Journal of Experimental Botany, Vol. 74, No. 3 pp.

1090–1106, 2023
https://doi.org/10.1093/jxb/erac457  Advance Access Publication 19 November 2022

RESEARCH PAPER

Time-course transcriptome analysis reveals regulation of

Arabidopsis seed dormancy by the transcription factors
WOX11/12

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Jiakai Liao1,2,†, Ban Deng3,†, Xinyu Cai1, Qixin Yang1,4, Bangping Hu1,4, Jiajing Cong2, Yuxiang Zhang2,
Gang Wang1, Guiliang Xin1, Yuting Li1, Li Yang1, Daizhen Zhang1, Jin Zhang5 and Bobin Liu1,2,4,*,
1
Jiangsu Key Laboratory for Bioresources of Saline Soils, Jiangsu Synthetic Innovation Center for Coastal Bio-agriculture, School of
Wetlands, Yancheng Teachers University, Yancheng 224007, China
2
Basic Forestry and Proteomics Research Center, College of Life Science, Fujian Agriculture and Forestry University, Fuzhou 350002,
Fujian, China
3
Center for Genomics and Biotechnology, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian, China
4
Fujian Colleges and Universities Engineering Research Institute of Conservation and Utilization of Natural Bioresources, College of
Forestry, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian, China
5
State Key Laboratory of Subtropical Silviculture, School of Forestry and Biotechnology, Zhejiang A&F University, Hangzhou, Zhejiang
311300, China

†
These authors contributed equally to this work.
* Correspondence: liubb@yctu.edu.cn

Received 5 August 2022; Editorial decision 8 November 2022; Accepted 18 November 2022

Editor: Steve Penfield, John Innes Centre, UK

Abstract
The induction of seed dormancy and its release involve a finely regulated genetic program controlled by various en-
vironmental and developmental cues that are critical for plant survival and population expansion. Light plays a key
role in seed dormancy and germination, but the molecular mechanisms underlying the control of dormancy are un-
clear. In the present study, high-resolution temporal RNA-seq in Arabidopsis identified WOX11 as encoding a hub
transcription factor during the seed dormancy induction and release stages. This gene might have evolved from
gymnosperms and expanded in angiosperms with highly conserved expression patterns in seeds. WOX11 and its ho-
molog WOX12 were highly expressed from 2 d after pollination, and mRNA abundance was greatly increased during
the seed dormancy induction and release stages. Further, we found that WOX11 plays a role in the regulation of seed
dormancy downstream of phytochrome B (PHYB)-mediated red-light signaling during the induction stage, indicating
that WOX11/12 are newly identified components of red-light signaling transduction. Taken together, our results sug-
gest that WOX11/12-mediated PHYB signaling regulates seed dormancy in Arabidopsis, and provide insights into the
developmental regulation and evolutionary adaptation of plants to changes in the light environment.

Keywords:   Arabidopsis, light signaling, PHYB, seed dormancy, red light, transcriptome, WOX11.

Abbreviations:  COP1–SPA, PHOTOMORPHOGENIC1–SUPPRESSOR OF PHYA-105; DAP, days after pollination; PHY, phytochrome; PIF, PHYTOCHROME
INTERACTING FACTOR; WOX, WUSCHEL-RELATED HOMEOBOX.
© The Author(s) 2022. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email:
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 Regulation of Arabidopsis seed dormancy by WOX11/12  |  1091
Introduction
Because of their sessile nature, plants must sense and adapt to
the surrounding environment. Among the various cues, the
light environment is one of the most important because it di-
rectly influences the efficiency of energy acquisition by pho-
tosynthesis.To sense the light environment, plants have evolved
several photoreceptors such as phytochromes (PHYs), cryp-
tochromes, phototropins, UV RESISTANCE LOCUS 8, and
LOV-F-box/Kelch domain proteins. These photoreceptors
perceive the dynamic light environment and regulate major
developmental processes throughout the plant life cycle (Sul-
livan and Deng, 2003; Kami et al., 2010; Jenkins, 2017). Of
these photoreceptors, PHYs and cryptochromes, which absorb
red/far-red light and blue light, respectively, have been found
to regulate major plant growth and development processes in
Arabidopsis, including de-etiolation, chloroplast development,
cotyledon expansion, shade avoidance, stomatal development,
stomatal opening, and photoperiodic flowering (Kami et al.,
2010). Photoreceptor-mediated light signaling plays a role in
a number of important agronomic traits (Wang et al., 2014;
Gururani et al., 2015; Yang et al., 2017). Hence, elucidation of
the light-signal transduction mechanism is the key to under-
standing how plants adapt to their light environment and has
important practical implications.
Phytochromes originated from streptophytes and are
widely distributed and highly conserved in land plants (Li
et al., 2015).They play a significant role throughout the entire
plant life cycle from seed germination to flowering (Franklin
and Quail, 2010), and they mediate physiological responses
via controlled downstream gene transcription. According to
current research, PHYs have two general signaling pathways.
The first includes the interaction between PHYs and the
CONSTITUTIVE PHOTOMORPHOGENIC1–SUP-
PRESSOR OF PHYA-105 (COP1–SPA) complex, which is
an E3 ligase complex that degrades certain sets of transcrip-
tion factors (Lu et al., 2015; Sheerin et al., 2015). The second
involves the binding of PHYTOCHROME INTERACT-
ING FACTORs (PIFs), a subset of bHLH transcription fac-
tors, to influence downstream target gene expression (Leivar
and Quail, 2011). The active form of PHYs (Pfr) binds with
the COP1–SPA complex to inactivate its E3 ligase activity, and
transcription factors such as ELONGATED HYPOCOTYL
5 and LONG HYPOCOTYL IN FAR-RED1, which are
positive regulators of photomorphogenesis, accumulate in the
nucleus to promote light responses (Lu et al., 2015; Sheerin
et al., 2015). In addition, Pfr binds with PIFs, which sup-
press the photomorphogenic response in the dark, to inacti-
vate their activity via rapid degradation and/or sequestration
away from their target DNA sequences (Leivar and Quail,
2011; Park et al., 2012). Hence, PHYs manage complicated
systems that regulate numerous elements of light-responsive
photomorphogenesis via the COP1–SPA pathway or the PIF
pathway. However, these core regulatory mechanisms do not
fully explain the light-regulated gene expression network.
Other internal factors such as the circadian clock, phytohor-
mones, and other signals modify the photoreceptor-regulated
transcriptional network to fine-tune plant fitness to the light
environment (Leivar and Quail, 2011; Fraser et al., 2016).
Seed dormancy, in which seeds do not germinate under suit-
able conditions, prevents preharvest sprouting and helps plants
overcome unfavorable natural habitats (Willis et al., 2014; Shu
et al., 2016; Nonogaki, 2019). Physiological dormancy is the
most common type and is induced during maturation drying
following the completion of embryogenesis (Angelovici et al.,
2010; Penfield, 2017). Over time, the dormancy level of seeds
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will gradually decrease in the desiccation stage until they are
ready for germination (Angelovici et al., 2010). Generally, dor-
mancy induction, maintenance, and release, and seed germina-
tion are the four main seed statuses (Yang et al., 2020b). PHYs
have been reported to promote seed dormancy release and
seed germination by absorbing red light while repressing seed
germination by absorbing far-red light (Borthwick et al., 1952;
Seo et al., 2006). PHYB plays fundamental roles in dormancy
release and germination by modifying the ratio of abscisic acid
(ABA) and gibberellin (GA), which determines seed fate: either
to maintain a dormant state or to germinate (Shu et al., 2016;
Penfield, 2017). Under low Red/Far-red conditions, PIF1
accumulates in the nucleus to directly or indirectly repress GA
biosynthesis and activate ABA biosynthesis, or to block GA sig-
naling and stimulate ABA signaling (Yang et al., 2020b). How-
ever, under a high Red/Far-red environment, PHYB moves
into the nucleus and interacts with PIF1, triggering PIF1 deg-
radation to reverse the ABA and GA balance, and resulting in
seed germination (Yang et al., 2020b). During seed dormancy
release and germination, red light can activate PHYB in Ara-
bidopsis to restrict REVEILLE1 mRNA transcription, which
inhibits GA biosynthesis, resulting in an increase in bioactive
GA content (Jiang et al., 2016). In dormant seeds, PHYB acts as
a negative regulator by suppressing the transcription of REV-
EILLE1 (Jiang et al., 2016). Based on analysis of Arabidopsis,
SPATULA is another transcription factor that is downstream of
PHYB and is involved in seed dormancy maintenance (Vaistij
et al., 2013). ABA is necessary for seed dormancy induction at
the embryo maturation stage, whilst GA inhibits ABA activity
(Sawers, 2016). DELAY OF GERMINATION 1 (DOG1) is a
major dormancy regulator, similar to ABA, that affects the de-
gree of dormancy during seed maturation and in response to
the environment once seeds have been dispersed (Footitt et al.,
2020). However, the mechanism of seed dormancy induction,
which is a key process that determines the level of dormancy
based on light signaling, remains unclear.
In the present study, we employed high-resolution tem-
poral RNA-seq during seed development and germination in
Arabidopsis to explore how developmental signaling modifies
the light-mediated transcriptional network. Using bioinfor-
matic methods, we identified WUSCHEL-RELATED HO-
MEOBOX 11 (WOX11), which is a key transcription factor
1092 | Liao et al.
participating in root and shoot meristem regeneration (Zhao
et al., 2009; J. Liu et al., 2014; 2018; B. Liu et al., 2018), as a hub
transcription factor that potentially regulates seed dormancy
induction and release in Arabidopsis. In addition, we show that
WOX11 and its homolog WOX12 act downstream of PHYB
to regulate the seed dormancy, and that this might be an evo-
lutionarily conserved role acquired in angiosperms. Our results
shed light on the regulation of dormancy as plants adapt to
changes in the light environment and on the mechanism un-
derlying the expansion of angiosperms to land.
Materials and methods
Plant materials and growth conditions
All the mutant and transgenic plants were produced in the Arabidop-
sis thaliana Col-0 background. The mutants wox11-2 (SALK_004777),
wox12-1 (SALK_087882), the double-mutant wox11 wox12, and phyB-
9 (Reed et al., 1993) and the transgenic WOX11-overexpressing line
WOX11-OE and PWOX11::GUS line have been described previously (J.
Liu et al., 2014). The phyB-9 mutant was crossed with the WOX11-OE
line, and the resulting T2 progeny were screened under red light. The
T3 progeny were then checked by RT-qPCR to obtain homozygous
T3 lines.
All the plants were grown in a vermiculite and peat soil mixture (1:3)
with a regular water supply in a growth room at 22 °C, 60–70% humidity,
and long-day conditions (16/8  h light/dark, 80 µmol m–2 s–1). Mature
seeds were all freshly collected at the same time and immediately used
for experiments. Seeds were first surface-sterilized with 75% ethanol, and
sown on moist filter paper that was then placed on half-strength MS me-
dium plates containing ½× Murashige and Skoog salt mix, 1% sucrose,
2.5 mM MES (pH 5.8), and 0.8% agar. Seeds were incubated in a growth
chamber (HiPoint 740F-LED) at 22 °C, 60–70% humidity, and contin-
uous white light (60 µmol m–2 s–1).
Light sources
Monochromatic red light (peak at 660 nm; half-bandwidth of 20 nm) and
far-red light (peak at 730 nm; half-bandwidth of 20 nm) were produced
from an LED light. A LI-250A light meter equipped with an LI-190R
Quantum sensor (both LI-COR Biosciences) was used to measure light
intensity.
Dormancy and germination assays
To measure dormancy, all the seeds were freshly harvested 18 d after pol-
lination (DAP) (Bentsink et al., 2006; Nakabayashi et al., 2012), surface-
sterilized with 75% ethanol and then sown on to moist filter paper as
described above. Seeds were either stratified first at 4 °C in the dark for
3 d or those without stratification were directly incubated in a growth
chamber under white light as described above. The dormancy level of
each seed batch was indirectly measured by germination tests, with ger-
minated seeds being defined as having protruded radicals (Hilhorst, 2011;
Graeber et al., 2012). Seed germination was checked every 24 h. Three
replicates of 50 seeds for each genotype were obtained from four plants,
and used for one biological experiment. All experiments were carried out
three times with similar results, and all data are shown as the mean (±SE)
of these three independent experiments.
In addition, to examine whether WOX11 and WOX12 are genetically
downstream of PHYB, freshly harvested seeds without cold stratification
were incubated under white light for 1 h, and then exposed for 5 min to
either far-red light (PHYB-off) or red light after the phyB-off (PHYB-
on). The seeds were then kept in the dark for 4 d and their germination
rates were recorded.
GUS histochemical analysis
The PWOX11::GUS homozygous transgenic seeds were harvested at dif-
ferent stages of development and at different imbibition times. The GUS
staining procedure was described previously (B. Liu et al., 2014), and a
stereomicroscope and digital camera (Leica M205FA) were used to image
the results.
RNA extraction and RT–qPCR assays
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Total RNA from Arabidopsis siliques or imbibed seeds at different devel-
opmental stages was isolated using a RNAprep Pure Plant Kit (Tiangen).
A NanoDrop 2000 Spectrophotometer (ThermoFisher) was used to an-
alyse the RNA quantity and purity. First-stand cDNA was synthesized
using 1 µg of total RNA using a PrimeScript™ RT Reagent Kit with
gDNA Eraser (Perfect Real Time, TaKaRa), following the manufacturer’s
instructions. Quantitative PCR was performed using QuantStudio™ 6
Flex (ThermoFisher Scientific) with GoTaq® qPCR Master Mix (Pro-
mega) following the manufacturers’ instructions.Three technical replicates
were performed for each reaction, and the expression levels were normal-
ized to those of the reference genes PP2A and ACTIN2 (Czechowski
et al., 2005) by using the 2–∆∆CT method (Livak and Schmittgen, 2001).
Each experiment was performed with at least three biological replicates.
The primers are listed in Supplementary Table S1.
RNA-seq and data analysis
Total RNA was extracted as described above from freshly harvested
dry seeds and from seeds imbibed for 3 h, and the quality of the RNA
samples was analysed with a Thermo Scientific™ NanoDrop™ One
Spectrophotometer and an Agilent Bioanalyzer 2100 (RNA 6000
Nano Kit). Barcoded cDNA libraries were constructed using Illumina
Poly-A Purification TruSeq library reagents and protocols. Three bio-
logical replicates were sequenced on an Illumina NovaSeq6000 (paired-
end 150-bp run). The clean reads for each sample were filtered from
the raw reads, and were mapped to the Arabidopsis reference genome
(TAIR10) using HISAT2 (Kim et al., 2019). The RNA-seq results are
summarized in Supplementary Table S2. The P-value and false-dis-
covery rate (FDR) were calculated using the edgeR package in Bio-
conductor (Robinson et al., 2010). Genes that showed a greater than
2-fold difference (log2|FoldChange|>1) in relative mRNA abundance
with P<0.01 and FDR<0.05 were considered differentially expressed.
Metascape (http://metascape.org/) was used for Gene Ontology (GO)
analysis (Zhou et al., 2019), and Cytoscape (Shannon et al., 2003) was
used to display the results.
Seed development, maturation, and germination RNA-seq data gen-
erated previously by Klepikova et al. (2016) were downloaded from the
NCBI SRA database (https://www.ncbi.nlm.nih.gov/bioproject, ID
PRJNA314076) and used for further analysis. The gene expression levels
were normalized as transcripts per kilobase of exon model per million
mapped reads (TPM) using the Salmon algorithm (Patro et al., 2017).
A dendrogram was generated by hierarchical cluster analysis (Datta and
Datta, 2003).The co-expression modules were grouped via k-means clus-
tering and hub transcription factors were identified using the weighted
gene correlation network analysis (WGCNA) package (Langfelder and
Horvath, 2008) by calculating the correlation coefficient and expression
similarity of gene pairs. Analyses of GO terms were performed using GO
Term Enrichment for Plants (https://www.arabidopsis.org/tools/go_
term_enrichment.jsp), and the results were displayed using Cytoscape.
 Regulation of Arabidopsis seed dormancy by WOX11/12  |  1093
Phylogenetic analysis
The full-length of amino acid residue sequences of WOX11 and WOX8/9
used in this study were aligned using ClustalX2.1 and then submitted
to IQ-Tree (http://iqtree.cibiv.univie.ac.at/) for phylogenetic tree con-
struction using the maximum-likelihood algorithm. The tree was visu-
alized using EvolView (https://www.evolgenius.info/evolview/#login).
The expression patterns of WOX11s and WOX8/9s were obtained from
Phytozome (https://phytozome-next.jgi.doe.gov/). Detailed informa-
tion for the sequences used in phylogenetic analysis is listed in Supple-
mentary Table S3
Statistical analysis
Significant differences between pairs of means were determined using
Student’s t-test using GraphPad Prism v. 7.0a for Mac (www.graphpad.
com).
Accession numbers
WOX11 (AT3G03660); WOX12 (AT5G17810); PHYB (AT2G18790);
PP2A (AT1G13320); ACTIN2 (AT3G18780).
Results
High-resolution temporal profiling of transcripts
associated with seed dormancy and germination
Most previous molecular studies of seed dormancy and ger-
mination have focused on mutant screening and mapping of
quantitative trait loci, whilst the transcriptional regulatory
mechanism underlying seed dormancy and its release remains
largely unexplored. To obtain a detailed picture of the regu-
lation of seed dormancy and germination, we downloaded
publicly available, high-resolution time-course RNA-seq data
for seed development, maturation, and germination produced
using an Illumina sequencing platform (Klepikova et al., 2016).
The datasets contained 30 samples from fertilization to germi-
nation at 15 time points from ovules before pollination through
to germinating seeds after 3 d of soaking, all with two biolog-
ical replicates. A total of 950 million high-quality reads were
used for bioinformatics analysis, and we used the average TPM
value of the two replicates to calculate the expression level at
each time point. To limit the impact of noise, we characterized
a gene as expressed if its TPM value was greater than 1.
We then carried out an unsupervised hierarchical cluster-
ing (HC) analysis (Fig. 1A) and principal component analysis
(PCA, Fig. 1B) for the 15 time-series samples. The HC of the
transcriptomic data separated the sample set into two groups.
One of these could be divided into two subgroups to sepa-
rate germination processes and different development stages,
which in turn was further divided into two trees (Fig. 1A).
Thus, the 15 time points were grouped into four phases: Ph1
(ovules and seeds from young silique) represented the morpho-
genesis phase, when the seed was undergoing rapid cell divi-
sion and differentiation; Ph2 (seeds from siliques) represented
the seed maturation stage, when dormancy is induced; Ph3
(yellow and dry seed) represented the desiccation phase, when
the seed dormancy was being released; and Ph4 (germinating
seeds) represented the germination stage. A similar grouping
was exhibited by the PCA (Fig. 1B). These results indicated
that the seed development and germination processes could be
roughly grouped into four phases based on the mRNA tran-
scription levels.
To provide further insights into the dynamic transcriptional
changes and functional shifts occurring during seed develop-
ment and germination, we subjected the genes to hierarchical
clustering analysis with default parameters and found that they
were grouped into eight clusters, denoted as C1 to C8 (Fig. 1C,
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D). The expression patterns of genes in C3 (5686), C4 (3667),
and C8 (4666) increased during the seed dormancy-release
and germination stages, whereas in contrast the transcript lev-
els of genes in C5 (4314) and C6 (5707) decreased during
these stages. We next performed GO term enrichment analysis
for each cluster and found that genes in C3, C4, and C8 were
mainly involved in abiotic stimulus responses (such as osmotic
stress, salt stress, chitin, and light signaling) and responses to
abscisic acid and karrikin, as well as RNA processing, response
to oxygen-containing compounds, seed development, and seed
dormancy processes (Fig. 1E). These results suggested that C3,
C4, and C8 participated in environmental responses and seed
development processes.
Identification of hub transcription factors using gene
co-expression network analysis
Because the induction and release of seed dormancy is accom-
panied by genome-wide changes in gene expression, dynamic
activation of transcription factors (TFs) is assumed to be a cru-
cial regulatory mechanism that governs seed dormancy and
affects thousands of genes. To obtain a comprehensive under-
standing of the enhancement of gene expression during the
seed maturation and germination stages that could be highly
associated with dormancy and release, we first constructed a
co-expression network of the C3, C4, and C8 groups using
WGCNA, and 18 distinct color-coded modules were identi-
fied (Fig. 2A).The largest module (blue) contained 7249 genes,
the smallest (dark orange) contained only 33 genes, and nine
ungrouped genes were assigned to the gray module (see also
Supplementary Table S4). To further investigate the biolog-
ical significance of each module, GO enrichment analysis was
performed. Significant GO enrichment was obtained only for
10 modules whose genes were related to processing of non-
coding (nc)RNA, translational termination, single-organism
process, response to heat, biosynthetic process, plastid organi-
zation, cellular response to stimulus, single-organism process,
mRNA processing, and cellular respiration (Fig. 2B; Supple-
mentary Table S4). It was interesting that the green module
was enriched not only in the response to heat but also in the
responses to light, salt, osmotic stress, and temperature, to the JA,
GA, and ABA responses, and to the dormancy process. Given
1094 | Liao et al.

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Fig. 1.  Transcriptomic landscape of seed formation, desiccation, and germination in Arabidopsis. (A) Hierarchical clustering analysis of high-resolution
time-course RNA-seq for seed development, maturation, and germination data from Klepikova et al. (2016), showing that the developmental process
is separated into four phases. Ovule, ovules before pollination; SD.y1–5, seeds from young siliques 1–5 when the flowers had first fallen from each;
SD1/3/5/7, seeds from siliques 1/3/5/7 when they were green and the length of each was up to 1.5 cm; SD.sn1 seeds from silique 1 when it was
senescent; SD.d, dry seeds from silique 1; SD.g1–3, germinating seeds at days 1–3 after soaking. (B) Principal component analysis of the transcription
data described in (A). Each dot represents the mean value of a time point. (C) Heatmap of gene expression at the different time points. Hierarchical
k-means clustering divides the genes into eight clusters. (D) Graphical representation of the expression data in (C) showing the expression patterns of the
genes in each cluster with time. (E) The major GO terms associated with each cluster.

that seed dormancy is regulated by various environmental and
hormone signaling pathways (Wang et al., 2018; L. Yang et al.,
2020), we defined the green module as the ‘dormancy regula-
tion module’. Furthermore, 54 out of 55 TFs were identified
as hub TFs (Fig. 2C). Intriguingly, PIF6 (Penfield et al., 2010),
TZF5 (Bogamuwa and Jang, 2013), AGL67 (Narro-Diego
et al., 2017), FLC (Chen and Penfield, 2018), and RVE7 (Liu
et al., 2021), which are characterized as dormancy regulators,
were categorized as hub TFs in the green module. Most of the
hub TFs were environmental response factors, such as HSF and
ERF family members, that might be involved in seed dormancy
regulation. Transcription factors related to meristem develop-
ment were also present, such as PLT3, WOX2, and WOX11. It
was interesting that WOX11, which is specifically a wounding-
response TF (J. Liu et al., 2014), was highly expressed during
the seed maturation and germination stages (Supplementary
Fig. S1), suggesting that this gene might have functions in seed
dormancy and germination.
To study the potential functions that the WOX11/12 genes
have acquired during evolution, we constructed an evolu-
tionary tree and mapped the dynamic changes in their ex-
pression patterns (Fig. 2D).The tree contained all intermediate
clade proteins from one lycophyte, one monilophyte, four
gymnosperms, and six angiosperms, including four dicots and
three monocots. The tree showed that the WOX11/12 sub-
clade is specific to angiosperms and might have originated from
 Regulation of Arabidopsis seed dormancy by WOX11/12  |  1095

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Fig. 2.  Identification of hub transcription factors of seed dormancy in Arabidopsis as determined using co-expression network analysis. (A)
Co-expression modules were identified using weighted gene correlation network analysis (WGCNA) and are shown in a hierarchical clustering tree. Each
gene is represented by a leaf on the tree. The principal tree branches are divided into 18 modules, each of which is identified with a different color. (B)
GO term enrichment analysis of the genes from the 18 modules as visualized using Cytoscape. The functions of the modules are summarized based
on the GO terms. The full results of the GO analysis are given in Supplementary Table S4. (C) Identification of hub transcription factors (TFs). Correlation
coefficients were calculated using the WGCNA tool for R software with default values. Hub TFs with a correlation coefficient greater than 0.4 were chosen
for visualization using Cytoscape. (D) Phylogenetic tree and expression patterns of the intermediate subclades of WOX family genes from a lycophyte to
angiosperm plants. Gray boxes indicate that there are no expression data available. Note that WOX11 and WOX12 are angiosperm-specific with high
expression in seeds. Lycophyte: Sk, Selaginella kraussiana. Monilophyte: Cr, Ceratopteris richardii. Gymnosperms: Gmo, Gnetum montanum; Gg, G.
gnemon; Gb, Ginkgo biloba; and Pa, Picea abies. Angiosperm dicots: Amt, Amborella trichopoda; Ah, Amaranthus hypochondriacus; Gm, Glycine max;
and At, Arabidopsis thaliana. Angiosperm monocots: Zm, Zea mays; Os, Oryza sativa; and Sb, Sorghum bicolor.

gymnosperm plants. The WOX11/12 subclade has also under-
gone gene expansion in angiosperm plants. To gain insights
into the potential functions of WOX11/12 members, we first
checked the expression levels of WOX11 and WOX12 in roots,
stems, leaves, flowers, and seeds by using publicly available data.
The resulting heatmap clearly showed that intermediate clade
members were highly expressed in the seeds of angiosperm
plants, suggesting that they might have conserved roles in seed
dormancy or developmental regulation (Fig. 2D). Based on the
results of the co-expression analysis and the similar expression
patterns in seeds, we next investigated the physiological func-
tions of WOX11.
1096 | Liao et al.

WOX11/12 redundantly suppress seed dormancy in
Arabidopsis
To better understand the functions of WOX11 and WOX12
in seed dormancy, we first examined the dormancy levels of
WOX11/12 loss-of-function and WOX11 gain-of-function
lines in freshly harvested seeds using germination tests. We
found that seeds from the wox11-2 and wox12-1 single-mutants
exhibited similar germination compared with that of the wild-
type Col-0 (20%) whilst the wox11 wox12 double-mutant had
germination of only 8% (Fig. 3A). Germination of the trans-
genic WOX11-overexpression (-OE) seeds was 60%. After 3
than that of WOX11-OE (Fig. 3D). Complete dormancy re-
lease by after-ripening was reached after 6 months for all three
genotypes. Taken together, these results indicated that WOX11
and WOX12 redundantly suppress seed dormancy and pro-
mote its release in Arabidopsis.
WOX11/12 expression patterns in Arabidopsis
To clarify the function of WOX11/12 in seed dormancy and
germination, we first checked their expression patterns in the
TraVA database (Transcriptome Variation Analysis, http://tra-

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d of imbibition we found that only 10% of the wox11 wox12
seeds germinated compared with 50% of Col-0 seeds, whereas
more than 90% of WOX11-OE seeds germinated (Fig. 3B).
Maximum germination was reached after 5 d of imbibition
and was 60% for Col-0, 15% for wox11 wox12, and almost
100% for WOX11-OE. We checked the germination rate of
similar seeds that were treated with 3 d of cold stratification,
and all three genotypes showed 100% germination after 2 d of
imbibition (Fig. 3C). This indicated that there were no differ-
ences in the vigor and germination ability among the seeds.
Next, we examined the dormancy release time for the seeds.
During seed storage, the germination of Col-0 seeds increased
faster than that of the wox11 wox12 double-mutant but slower
vadb.org/) and found that expression was high in seeds from
the first yellowing siliques but not in the silique tissue (silique
1), and high in the second yellowing silique still containing
seeds (silique 2) (Fig. 4A).There was little expression in the root
apex.The WOX11/12 transcript levels were also increased at 1
d after the start of imbibition (days after soaking, DAS).We also
checked the Arabidopsis eFP Browser and found that the ex-
pression level of WOX11 increases in globular-stage seeds and
reaches its peak in seeds at the mature-green stage (Supple-
mentary Fig. S1A, B). WOX11 was also highly transcribed in
imbibed seeds, especially in the radicle and micropylar endo-
sperm (Supplementary Fig. S1B, C). To confirm this, we used
RT-qPCR to check whether WOX11/12 were specifically

Fig. 3.  WOX11/12 suppress seed dormancy in Arabidopsis. (A) Germination of freshly harvested seeds of Col-0, wox11, wox12, the double-mutant
wox11 wox12, and the transgenic WOX11-overexpression (OE) line incubated under white light for 2 d without stratification. (B, C) Germination over
time of seeds of Col-0, wox11 wox12, and WOX11-OE kept under white light either without (B) or with (C) stratification at 4 °C for 3 d in the dark. (D)
Germination of seeds following different storage times, after which they were incubated under white light for 5 d without stratification. All data are means
(±SE), n=3. Significant differences compared with Col-0 were determined using Student’s t-test: *P≤0.05, ***P≤0.001, ****P≤0.0001; n.s., not significant.
 Regulation of Arabidopsis seed dormancy by WOX11/12  |  1097

A B

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C D

E F G H

I J K L

Fig. 4.  Expression levels of WOX11/12 are highly induced during seed development and germination in Arabidopsis. (A) Heatmap of the relative
expression ratios (log2) of WOX11 and WOX12 in the root apex and shoot apical meristem (SAM) of 7-day-old plants, in the petiole and leaf blade of
the third leaf, flowers, seeds from the first yellowing silique, silique 1 (first yellowing silique without seeds), silique 2 (second yellowing silique including
seeds), dry seeds, and seeds at 1–3 d after soaking (DAS). Data were downloaded from TraVA (http://travadb.org/). (B) Relative expression of WOX11
1098 | Liao et al.
in seeds and silique tissue (i.e. without seeds) at 18 d after pollination (DAP), as determined by RT-qPCR. The mRNA levels were first normalized to that
of the PP2A reference gene, and expression is relative to that in the seeds, the value of which was set as 1 relative expression unit (REU). (C) Relative
expression of WOX11 and WOX12 in seeds at 2–20 DAP (see Supplementary Fig. S1A). Expression is relative to that at 2 DAP, the values of which were
set as 1. (D) Relative expression of WOX11 and WOX12 in freshly harvested seeds after different times of imbibition. Expression is relative to that at 0 h,
the values of which were set as 1. All data are means (±SE), n=3. In (B), the significant difference was determined using Student’s t-test: ****P≤0.0001.
Results corresponding to (B–D) using ACTIN2 as an alternative reference gene are shown in Supplementary Fig. 2. (E–L) Temporal and spatial expression
of WOX11 as determined using the PWOX11::GUS reporter construct with GUS staining for 4 h. (E) Freshly harvested seeds (14 DAP) and (F) dry seeds (20
DAP). (G–L) Seeds after (G) 1 h, (H) 3 h, (I) 6 h, (J) 24 h, (K) 48 h, and (L) 72 h of imbibition.

expressed in seeds and found that this was indeed the case, with genes (hereafter referred to as w11w12D-up-regulated and
no signal being detected in pods (Fig. 4B; Supplementary Fig. w11w12D-down-regulated genes, respectively) (Fig. 5A). We
S2A). We further analysed transcription levels during the seed then performed GO analysis and functionally clustered the

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development and imbibition processes and found that WOX11
and WOX12 transcript abundance gradually increased from 4
DAP, and at 8–14 DAP during the seed maturation stage when
dormancy is induced (Le et al., 2010; L. Yang et al., 2020) it
was 30 times higher compared to 2 DAP (Fig. 4C; Supple-
mentary Fig. S2B). After 14 DAP, the expression levels of both
WOX11 and WOX12 sharply decreased. During seed imbi-
bition, WOX11 and WOX12 transcript abundance reached a
peak at 3 hours of imbibition and then decreased back to the
original levels (Fig. 4D; Supplementary Fig. S2C).
We also examined WOX11 expression patterns using the
PWOX11::GUS transgenic line with β-glucuronidase (GUS)
under the control of the WOX11 promoter. Consistent
with the results reported above, Arabidopsis plants harboring
PWOX11::GUS showed strong expression of WOX11 in the rad-
icle and plumular axis of the embryo 14 DAP (Fig. 4E) whilst
GUS staining was focused on the radicle pericycle at 20 DAP
(Fig. 4F). During the imbibition stage, the GUS staining grad-
ually increased in the pericycle after 1 h of imbibition (Fig. 4G)
and expanded into all radicles after 3 h (Fig. 4H). The expres-
sion level of WOX11 then gradually decreased, and only weak
staining in the hypocotyl was apparent after 72 h of imbibition
(Fig. 4I–L). The dynamic patterns of expression exhibited by
WOX11 and WOX12 during the seed dormancy induction
and release stages strongly indicated that they play roles in these
processes.
WOX11/12 might be involved in red-light signaling
during the regulation of seed dormancy
To understand their functions in seed dormancy induction
and release in Arabidopsis, we examined WOX11/12-reg-
ulated transcriptomic changes using RNA-seq analysis. We
used freshly harvested dry seeds (18 DAP) and seeds imbibed
for 3 h of the Col-0 wild type and the wox11 wox12 double-
mutant, and obtained at least 6 Gb clean data containing >20
million clean reads for each sample. A total of 287 million
high-quality reads were mapped to the Arabidopsis reference
genome with a mapped ratio of more than 99% (Supplemen-
tary Table S2). Comparing the freshly harvested seeds dry
seeds of the wox11 wox12 double-mutant with those of Col-
0, we identified 312 up-regulated and 453 down-regulated
results using Metascape (Zhou et al., 2019). This revealed that
the w11w12D-down-regulated genes are preferentially asso-
ciated with xyloglucan and flavonoid biosynthesis, responses
to environmental stress such as osmotic, light, and oomycete
stress, and phytohormones including ethylene, salicylic acid,
jasmonic acid, and karrikin (Fig. 5B). The w11w12D-up-
regulated genes were specifically enriched in camalexin and
disaccharide biosynthesis, response to organonitrogen com-
pound, decreased oxygen level, wounding, and indole-con-
taining compound metabolic process (Fig. 5B).
After 3 h of imbibition, we identified 134 up-regulated and
932 down-regulated genes in the wox11 wox12 double-mutant
seeds compared with the Col-0 seeds (hereafter referred to as
w11w12I-up-regulated and w11w12I-down-regulated genes,
respectively) (Fig. 5C). The presence of more down-regulated
genes in imbibed wox11 wox12 seeds than in those of Col-0
indicated that many genes are not activated, perhaps because
WOX11 and WOX12 are transcriptional activators (Lin et al.,
2013). The w11w12I-down-regulated genes were specifically
involved in the red-light response, DNA replication initiation,
root development, and polysaccharide metabolic processes
(Fig. 5D). The w11w12I-up-regulated genes were specifically
enriched in camalexin biosynthesis, indole-containing com-
pound metabolism, toxin metabolism, and pectin degradation.
Interestingly, 63.9% (596 out of 932) of the w11w12I-down-
regulated genes were up-regulated in Col-0 seeds after 3 h of
imbibition comparing with the Col-0 dry seeds. (Supplemen-
tary Fig. 3A). DNA replication, cell wall organization, red-light
response, xylan metabolic, gibberellic, and brassinosteroid sig-
naling pathways, and plasmodesmata-mediated intercellular
transport genes were highly enriched (Supplementary Fig. 3B).
Because of the seed-specific expression patterns of
WOX11/12, we wondered whether they regulated seed dor-
mancy induction and release by mediating light signaling. Red
light is a critical environmental factor in the regulation of seed
dormancy and germination, and genes involved in seed ger-
mination that are up- and down-regulated by it have been
identified previously (Shi et al., 2013). We found that 12.3%
of w11w12D-down-regulated and 25.1% w11w12I-down-
regulated genes overlapped with the genes that have previously
been shown to be up-regulated in wild-type plants under
red light (Fig. 5E). Some genes that did not overlap with the
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Fig. 5.  WOX11/12 regulate transcripts involved in light-signal transduction during seed dormancy and germination in Arabidopsis. (A) Volcano plot
showing differentially expressed (DE) genes between dry seeds of Col-0 and the wox11 wox12 double-mutant at 18 d after pollination (DAP). (B) GO term
enrichment analysis of the up- and down-regulated genes in wox11 wox12 shown in (A). Red indicates GO terms enriched for down-regulated genes
and blue indicates GO terms enriched for up-regulated genes. (C) Volcano plot showing DE genes between Col-0 and wox11 wox12 seeds after 3 h of
imbibition. (D) GO enrichment analysis of up- and down-regulated genes in wox11 wox12 shown in (C). Red and blue indicate GO terms enriched for
1100 | Liao et al.
down- and up-regulated genes, respectively. (E) A Circos plot showing the overlap among the DE genes in dry seeds that were down-regulated in the
double-mutant (w11w12D down-regulated), DE genes in imbibed seeds that were down-regulated in the double-mutant (w11w12I down-regulated), and
DE genes that have previously been shown to be up-regulated in Col-0 in response to red light (Shi et al., 2013). Purple lines link genes that are common
to the different categories, and blue lines link different genes with the same GO term. (F) Enrichment analysis of the genes shown in (E).
­ p-regulated genes under red light fell into the same ontology
u expression peaked, with levels in the phyB mutant were only a
term. GO enrichment showed that the WOX11/12-mediated
transcriptome in seeds after 3 h of imbibition was similar to
that under red light (Fig. 5F). In the dry seeds collected at 18
DAP, four GO terms were the same as those found for red light
stimulation, namely response to light stimulus, secondary met-
abolic process, hemicellulose metabolic process, and response
to karrikin. These results strongly suggested that WOX11/12
mediated red-light signaling in seed dormancy induction and
release.
Interestingly, GO terms related to light signaling were
enriched in the w11w12D-down-regulated and w11w12I-
down-regulated gene sets (Fig. 5). In addition, during the im-
bibition process, WOX11/12 also affected the expression of
genes related to light signaling (Supplementary Fig. S3A, B).
Light represses hypocotyl elongation and promotes hook ex-
pansion, and we found that the hypocotyl length of WOX11-
OE plants was significantly shorter than that of Col-0 under
dark conditions, while there was no difference between wox11
wox12 and Col-0 (Supplementary Fig. S3C, G). In red, blue, and
far-red light environments, the hypocotyl length of WOX11-
OE plants was significantly shorter than that of Col-0, whereas
that of the wox11 wox12 double-mutant was slightly longer
(Supplementary Fig. S3D–G). Overexpression of WOX11 also
promoted hook expansion, which is also regulated by light sig-
naling, while there was no difference between wox11 wox12
and Col-0 (Supplementary Fig. S3H, I). Given that WOX11
was highly expressed in seeds and expressed at low levels in
the hypocotyl after germination, these results suggested that
WOX11/12 are the red-light signaling components that might
specifically function in seed dormancy and germination.
WOX11/12 function downstream of PHYB to regulate
seed dormancy and germination
PHYB is the major phytochrome that senses red light and FR
light signaling during seed dormancy regulation. We found
that under white light, freshly harvested seeds of the phyB mu-
tant showed a strong dormancy phenotype after imbibition
without stratification (Supplementary Fig. S4), consistent with
a previous report (Jiang et al., 2016). To determine whether
the enhanced seed dormancy of the phyB mutant is associated
with the transcription levels of WOX11 and WOX12, we car-
ried out RT-qPCR analysis to quantify their relative mRNA
abundance in comparison with Col-0. During seed develop-
ment, the expression levels of WOX11 and WOX12 were sig-
nificantly lower in the phyB mutant than in Col-0 (Fig. 6A, B;
Supplementary Fig. S2D, E), especially from 12–14 DAP when
third to a quarter of those in Col-0.
To examine whether WOX11 and WOX12 are genetically
downstream of PHYB, freshly harvested seeds without cold
stratification were exposed to different light conditions (Jiang
et al., 2016). When kept in the dark, the germination rate of
Col-0 seeds was 45%, the wox11 wox12 double-mutant was
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18%, and the WOX11-OE transgenic line was 70% (Fig. 6C).
When exposed to red light (the PHYB-on condition), the
germination rate of Col-0 was 65%, wox11 wox12 was 20%,
and WOX11-OE was 90%.When exposed to far-red light (the
PHYB-off condition), Col-0 and wox11 wox12 seeds failed to
germinate, whereas the WOX11-OE seed germination rate
was close to 20%. These results suggested that the function of
WOX11/12 is downstream of the activated form of PHYB.
We next constructed transgenic plants overexpressing WOX11
in the phyB-9 mutant background (phyB-9 WOX11-OE) and
used homozygous seeds to examine the genetic relationship
between PHYB and WOX11. Overexpression of WOX11
complemented the dormancy phenotype of the phyB mutant
(Fig. 6D), suggesting that WOX11 acts genetically downstream
of PHYB.
The balance of ABA and GA levels is primarily respon-
sible for seed dormancy and germination. We found that
GA-regulated genes encoding expansins (EXPs) and xyloglu-
can endo-transglycosylase/hydrolases (XTHs) (Graeber et al.,
2014) were not activated in imbibed seeds of wox11 wox12
except for XTH18 (Fig. 7A). GA 20-oxidase and GA 3-oxidase
(GA3ox) catalyse GA biosynthesis to generate bioactive hor-
mones, which are in turn deactivated by a variety of enzymes,
including GA 2-oxidase (Yamaguchi, 2008). In freshly har-
vested dormant seeds, the expression levels of GA20ox1 and
GA20ox2 were significantly decreased in wox11 wox12 com-
pared with those in Col-0 (Fig. 7B; Supplementary Fig. S2F),
whereas in the WOX11-OE line the expression levels of
GA20ox1, GA20ox3, GA3ox1, and GA3ox2 were increased by
between 2- and 50-fold.These results implied that WOX11/12
modulate the expression of GA biosynthesis genes. In addition,
we found that treating freshly harvested dormant wild-type
seeds with 1 μM GA3 led to 100% germination compared to
only 65% in untreated seeds (Fig. 7C, D), whereas even 20 μM
GA3 treatment did not increase germination of wox11 wox12
seeds. On the other hand, stratification treatment was able to
break dormancy in both the double-mutant and the wild-type
seeds, increasing germination to 100% in both cases. Thus, the
dormant wox11 wox12 seeds were viable but insensitive to GA
treatment. This suggested that WOX11/12 positively controls
dormancy and germination via GA signaling.
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Fig. 6.  Phytochrome B promotes the expression of WOX11/12 to suppress primary seed dormancy in Arabidopsis. RT-qPCR analysis of the expression
levels of (A) WOX11and (B) WOX12 in siliques at different times during seed development (see Supplementary Fig. 1A) in Col-0 and the phyB-9 mutant.
The WOX11/12 mRNA levels were first normalized to the PP2A reference gene, and expression is relative to that at 8 d after pollination, the values
of which were set as 1 relative expression unit (REU). Results corresponding to (A, B) using ACTIN2 as an alternative reference gene are shown in
Supplementary Fig. 2. (C) The effects of different light treatments on germination of seeds of the Col-0 wild type, the wox11 wox12 double-mutant,
and the transgenic WOX11-overexpression (OE) line. Freshly harvested seeds without stratification were incubated under white light for 1 h and then
either kept in the dark, or exposed to far-red light for 5 min (phytochrome B off conditions, phyB-off) or red light for 5 min after the phyB-off (phyB-on
conditions). After that, the seeds were kept in the dark for 4 d and the germination rate was determined. (D) Germination rates of freshly harvested seeds
without stratification of Col-0, the phyB-9 mutant, the transgenic WOX11-OE line, and a homozygous T3 line of a cross between phyB-9 and WOX11-OE
under white light for 3 d. All data are means (±SE), n=3. Significant differences compared with Col-0 were determined using Student’s t-test: *P≤0.05,
**P≤0.01, ***P≤0.001, ****P≤0.0001; n.s., not significant.

Discussion factors interact to affect dormancy and germination. The dor-

Our study reveals the presence of a PHYB–WOX11/12 signal-
ing pathway in the regulation of seed dormancy in Arabidopsis.
Our results support the following model for the mechanism that
guides the regulation of dormancy levels during the dormancy
induction and release stages. First, WOX11/12 are expressed in
the early stages of seed development and are highly activated
in the seed dormancy induction stage by PHYB-mediated
red-light signaling. WOX11 and WOX12 then act redundantly
downstream of PHYB to regulate seed dormancy by modulat-
ing GA signaling. The PHYB–WOX11/12 molecular module
determines the level of dormancy during the dormancy in-
duction stage in Arabidopsis, and could be a conserved regula-
tory mechanism in angiosperms.
A new role for WOX11/12 in regulation of seed
dormancy
Physiology dormancy is an important and adjustable process
that is widely present in seed plants, and various environmental
mancy level is determined during the induction stage, and this
determines the duration until dormancy release (Finch-Savage
and Footitt, 2017). ABA is a critical phytohormone for dor-
mancy induction, and the impairment of its biosynthesis and
signaling pathways affects seed dormancy. GA functions as an
antagonist of ABA in the regulation of dormancy (Shu et al.,
2016). DOG1 is a pivotal protein in seed dormancy induc-
tion, and its mRNA level determines the dormancy level in
Arabidopsis (Nakabayashi et al., 2012). In this study, we identi-
fied WOX11/12 as hub transcription factors during the seed
dormancy release and germination stages (Fig. 2). WOX11/12
were highly expressed during the period 8–14 DAP (Fig. 4C;
Supplementary Fig. S2B), which covers the seed maturation
and dormancy induction stages (Le et al., 2010; L. Yang et al.,
2020). The double-mutant wox11 wox12 showed a deeper
dormancy status, whereas the WOX11-OE transgenic line
exhibited a lost dormancy phenotype (Fig. 3). These results
strongly suggested that WOX11/12 are the negative regula-
tors of dormancy during the induction stage. It was interesting
that WOX11/12 might regulate GA signaling during seed
1102 | Liao et al.

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Fig. 7.  WOX11/12 are involved in GA signal transduction in Arabidopsis seeds. (A) Heatmap showing the relative expression of expansins (EXPs) and
xyloglucan endo-transglycosylase/hydrolases (XTHs), which are regulated by GA and function in cell wall remodeling, in dry (D) and imbibed (I) seeds of
Col-0 and the wox11 wox12 double-mutant. (B) Relative expression of the GA biosynthesis and catabolism genes in freshly harvested dry seeds of Col-
0, wox11 wox12, and the transgenic WOX11-overexpression (OE) line, as determined by RT-qPCR. The mRNA levels were first normalized to the PP2A
reference gene, and expression is relative to that of Col-0, the values of which were set as 1 relative expression unit (REU). Results using ACTIN2 as an
alternative reference gene are shown in Supplementary Fig. 2. (C) Germination of freshly harvested unstratified seeds of Col-0 and wox11 wox12 treated
with different GA concentrations, compared with stratified seeds (4 °C) with no GA under white light for 5 d. (D) Germination rates for the seeds shown
in (C). All data are means (±SE). Significant differences compared with Col-0 were determined using Student’s t-test: *P≤0.05, **P≤0.01, ***P≤0.001,
****P≤0.0001; n.s., not significant.

­dormancy (Fig. 7). Hence, WOX11/12 are the newly identi-
fied antagonistic factors of ABA and DOG1 that regulate seed
dormancy.
The members of the WOX protein family are well known
for their roles in meristem maintenance and regeneration, and
they regulate a series of developmental processes, including
embryogenesis, meristem cell division and differentiation, leaf
blade morphogenesis, and plant architecture formation (van
der Graaff et al., 2009; Zhang et al., 2020; Kawai et al., 2022).
WOX11–LBD16/29 and WOX11/12–WOX5 have previ-
ously been reported to be involved in the first step of the tran-
sition of stem cell fate and the initiation of root primordia,
respectively, as part of adventitious root formation in Arabi-
dopsis (J. Liu et al., 2014; Hu and Xu, 2016). In the adventitious
shoot induction stage, WOX11–LBD16 signaling is involved in
callus pluripotency acquisition (J. Liu et al., 2018). LBD16 and
LBD29 also function in lateral root initiation and trigger callus
induction (Lee et al., 2009; Fan et al., 2012), and cell wall loss
factors such as EXPANSIN14 are the direct targets (Lee et al.,
2013; Xu et al., 2018). Cell wall remodeling is also a key stage
in seed dormancy release and germination (Nonogaki, 2019).
Our results support WOX11/12 having roles in the regulation
of seed dormancy (Fig. 3), as the expression of WOX11/12
was mainly activated during seed maturation and later in the
seed germination stage (Fig. 4C, D; Supplementary Fig. S2B,
C), which is distinct from organ regeneration. Interestingly, we
found that cell wall remodeling genes were downregulated in
wox11 wox12 double-mutant seeds (Fig. 7A), which might be
 Regulation of Arabidopsis seed dormancy by WOX11/12  |  1103
the main reason for their longer dormancy compared to the
wild type. Regulation of genes for cell wall modification is a
conserved function, because WOX13, an ancient subclade, is
also reported to regulate cell wall modifications in Arabidop-
sis and mosses (Sakakibara et al., 2014; Ikeuchi et al., 2022).
Overall, these results indicate that WOX11/12 have a newly
identified function in regulating seed dormancy and germina-
tion based on their specific expression patterns.
WOX11/12 function downstream of red-light signaling
to regulate seed dormancy
By altering hormone contents and signaling, light modulates
all aspects of seed dormancy from induction to release (L.Yang
et al., 2020). PHYB-mediated red-light signaling plays a role in
seed dormancy and germination by modifying GA biosynthesis
or signaling (Jiang et al., 2016). REVEILLE1/2 (RVE1/2) accu-
mulates during the seed maturation stage to induce and main-
tain dormancy by directly binding to the promoter of GA3ox2
to repress its expression, and PHYB activated after imbibition
reverses this process to promote seed germination (Jiang et al.,
2016). However, the relationship between red-light signaling
and RVE1/2 during the dormancy induction stage remains
unclear. The first quantitative characteristic locus for seed dor-
mancy to be discovered was DOG1 and it determines the level
of dormancy at the end of seed maturity (Bentsink et al., 2006).
More recently, light cues have been shown to modulate DOG1
transcription to regulate seed dormancy via a mechanism that
is dependent on the evening complex of the circadian clock
(Zha et al., 2020). In the present study, WOX11/12 mRNA
was detected during the very early stages of embryogenesis
by RT-qPCR and GUS staining (Fig. 4; Supplementary Fig.
S2B). Interestingly, the expression levels of WOX11 and its ho-
molog WOX12 were positively regulated by PHYB during
seed development and the dormancy induction stages (Fig.
6A, B; Supplementary Fig. S2D, E), suggesting that red-light
signaling can further activate their expression. Under PHYB-
off conditions (exposure to far-red light), WOX11 overexpres-
sion was sufficient to break the seed dormancy status and it
could rescue the phenotype of the phyB mutant (Fig. 6C, D),
indicating that WOX11 works as a red-light signaling com-
ponent that specifically functions in seed dormancy regula-
tion. Because PHYB accumulates and inputs light signaling
to regulate seed dormancy (L. Yang et al., 2020), we proposed
that red light positively regulates WOX11/12 expression to
modify dormancy during the seed maturation stage. Therefore,
WOX11/12 are the newly identified regulators downstream
of PHYB that function in seed dormancy induction and they
might work together with RVE1/2, DOG1, and other light-
signaling components to maintain dormancy. Our results also
suggested that light signaling interacted with developmental
signaling to fine-tune plant growth during adaptation to envi-
ronmental changes.
Evolutionarily conserved function of WOX11/12
The ancestral dormancy type among gymnosperms and
angiosperms is morphological dormancy, in which the em-
bryo requires time to mature and germinate (Linkies et al.,
2010). The increasing relative size of the embryo has occurred
not only in angiosperms but also in gymnosperms, and it is
thought to be one of the main factors driving the evolution
of physiological dormancy (Forbis et al., 2002; Linkies et al.,
2010). The intermediate clade of the WOX gene family is di-
vided into the WOX8/9 and WOX11 subclades, and we found
that they had seed-specific expression patterns (Fig. 2D). The
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members of the WOX8/9 clade are reported to be involved in
embryogenesis (Palovaara et al., 2010; Ueda et al., 2011) and
meristem maintenance (Wu et al., 2005) in angiosperms and
gymnosperms. Interestingly, the WOX11 clade originated from
the WOX8/9 clade, expanded in angiosperms, and had a con-
served seed-specific expression pattern (Fig. 2D). Given that
WOX11/12 have conserved roles in shoot and root organs de
novo (Zhao et al., 2009; J. Liu et al., 2014, 2018; B. Liu et al.,
2018 ), we propose that they might have a shared dormancy
regulating function in angiosperm plants.The PHYB–WOX11
pathway might be angiosperm-specific and could have been
acquired after the evolutionary separation between gymno-
sperms and angiosperms.
The acquisition of seed dormancy will help plants pass on
genes under harsh environmental stresses, such as cold tem-
peratures and drought (Penfield, 2017). Seed production is
critical for higher plant life cycles, and they can be dispersed
worldwide by wind, water, and animals, and can survive in
various environments after being shed from the plant (For-
bis et al., 2002; Shu et al., 2016). Hence, it is very impor-
tant to understand what triggers seed germination in order
to be able to stop the spread of certain plants and to de-
termine the location and timing of the generation of new
plants (Penfield, 2017). Dormancy is a property that prevents
seed germination, so uncovering the general mechanism of
dormancy regulation will help us to understand how higher
plant expansion occurs worldwide in various environments.
In this study, we found that WOX11/12 were activated by
PHYB-mediated red-light signaling during the seed matura-
tion and the germination stage, and played roles in alleviating
seed dormancy by modulating GA signaling (Figs 3, 6, 7).
These results suggest that the red-light environment during
seed maturation and germination is critical for the dormancy
level and determines the germination time. During the mat-
uration stage, the environment surrounding each pod is dif-
ferent because of the climate, weather, light environment, and
location, as well as other factors affecting red-light irradia-
tion (Hernando et al., 2021). These differences can produce
seeds with a range of dormancy characteristics, which will
make them germinate at different times of the year or in
response to ecosystem disturbances. Our results suggest that
1104 | Liao et al.
WOX11/12 are key dormancy regulators that might finely
regulate the level of dormancy and help with seed distribu-
tion and survival in new environments.
In summary, we show that WOX11 and its homolog
WOX12 have a newly identified role in the control of seed
dormancy and germination and that they are new components
downstream of PHYB that modulate dormancy during the
induction stage. Our results provide insights into the regula-
tory mechanism of red-light signaling during plant growth and
development, which is critical for the adaptation of plants to
changes in the light environment.
Supplementary data
The following supplementary data are available at JXB online.
Table S1. The primers used in the study.
Table S2. Summary of RNA-seq read mapping results.
Table S3. Details of the intermediate clade of the WOX gene
family in Fig. 2D.
Table S4. GO term analysis of the modules shown in Fig. 2B.
Fig. S1. Expression patterns of WOX11 during seed
development.
Fig. S2. Results of RT-qPCR analyses shown in Figs 4, 6,
and 7 using the alternative reference gene ACTIN2.
Fig. S3. Data supporting the involvement of WOX11/12
light signal transduction.
Fig. S4. Germination of seeds of Col-0 and the phyB-9 mu-
tant showing that PHYB suppresses seed dormancy.
Acknowledgements
We very much appreciate constructive suggestions on the data analysis
made by Prof. Yoshito Oka (Basic Forestry and Proteomics Research
Center) and Prof. Zhong-jian Liu (College of Landscape Architecture,
Fujian Agriculture and Forestry University), and we thank Dr Zhaohe
Yang (Basic Forestry and Proteomics Research Center) for phenotype
analysis and seed storage. We also thank Dr Yu Li (Institute of Botany,
Chinese Academy of Sciences) and Prof. Lianfeng Gu (Basic Forestry
and Proteomics Research Center) for their expert assistance in handing
samples and RNA-seq analysis, respectively.
Author contributions
BL: conceptualization; BL, JL, BD, XC, QY, BH, GW, JC, and YZ: in-
vestigation; BL and JL: writing—original draft; BL, JL, GX, YL, LY, DZ,
and JZ: writing—review & editing; BL: funding acquisition; BL and JL:
project administration.
Conflict of interest
The authors declare that they have no conflict of interest in relation to
this work.
Funding
This work was supported by the Natural Science Foundation of Jiangsu
Province (BK20221408), the National Natural Science Foundation of
China (31870661, 31770325), start-up funds for scientific research of
Yancheng Teachers University (204670015), and the Opening Foundation
of Jiangsu Key Laboratory for Bioresources of Saline Soils (206670144).
Data availability
RNA-seq data for transcriptomic analysis of the wox11 wox12 double
mutant are available at the National Genomics Data Center (https://
ngdc.cncb.ac.cn/) under the accession number CRA007485. In addition,
Downloaded from https://academic.oup.com/jxb/article/74/3/1090/6833648 by guest on 28 March 2023
the data supporting the findings of this study are available from the cor-
responding author, Bobin Liu, upon request.
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