: a nature-inspired sun blocker
for broad-spectrum sunscreens. Green Chem. 17:320–324. https://doi.
org/10.1039/c4gc01333f.
Quentin, M., Allasia, V., Pegard, A., Allais, F., Ducrot, P.H., Favery, B.,
Levis, C., Martinet, S., Masur, C., Ponchet, M., et al. (2009).
Imbalanced lignin biosynthesis promotes the sexual reproduction of
homothallic oomycete pathogens. PLoS Pathog. 5, e1000264.
https://doi.org/10.1371/journal.ppat.1000264.
Quilichini, T.D., Samuels, A.L., and Douglas, C.J. (2014). ABCG26-
mediated polyketide trafficking and hydroxycinnamoyl spermidines
contribute to pollen wall exine formation in Arabidopsis. Plant Cell
26:4483–4498. https://doi.org/10.1105/tpc.114.130484.
Ralph, J., Lapierre, C., and Boerjan, W. (2019). Lignin structure and its
engineering. Curr. Opin. Biotechnol. 56:240–249. https://doi.org/10.
1016/j.copbio.2019.02.019.
Renault, H., Werck-Reichhart, D., and Weng, J.K. (2019). Harnessing
lignin evolution for biotechnological applications. Curr. Opin.
Biotechnol. 56:105–111. https://doi.org/10.1016/j.copbio.2018.10.011.
Rinaldi, R., Jastrzebski, R., Clough, M.T., Ralph, J., Kennema, M.,
Bruijnincx, P.C.A., and Weckhuysen, B.M. (2016). Paving the way
Plant Communications 4, 100682, November 13 2023 ª2023 The Author(s). 15
VAMP726 confers pollen resistance to heat and UV Plant Communications
for lignin valorisation: recent advances in bioengineering, biorefining
and catalysis. Angew. Chem., Int. Ed. Engl. 55:8164–8215. https://
doi.org/10.1002/anie.201510351.
Sanderfoot, A.A., Assaad, F.F., and Raikhel, N.V. (2000). The
Arabidopsis genome. An abundance of soluble N-ethylmaleimide-
sensitive factor adaptor protein receptors. Plant Physiol. 124:1558–
1569. https://doi.org/10.1104/pp.124.4.1558.
Scholl, R.L., May, S.T., and Ware, D.H. (2000). Seed and molecular
resources for Arabidopsis. Plant Physiol. 124:1477–1480. https://doi.
org/10.1104/pp.124.4.1477.
Shi, J., Cui, M., Yang, L., Kim, Y.J., and Zhang, D. (2015). Genetic and
biochemical mechanisms of pollen wall development. Trends Plant
Sci. 20:741–753. https://doi.org/10.1016/j.tplants.2015.07.010.
Shigeto, J., Nagano, M., Fujita, K., and Tsutsumi, Y. (2014). Catalytic
profile of Arabidopsis peroxidases, AtPrx-2, 25 and 71, contributing
to stem lignification. PLoS One 9, e105332. https://doi.org/10.1371/
journal.pone.0105332.
Shigeto, J., Kiyonaga, Y., Fujita, K., Kondo, R., and Tsutsumi, Y.
(2013). Putative cationic cell-wall-bound peroxidase homologues in
Arabidopsis,AtPrx2,AtPrx25, and AtPrx71, are involved in
lignification. J. Agric. Food Chem. 61:3781–3788. https://doi.org/10.
1021/jf400426g.
Shigeto, J., Itoh, Y., Hirao, S., Ohira, K., Fujita, K., and Tsutsumi, Y.
(2015). Simultaneously disrupting AtPrx2,AtPrx25 and AtPrx71 alters
lignin content and structure in Arabidopsis stem. J. Integr. Plant Biol.
57:349–356. https://doi.org/10.1111/jipb.12334.
Stapleton, A.E. (1992). Ultraviolet radiation and plants: burning questions.
Plant Cell 4:1353–1358. https://doi.org/10.1105/tpc.4.11.1353.
Sun, Q., Liu, X., Yang, J., Liu, W., Du, Q., Wang, H., Fu, C., and Li, W.X.
(2018). MicroRNA528 affects lodging resistance of maize by regulating
lignin biosynthesis under nitrogen-luxury conditions. Mol. Plant
11:806–814. https://doi.org/10.1016/j.molp.2018.03.013.
Terrett, O.M., and Dupree, P. (2019). Covalent interactions between
lignin and hemicelluloses in plant secondary cell walls. Curr. Opin.
Biotechnol. 56:97–104. https://doi.org/10.1016/j.copbio.2018.10.010.
Tobimatsu, Y., and Schuetz, M. (2019). Lignin polymerization: how do
plants manage the chemistry so well? Curr. Opin. Biotechnol.
56:75–81. https://doi.org/10.1016/j.copbio.2018.10.001.
Tronchet, M., Balague
´, C., Kroj, T., Jouanin, L., and Roby, D. (2010).
Cinnamyl alcohol dehydrogenases-C and D, key enzymes in lignin
biosynthesis, play an essential role in disease resistance in
Arabidopsis. Mol. Plant Pathol. 11:83–92. https://doi.org/10.1111/j.
1364-3703.2009.00578.x.
Uemura, T., Ueda, T., Ohniwa, R.L., Nakano, A., Takeyasu, K., and
Sato, M.H. (2004). Systematic analysis of SNARE molecules in
Arabidopsis: dissection of the post-Golgi network in plant cells. Cell
Struct. Funct. 29:49–65. https://doi.org/10.1247/csf.29.49.
Uemura, T., Nakano, R.T., Takagi, J., Wang, Y., Kramer, K.,
Finkemeier, I., Nakagami, H., Tsuda, K., Ueda, T., Schulze-Lefert,
P., and Nakano, A. (2019). A Golgi-released subpopulation of the
trans-Golgi network mediates protein secretion in Arabidopsis. Plant
Physiol. 179:519–532. https://doi.org/10.1104/pp.18.01228.
Wang, K., Guo, Z.L., Zhou, W.T., Zhang, C., Zhang, Z.Y., Lou, Y.,
Xiong, S.X., Yao, X.Z., Fan, J.J., Zhu, J., and Yang, Z.N. (2018).
The regulation of sporopollenin biosynthesis genes for rapid pollen
wall formation. Plant Physiol. 178:283–294. https://doi.org/10.1104/
pp.18.00219.
Wellman, C.H., Osterloff, P.L., and Mohiuddin, U. (2003). Fragments of
the earliest land plants. Nature 425:282–285. https://doi.org/10.1038/
nature01884.
Weng, J.K., and Chapple, C. (2010). The origin and evolution of lignin
biosynthesis. New Phytol. 187:273–285. https://doi.org/10.1111/j.
1469-8137.2010.03327.x.
Wheeler, T., and von Braun, J. (2013). Climate change impacts on global
food security. Science 341:508–513. https://doi.org/10.1126/science.
1239402.
Xue, J.S., Zhang, B., Zhan, H., Lv, Y.L., Jia, X.L., Wang, T., Yang, N.Y.,
Lou, Y.X., Zhang, Z.B., Hu, W.J., et al. (2020). Phenylpropanoid
derivatives are essential components of sporopollenin in vascular
plants. Mol. Plant 13:1644–1653. https://doi.org/10.1016/j.molp.
2020.08.005.
Yang, X., Gao, S., Xu, S., Zhang, Z., Prasanna, B.M., Li, L., Li, J., and
Yan, J. (2010). Characterization of a global germplasm collection and
its potential utilization for analysis of complex quantitative traits in
maize. Mol. Breed. 28:511–526. https://doi.org/10.1007/s11032-010-
9500-7.
Yu, K., Yang, W., Zhao, B., Wang, L., Zhang, P., Ouyang, Y., Chang, Y.,
Chen, G., Zhang, J., Wang, S., et al. (2022). The Kelch-F-box protein
SMALL AND GLOSSY LEAVES 1 (SAGL1) negatively influences
salicylic acid biosynthesis in Arabidopsis thaliana by promoting the
turn-over of transcription factor SYSTEMIC ACQUIRED
RESISTANCE DEFICIENT 1 (SARD1). New Phytol. 235:885–897.
https://doi.org/10.1111/nph.18197.
Yue, F., Lu, F., Sun, R.C., and Ralph, J. (2012). Syntheses of lignin-
derived thioacidolysis monomers and their uses as quantitation
standards. J. Agric. Food Chem. 60:922–928. https://doi.org/10.
1021/jf204481x.
Yun, H.S., Sul, W.J., Chung, H.S., Lee, J.H., and Kwon, C. (2022).
Secretory membrane traffic in plant-microbe interactions. New
Phytol. 237:53–59. https://doi.org/10.1111/nph.18470.
Zhang, B., Karnik, R., Wang, Y., Wallmeroth, N., Blatt, M.R., and
Grefen, C. (2015). The Arabidopsis R-SNARE VAMP721 interacts
with KAT1 and KC1 K
+
channels to moderate K+ current at the
plasma membrane. Plant Cell 27:1697–1717. https://doi.org/10.1105/
tpc.15.00305.
Zhang, D., Liang, W., Yin, C., Zong, J., Gu, F., and Zhang, D. (2010).
OsC6, encoding a lipid transfer protein, is required for postmeiotic
anther development in rice. Plant Physiol. 154:149–162. https://doi.
org/10.1104/pp.110.158865.
Zhang, J., Liu, Y., Li, C., Yin, B., Liu, X., Guo, X., Zhang, C., Liu, D.,
Hwang, I., Li, H., and Lu, H. (2022). PtomtAPX is an autonomous
lignification peroxidase during the earliest stage of secondary wall
formation in Populus tomentosa Carr. Nat. Plants 8:828–839. https://
doi.org/10.1038/s41477-022-01181-3.
16 Plant Communications 4, 100682, November 13 2023 ª2023 The Author(s).
Plant Communications VAMP726 confers pollen resistance to heat and UV
