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Stress Biology
Single-cell transcriptomic analysis
reveals thedevelopmental trajectory
andtranscriptional regulatory networks
ofquinoa salt bladders
Hao Liu1†, Zhixin Liu1†, Yaping Zhou1†, Aizhi Qin1, Chunyang Li1, Yumeng Liu1, Peibo Gao1, Qianli Zhao1,
Xiao Song1, Mengfan Li1, Luyao Kong1, Yajie Xie1, Lulu Yan1, Enzhi Guo1 and Xuwu Sun1*
Abstract
Salt bladders, specialized structures on the surface of quinoa leaves, secrete Na+ to mitigate the effects of the plant
from abiotic stresses, particularly salt exposure. Understanding the development of these structures is crucial for elu-
cidating quinoa’s salt tolerance mechanisms. In this study, we employed transmission electron microscopy to detail
cellular differentiation across the developmental stages of quinoa salt bladders. To further explore the developmental
trajectory and underlying molecular mechanisms, we conducted single-cell RNA sequencing on quinoa proto-
plasts derived from young leaves. This allowed us to construct a cellular atlas, identifying 13 distinct cell clusters.
Through pseudotime analysis, we mapped the developmental pathways of salt bladders and identified regulatory
factors involved in cell fate decisions. GO and KEGG enrichment analyses, as well as experimental results, revealed
the impacts of salt stress and the deprivation of sulfur and nitrogen on the development of quinoa salt bladders.
Analysis of the transcription factor interaction network in pre-stalk cells (pre-SC), stalk cells (SC), and epidermal blad-
der cells (EBCs) indicated that TCP5, YAB5, NAC078, SCL8, GT-3B, and T1P17.40 play crucial roles in EBC development.
Based on our findings, we developed an informative model elucidating salt bladder formation. This study provides
a vital resource for mapping quinoa leaf cells and contributes to our understanding of its salt tolerance mechanisms.
Keywords Quinoa, Salt bladders, Single-cell RNA sequencing, Developmental trajectory, Salt tolerance
Introduction
Soil salinity has a serious impact on crop productivity,
and improving salt tolerance has become an important
strategy for genetic breeding to maintain the world’s
food supply, improve land use efficiency, and maintain
crop yields (Elhindi etal. 2017). Quinoa (Chenopodium
quinoa Willd.) is an annual herb that can grow under
adverse abiotic stresses such as high salinity, drought,
and cold (Jacobsen et al. 2003). Originally from the
Andean region of South America, quinoa is a food crop
with high nutritional and commercial value (Angeli etal.
2020). Quinoa contains a large number of phytochemi-
cals with health benefits, including amino acids, fibers,
polyunsaturated fatty acids, vitamins, minerals, saponins,
phytosterols, phytoalexins, phenols, betaine, and glycine
betaine (Graf etal. 2015). In addition, quinoa is gluten-
free, making it particularly suitable for people with wheat
allergies or intolerances (Bian etal. 2023). Quinoa is a
Handling editor: Dr. Kunpeng Jia.
† Hao Liu, Zhixin Liu and Yaping Zhou contributed equally to this work.
*Correspondence:
Xuwu Sun
sunxuwu@henu.edu.cn
1 National Key Laboratory of Cotton Bio-Breeding and Integrated
Utilization, State Key Laboratory of Crop Stress Adaptation
and Improvement, School of Life Sciences, Henan University,
Kaifeng 475001, China
Page 2 of 23
Liuetal. Stress Biology (2024) 4:47
dicotyledonous C3 salt plant, but it is often mistaken for
a cereal such as maize and wheat (monocotyledons of the
grass family), hence the term ‘pseudo-cereal’ (Graf etal.
2015). Similar to most leaves, the adaxial and abaxial leaf
surfaces of quinoa contain many epidermal bladder cells
(EBCs), the density of which varies significantly with leaf
age, with the highest density in young leaves (L. Shabala
et al. 2012). Epidermal bladder cells are modified tri-
chomes, spherical in shape, and each EBC is ten times
larger in diameter than an epidermal cell (EC), thus able
to absorb up to 1,000 times more Na+ than an EC (Sha-
bala etal. 2014). Studies suggest that quinoa salt blad-
ders protect plant cells from UV damage and that their
accumulated organic osmoregulators scavenge reactive
oxygen species and reduce water loss (Adolf etal. 2013;
Shabala etal. 2012).
e EBCs on the surface of quinoa leaves form the EC-
SC-EBC complex together with the stalk cell (SC) and
the epidermal cell (EC) at the base (Shabala etal. 2014).
SCs contain most organelles such as mitochondria, chlo-
roplasts, endoplasmic reticulum, Golgi apparatus, and
plasmodesmata often appear in close contact to vesicu-
lar structures (Bazihizina et al. 2022). By analogy with
the pattern of epidermal cells and trichome formation in
Arabidopsis, a simplified model of salt bladder organo-
genesis was proposed: during vesicle complex formation,
the ‘doomed’ epidermal cell extends slightly to divide into
two cells, giving rise to a lower and upper segment. e
upper segment then extends outwards and forms the tri-
chome initial, which divides again to form the apical and
basal cells. e apical cell increases in size and develops a
large central vesicle, which eventually turns into a spheri-
cal vesicle, while the basal cell expands a little and devel-
ops into a stalk cell (Shabala etal. 2014). In a recent study,
Zhang et al. studied the developmental process of salt
bladders by paraffin sectioning of Chenopodium album
leaves at different developmental stages, and the results
were generally consistent with the simplified model of
salt bladder histogenesis described above (Zhang etal.
2022).
To regulate the osmotic potential balance between the
cytoplasm and the vesicles and to maintain the normal
function of the organelles, plants accumulate soluble sub-
stances in the cytoplasm that do not affect the enzymatic
activity and the structure and function of biomolecules
to reduce the osmotic potential in the cytoplasm. e
main osmolytes reported to be involved in osmoregu-
lation in quinoa include proline, inositol, and glycine
betaine (Jacobsen et al. 2007; Kiani‐Pouya et al. 2017;
Ruffino etal. 2009). Proline is an osmotic agent known
to be involved in plant salt responses, which can mitigate
the adverse effects of stressful environments in a vari-
ety of ways, including protecting cell structure, protein
integrity, and enhancing enzyme activity (Szabados &
Savouré 2010). Inositol also plays an important role in
plant osmoregulation, and inositol transport may be
closely coupled to Na+ transport (Adams etal. 2005). In
addition to its direct involvement in the regulation and
protection of vesicle-like membranes, glycine betaine
may also indirectly protect cells from environmental
stress through its action in signal transduction (Ashraf
& Foolad 2007). A metabolic study of EBCs from quinoa
under various abiotic stresses revealed that some primary
metabolites showed significant metabolic responses
under heat, cold, and intense light stresses, with little
change in secondary metabolites. Under salt and heat
stress, the lipid composition in EBCs changed signifi-
cantly (Otterbach etal. 2021). Using high-performance
liquid chromatography, it was found that UV treatment
induced a rapid accumulation of large amounts of beet-
root heme, flavonoids, and other substances in salt blad-
ders (Vogt etal. 1999). Betaine is a natural pigment in
plants and it is believed that the pigment in plant epi-
dermal cells absorbs UV light and reduces UV damage
to plants (Wang & Wang 2007). Flavonoids are second-
ary metabolites synthesized by plants that can absorb
UV light and resist peroxidation (Ichino etal. 2020). Salt
bladders can store up to 1 M NaCl within a giant vesi-
cle, and Na+ and Cl− need to cross the plasma membrane
(PM), traverse the cytoplasm, and be loaded into the
vesicle lumen (Böhm etal. 2018). EBCs can improve salt
tolerance through xylem Na+ loading, high reactive oxy-
gen species (ROS) tolerance, K+ homeostasis, and effec-
tive control of stomatal development. EBCs can improve
plant salt tolerance in a variety of ways, including
through xylem Na+ loading, high reactive oxygen species
(ROS) tolerance, K+ homeostasis, and effective control
of stomatal development (Adolf etal. 2013). e plasma
membrane ion transporter SOS1 (salt overly sensitive 1)
and HKT1 (high-affinity K+ transporters 1) systems are
the main players in plant Na+ transport across the PM
(Shi etal. 2002; Waters etal. 2013). SOS1 represents the
Na+/H+-antiporter protein that uses proton-powered
proton-motive-force (PMF) to move Na+ out of the cell
(Qiu etal. 2002).
One study reported high expression of genes homol-
ogous to the vesicular membrane Na+/H+ reverse
transporter protein NHX (Na+/H+ exchanger) in the
transcriptome data of quinoa salt bladders (Zou et al.
2017). When salt bladders accumulate salt to a certain
level, they will undergo autophagy, and apoptotic rup-
ture, thus avoiding or mitigating the damage caused by
stresses such as NaCl (Hu etal. 2020). e presence of
large vesicles of EBCs implies vesicle development and
transport of vesicle-associated proteins. Vesicular pro-
teins fuse with the vesicle membrane, thereby entering
Page 3 of 23
Liuetal. Stress Biology (2024) 4:47
the vesicle and participating in vesicle development
(Hu etal. 2020). During this process, the VPS4 (vacuo-
lar protein sorting 4) gene is involved in endocytosis for
transport to the vesicle and in the transport of vesicular
proteins in the late Golgi phase (Finken-Eigen etal. 1997;
Rothman & Stevens 1986). Echidna associated with green
V-ATPase is a vesicular H+ pump that fuels NHX in the
vesicular membrane, allowing Na+ transport into the
vesicles, improving vesicular osmoregulation and main-
taining Na+ and K+ homeostasis. Compared to other cell
types, vesicular ATPase activity is constitutively high in
the guard cell (GC) to meet the high and fast ion flux to
the vesicular membrane required for stomatal movement
(Bose et al. 2014; Rasouli etal. 2021). ere are many
GC proteins involved in resistance to salt stress, such
as auxin binding protein 19 (ABP19) that decreases GC
cytoplasmic pH and induces stomatal opening; dehydrin
early responsive to dehydration (ERD14) and LOW-TEM-
PERATURE-INDUCED 65 (LTI65) attract water into the
cell, regulate the osmotic potential and maintain water
status (Rasouli etal. 2021).
Single-cell transcriptome sequencing technology has
emerged as a powerful tool for plant research, allowing
a deeper understanding of cellular heterogeneity during
cell development and stress response. High-throughput
single-cell transcriptome sequencing was performed on
Arabidopsis root tissue protoplasts to resolve the devel-
opmental trajectories of root hair cells, and to confirm
the feasibility and effectiveness of high-throughput sin-
gle-cell sequencing in plants (Ryu et al. 2019). Regula-
tors of epidermal development under drought and salt
stress conditions were identified using single-cell RNA
sequencing, highlighting the sensitivity of epidermal cells
to environmental stress (Liu etal. 2022). By employing
single-cell transcriptome sequencing, the developmental
trajectory of pigment gland cells and the regulatory net-
work of transcription factors were analyzed, leading to
the proposal of a relatively complete model for the for-
mation of pigment glands (Sun etal. 2023). By construct-
ing a single cell resolution transcription map of cotton
root tips in response to salt stress, the cell heterogeneity,
root type difference and differentiation trajectory of plant
roots under salt stress were revealed, which laid a solid
foundation for elucidating the molecular mechanism of
plant resistance (Li etal. 2024).
EBCs, as one of the important structures of salt toler-
ance in quinoa, are of great interest in studying the mech-
anism of tolerance to salt stress in quinoa. Genes related
to membrane transporter proteins and those involved in
the response to salt stress have been identified through
RNA sequencing (RNA-seq) analysis of epidermal blad-
der cells (EBC) (Zou et al. 2017). In recent years, sin-
gle-cell transcriptome sequencing technologies have
significantly contributed to understanding the develop-
ment of specific plant tissues and cell types, revealing
new cell types, key regulatory genes for cell lineage devel-
opmental trajectories and fate determination, and molec-
ular mechanisms of environmental adaptation. However,
the molecular regulatory mechanisms underlying quinoa
salt bladder development are currently unclear and stud-
ies at the high-throughput single-cell level are lacking.
erefore, in this study, we constructed a cellular map
of young quinoa leaves using scRNA-seq. We classified
the major cell types, screened for differentially expressed
genes that are specifically expressed in different cell
types, and identified key regulators. e developmen-
tal trajectories of the different cell types were studied
by mimetic time series analysis. Using GO analysis of
DEGs, we found that genes involved in the regulation of
sulfur and nitrogen metabolism are highly expressed in
EBCs. Further physiological and cell biological analyses
showed that low sulfur (LS) treatment as salt treatment,
promotes EBC development, whereas low nitrogen (LN)
treatment inhibits EBC development. is suggests that
genes involved in the regulation of sulfur and nitrogen
metabolism could directly regulate EBC development
and thus further regulate quinoa tolerance to salt stress.
Our study provides a comprehensive resolution of the
developmental processes of epidermal bladder cells at the
single-cell level and sheds light on the pivotal role of soil
sulfur and nitrogen in enhancing the growth and salt tol-
erance of quinoa. ese findings offer novel insights into
the molecular mechanisms underlying the development
of salt bladders and establish a theoretical basis for the
development of salt-tolerant crop varieties.
Results
Structural anddevelopmental dynamics ofsalt bladders
inquinoa
As a typical feature of salt-tolerant plants, the salt blad-
ders on the leaf surface of quinoa are essential for regu-
lating its ion balance and cellular metabolism. Scanning
electron microscopy (SEM) of salt bladders on the upper
epidermis of quinoa leaves grown under normal con-
ditions for three weeks showed that mature salt blad-
ders consist of a short stalk and gourd-shaped bladders
that are narrower at the bottom and wider at the top
(Fig.1A and B). Mature salt bladders were much larger
than epidermal cells, with diameters around 100 μm,
and were scattered on the leaf surface (Fig.1A-B). Fig-
ure1C showed a schematic model of salt bladder devel-
opment, with the undifferentiated initial bladder cells
having divided by pericycle division to form pre-stalk
cells and anterior epidermal bladder cells. Subsequent
gradual expansion of the vesicles in the epidermal blad-
der cell pushes the major organelles to the cell edge. e
Page 4 of 23
Liuetal. Stress Biology (2024) 4:47
pre-stalk cell differentiates or divides further perpendicu-
larly to form a mature stalk cell that joins with the epider-
mal bladder cell above.
To observe the developmental process of the salt blad-
der in more detail, we used transmission electron micros-
copy (Rasouli, et al. 2021) on salt bladders on the true
leaves of two-week-old quinoa seedlings. e results
showed that the precursor cells divided and differentiated
twice to form bladder cells (Fig.2A-F). Furthermore, a
model for the development of salt bladder was proposed
(Fig.2G). In the initial bladder cell that just protrudes,
vesicles were dispersed (Fig. 2A). e epidermal cells
adjacent to the initial bladder cells transformed through
vesiculation, establishing a foundation for the absorption
and provisional salt retention (Fig.2B). Lysosomes within
the epidermal cells encapsulate chloroplasts and senes-
cent organelles, forming autophagic vesicles that digested
and integrated their contents into the vacuolar compart-
ment (Fig. 2B-C). During the development of bladder
cells, vacuoles progressively enlarged, pushing chloro-
plasts, Golgi apparatuses, and endoplasmic reticulums
to the cellular periphery, where they were subsequently
engulfed and digested by the expanding vacuolar system
(Fig.2E-F).
We also analyzed further the ultrastructure of the ini-
tiating bladder cells, EBCs, and mature stalk cells (Fig.
S1-S3). Initial bladder cells contained nuclei, Golgi appa-
ratus, ribosomes, mitochondria, and chloroplasts. e
small vesicles were not aggregated into large vesicles,
and there were also a large number of plasmodesmata
between initial bladder cells and neighboring epidermal
cells for the transport of insoluble substances into the
vesicles (Fig. S1). e cytoplasm of mature epidermal
cells and salt bladder cells was relatively dense. e salt
bladder consisted of EBCs and stalk cells, with the vesi-
cles in the EBCs occupying the vast majority of the space,
squeezing the organelles to the edges of the large vesicles
(Fig. S2). Chloroplasts, mitochondria, Golgi appara-
tus, and endoplasmic reticulum can be seen in mature
stalk cells. Among them, chloroplasts gradually shrank
and were subsequently endocytosed and digested by the
vesicles. ere were a large number of mitochondria and
Golgi bodies in the mature stalk cells, which may provide
energy for cellular activities (Fig. S3).
Construction ofasingle cell transcription atlas ofyoung
true leaves ofquinoa
To investigate the development of the salt bladder, we
performed single-cell RNA sequencing on the true
leaves of two-week-old quinoa seedlings to identify and
characterize the cell types and the expression patterns
of genes that are especially expressed in each of them.
Protoplasts were prepared by enzymatic digestion of
leaves, filtered and screened through a 40 μm pore cell
filter, and then used for sequencing library preparation.
After quality assessment of the sequencing data, 15,985
high-quality cells were obtained, with a mean number
of 8,766 UMI (unique molecular identifiers) per cell and
2,720 expressed genes per cell. e final number of cells
was 15,537 after eliminating double-cells, multicell, and
apoptotic cells, with a mean number of 8,138 UMI per
cell and 2,626 expressed genes per cell (TableS1). Subse-
quently, we employed Uniform Manifold Approximation
and Projection (UMAP) for dimensionality reduction
and executed cluster classification analysis on the fil-
tered single-cell sequencing data, which were classified
into 13 cell clusters based on the similarity of specifi-
cally differentially expressed genes (DEGs) in each cluster
(Fig.3A and TableS2). e correlation between differen-
tially expressed cell clusters was obtained by calculating
the Pearson correlation coefficients of the mean values
of gene expression between cell clusters. We found an
extremely strong correlation between clusters 1, 2, and
5, which implies that they most likely belong to similar
Fig. 1 Surface morphology and structural characteristics of salt bladders observed by SEM. A Horizontal view of epidermal salt bladders on the leaf
surface, showing their phenotype and distribution. B Vertical view of salt bladders on the upper epidermis of leaves, illustrating their phenotype
and distribution. Scale bar: 50 μm
Page 5 of 23
Liuetal. Stress Biology (2024) 4:47
cell types (Fig. 3B). e expression distribution char-
acteristics of representative marker genes in specific
cell clusters were illustrated, highlighting top five DEGs
expressed in specific cell cluster (Fig. 3C). To anno-
tate the cell types and identify the new marker genes in
quinoa leaves, we screened cluster-specific expressed
genes in each cluster (Fig.3D and TableS3). In plants,
especially quinoa, known marker genes for identifying
cell types remains limited and have been mainly iden-
tified in the model plant Arabidopsis thaliana so that
Fig. 2 Developmental analysis of quinoa salt bladders using transmission electron microscopy. A Development of the initial bladder cell
and the changes in internal organelles. B-D Division of the initial bladder cell into pre-stalk and epidermal cells. E Further division of pre-stalk
cells into stalk cells and epidermal bladder cells. F Formation of mature salt bladders and the changes in internal organelles. G Model illustrating
the formation and developmental progression of salt bladders. V: vacuole, N: nuclei, C: chloroplasts, P: plasmodesmata, PV: phagocytic vacuole.
Scale bar: 5 μm
(See figure on next page.)
Fig. 3 Identification of cell types and transcriptional profiles in two-week-old quinoa leaves using single-cell RNA sequencing. A UMAP plot
displaying 13 identified cell types, each coded with different colors. Red arrows indicate the developmental trajectories of stalk cells. B Heatmap
illustrating the correlation of gene expression between different cell clusters. The horizontal and vertical coordinates represent the different cell
clusters. The numbers on the graph are Pearson correlation coefficients, with darker colors indicating higher degrees of correlation. C Visualization
of known marker genes in the UMAP clustering maps. D Expression patterns of the top five differentially expressed genes (DEGs) with the highest
expression levels in each sub-cluster. The average expression level (color) and the proportion of cells expressing each gene (size) are shown for each
gene and cluster. Abbreviations: MPC, mesophyll cell; PC, pavement cell; MMC, meristemoid mother cell; pre-SC, pre-stalk cell; SC, stalk cell; GC,
guard cell; EBC, epidermal bladder cell; PP, phloem parenchyma; GMC, guard mother cell; u.k., unknown
Page 6 of 23
Liuetal. Stress Biology (2024) 4:47
Fig. 3 (See legend on previous page.)
Page 7 of 23
Liuetal. Stress Biology (2024) 4:47
homologous marker genes from other species can be
used for cell type identification. In addition, since some
known marker genes may be expressed in different cell
types, although at different levels, we utilized the expres-
sion patterns of multiple marker genes to collectively
identify different cell types in the true leaves of quinoa.
e Arabidopsis homologs basic helix-loop-helix (bHLH)
protein/LOC110732543, MUTE/LOC110727789, TOO
MANY MOUTHS (TMM/LOC110698887). SPEECHLESS
(SPCH/LOC110725693) are all specifically expressed in
cluster 4 and are considered marker genes for meriste-
moid mother cells (MMC) (Fig.3C) (Liu etal. 2020; Pil-
litteri et al. 2007). e Arabidopsis homolog SUGARS
WILL EVENTUALLY BE EXPORTED TRANSPORTERS 12
(SWEET12/LOC110703940) is a marker gene for phloem
parenchyma (PP) and is highly expressed in cluster 10
(Fig. 3C) (Kim et al. 2021). e Arabidopsis homolog
FAMA/LOC110716316, a marker gene for guard mother
cell (GMC), is specifically highly expressed in cluster
12, and AtFAMA has a role in controlling the transition
from GMC to guard cells (Fig.3C) (Ohashi-Ito & Berg-
mann 2006). e Arabidopsis homolog SLOW ANION
CHANNEL-ASSOCIATED 1 (SLAC1/LOC110702556) is
highly expressed in cluster 12, and AtSLAC1 has been
reported to encode a multi-transmembrane protein
involved in the regulation of stomatal lineage cell devel-
opment (Fig. 3C) (Deng et al. 2021). e Arabidopsis
homolog LIGHT HARVESTING CHLOROPHYLL A/B-
BINDING PROTEIN/LOC110715346, which is specifi-
cally expressed in clusters 1,2, and 5, has been reported
to encode a chloroplast protein and to be involved in the
regulation of stomatal development in the mesophyll
cell (MPC) with high expression (Liu etal. 2020). Prob-
able cation transporter HKT6/LOC110738464 is specifi-
cally expressed in clusters 3, 9, and 11, and is associated
with HKT1-type transporters (high-affinity potassium
transporter1) belonging to the HKT family of transcrip-
tion factors. AtHKT1 plays a key role in the dynamic
balance of Na⁺ and K⁺ under salt stress and contributes
to the reduction of Na⁺-specific toxicity in plants (Ali
et al. 2019). NITRATE TRANSPORTER 1 (NRT1/PTR
FAMILY 3.1-like/LOC110719467) is highly expressed
in clusters 3 and 9 (Fig.3C), and studies have reported
that NRT1 is involved in nitrate uptake, partitioning, and
storage in higher plants (Ali et al. 2019). ATP-binding
cassette family G25 (ABCG25/LOC110713974) is spe-
cifically expressed in clusters 6, 7, 9, 10 and 11 (Fig.3C).
AtABCG25 is a cytosolic ABA transporter protein, and at
the same time, ABCG transporter proteins are involved
in stratum corneum lipid transport (Kuromori et al.
2016; McFarlane et al. 2010). It has been shown that
SCs contain a large number of liposomes and a thick
stratum corneum enriched with genes related to ‘lipid
metabolism’ (Bazihizina et al. 2022). Short chain-dehy-
drogenase/reductases (SDR/LOC110736399) are specifi-
cally expressed in clusters 4, 6, and 9, and salt stress in
quinoa can be alleviated by up-regulated expression of
SDR genes (Al-Mushhin etal. 2021).
Annotation ofcell types based ontheanalysis ofGO
enrichment andKEGG
Due to the lack of studies on cell types in quinoa leaves
at the single-cell level, there is no resource of known
marker genes for identifying cell types. For those cell
types that could not be identified with available marker
genes, we tried to annotate the cell cluster with the infor-
mation of the Gene Ontology (GO) and Kyoto Encyclo-
pedia of Genes and Genomes (KEGG) pathway (Fig.4
and TableS4-5). Interestingly, we found that the DEGs of
clusters 1, 2, and 5 are enriched in very similar GO terms
(Fig.4A) and KEGG pathway (Fig.4B), consistent with
the Pearson correlation between cell populations from
the above analysis (Fig.3B). e GO terms enriched in
clusters 1, 2, and 5 include mainly the generation of pre-
cursor metabolites and energy, photosystem I assembly,
and chloroplast thylakoid membrane protein complex
(Fig. 4A); the KEGG pathways enriched in these clus-
ters are photosynthesis and porphyrin and chlorophyll
metabolism (Fig.4B). Similarly, cluster 8 is enriched for
GO terms related to photosystem I assembly and KEGG
pathways related to porphyrin and chlorophyll metabo-
lism. In contrast, clusters 4 and 12 are not enriched for
“photosynthesis-related”, which is consistent with the
lack of chloroplasts and photosynthetic functions in
MMC and GMC. e DEGs of cluster 9 are enriched for
endocytosis, a typical feature of EBC (Fig. S4A). In the
KEGG pathway analysis, it was observed that clusters
9 and 10 are enriched in similar pathways. e gigantic
vesicle in mature salt bladders caused chloroplast orga-
nelles to be pushed to the edge of the cell, resulting in a
deprivation of photosynthetically relevant capabilities.
Based on this observation, it is hypothesized that clus-
ter 10 consists of phloem parenchyma (PP) cells, which
are responsible for the carbon fixation function in pho-
tosynthetic tissues (Fig.4B). Cluster 9 is enriched to the
phagosome, proteasome, and ubiquitin-mediated prote-
olysis (Fig.4B). Clusters 6, 7, and 11 are enriched in the
similar KEGG pathway (Fig.4B), and their GO terms are
enriched in cytosolic ribosomes, suggesting that these
cell clusters are associated with protein translation in the
cytoplasm. is is consistent with our electron micros-
copy observation of dense cytoplasm, enriched cytosolic
ribosome, and vigorous protein translation activity in
SC. e KEGG pathway analysis of SC cluster 6 reveals
enrichment for protein processing in the endoplasmic
reticulum and protein export (Fig.4B). ese functions
Page 8 of 23
Liuetal. Stress Biology (2024) 4:47
Fig. 4 Enrichment analysis of differentially expressed genes (DEGs) in quinoa leaf cell clusters. A Gene Ontology (GO) enrichment analysis of DEGs
across 13 cell clusters. B Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of DEGs across 13 cell clusters
Page 9 of 23
Liuetal. Stress Biology (2024) 4:47
are typically associated with cells in the early stages of
development. In contrast, clusters 7 and 11 do not show
enrichment in these pathways. erefore, it is hypoth-
esized that cluster 6 represents pre-SC (pre-stalk cell),
while clusters 7 and 11 correspond to SC_7 and SC_11.
To distinguish between these two different stalk cells,
they are labeled with the cell cluster they belong to. In
summary, based on the marker genes screened in each
cell cluster, as well as the results of GO enrichment anal-
ysis and KEGG pathway analysis of these cell clusters,
we deduced that clusters 1, 2 and 5 belong to MPC_1,
MPC_2, MPC_5, cluster 4 belongs to MMC, cluster 8
belongs to GC, cluster 9 belongs to EBC (epidermal blad-
der cell), cluster 10 belongs to PP, and cluster 12 belongs
to GMC.
In the KEGG pathway analysis, we found that DEGs
associated with seleno-compound metabolism, Phago-
some, ubiquitin-mediated proteolysis, and Sulfur
metabolism are enriched in EBC (cluster 9) (Fig. 4B).
For example, phosphoglycerate mutase-like protein 4/
LOC110682269 was specifically expressed in EBC (clus-
ter 9) (Fig. S4B), and it was reported that its Arabidop-
sis homolog AT3G50520 encodes a phosphoglycerate
mutase-like family of proteins, which are involved in the
metabolic process in response to inorganic substances
and sulfur (Jedrzejas 2000). Similarly, cluster 3 is also
enriched for Selenocompound metabolism and Nitro-
gen metabolism. In GO analysis, we found that EBC
(cluster 9) and cluster 3 are enriched for similar func-
tions, including oxidative phosphorylation, copper ion
binding, s-adenosylmethionine metabolic process, and
carbon metabolism, phosphorylation, copper ion bind-
ing, S-adenosylmethionine metabolic process, and Car-
bon metabolism (Fig. 4A). Surprisingly, cluster 3 was
also enriched for the cytosolic ribosome-associated with
cytoplasmic solute transport. is suggests that cluster 3
may be a pavement cell (PC) adjacent to salt bladders. In
the GO analysis of DEGs (Fig.4A), we found that DEGs
in pre-SC (cluster 6) and PP (cluster 10) were enriched in
similar GO terms, copper ion binding, response to tem-
perature stimulus, carbon metabolism, oxidative phos-
phorylation, and significant enrichment in the nucleolus.
erefore, we hypothesized that the pre-stalk is closely
related to PP cells. Cluster 13 was named unknown (u.k.)
because there were no marker genes that could identify
the cell type in cluster 13 and the results of GO enrich-
ment analysis and KEGG pathway analysis differed
greatly from those of other clusters, making it impossible
to determine its cell type. We chose the marker genes of
MMC, pre-SC, SC, and EBC for feature map presentation
since this is the first instance of single-cell annotation of
quinoa salt bladder cell types (Fig. S5).
Analyzing thedevelopmental trajectory oftheEBC
intheleaves ofquinoa bypseudo‑time series analysis
To analyze the temporal distribution of the evaluated
single cells, a pseudo-time trajectory was employed to
visualize how cells from each cluster were distributed
along the main stem using the Monocle 2 (Trapnell
et al. 2014). e proposed temporal path of the qui-
noa leaf samples had three branches distributed along
a major developmental trajectory, with different clus-
ters of cells aligned more clearly at different branch
positions in the pseudo-temporal path (Fig. 5A, C).
Pseudotime analysis revealed that all single cells were
divided into five states of leaf cell development and dif-
ferentiation state (Fig.5B-C). Constructing a pseudo-
temporal trace of each cell cluster based on cell cluster
coloring can help in visualizing the developmental tim-
ing of each cluster (Fig.5D). Overall, the developmen-
tal progression of quinoa salt bladder cells was from
MMCs to EBCs and SCs. Interestingly, we found that
cell cluster 7 appeared earlier than cell cluster 11 on the
pseudotemporal curve, so the stalk cell cluster develop-
mental order was pre-SC_6 to SC_7 to SC_11 (Fig.5D).
Illustration of the pseudotime expression pattern of
the top 1 gene with highly variable expression showed
LOC110719982 exhibits high expression levels at the
onset of differentiation and remains highly expressed
in MMC (Fig.5E). In principle, the distribution charac-
teristics of different types of cells on the developmen-
tal trajectory can initially determine the relationship
between these cells at different developmental stages.
e distribution of GCs in the developmental trajec-
tory is relatively concentrated, but MPC_2 and PCs can
be found at several time points on the developmental
trajectory, suggesting that the development of MPCs
and PCs is more complex.
(See figure on next page.)
Fig. 5 Pseudotemporal analysis of quinoa leaf cell clusters. A-C Monocle2 was used to simulate the developmental trajectories and differentiation
states of quinoa leaf cell clusters over pseudotime, with each dot representing a single cell. D The distribution of various cell types along these
pseudotime trajectories is shown, with each dot color-coded by cell type. E The expression profiles of the most variable gene along pseudotime
were identified using Monocle2’s differential Gene Test function (q-value < 0.01). The black line represents the overall expression trend, with each
dot indicating a single cell. F A heatmap displays the genes with highly variable expression across three gene modules along pseudotime.
Additionally, a dot plot presents the GO enrichment analysis of these highly variable genes
Page 10 of 23
Liuetal. Stress Biology (2024) 4:47
Fig. 5 (See legend on previous page.)
Page 11 of 23
Liuetal. Stress Biology (2024) 4:47
e pseudo-time heatmaps of the top 300 genes with
highly variable expression show that their pseudo-time
patterns can be divided into three modules (Fig.5F). In
module 1, the expression of most genes first increases
and then decreases along the trajectory of pseudo-time
(Fig.5F). GO enrichment of module one reveals genes
involved in cell wall biogenesis, and response to gibber-
llin and brassinosteroid (Fig.5F and TableS6). In mod-
ule 2, the expression of all genes gradually decreases,
except for a small number of genes that reach high lev-
els of expression at the end of the trajectory of pseudo-
time (Fig.5F). In module 3, the expression of all genes
remains at low and stable levels but increases to its
highest level in the final stage of the trajectory (Fig.5F).
GO enrichment of module three revealed genes
involved in metal ion transport, and seed trichome
elongation (Fig.5F and TableS6).
To decode the developmental trajectory from MMC
to EBC, the cells of MMC, pre-SC, SC, and EBC were
extracted and re-analyzed in a quasi-chronological
manner. Cell developmental trajectories indicate that
MMC is situated at the starting point of differentia-
tion, and then differentiates into pre-SC, which further
differentiates into SC and EBC (Fig.6A-C). e quasi-
chronological expression profile of the top 2 genes with
hypervariable expression showed that LOC110720877
is highly expressed in EBC, and LOC110722270 in
MMC in the early stages of differentiation (Fig.6D). e
top 2,000 hypervariable genes were selected for quasi-
chronological cluster analysis, which was divided into
5 modules (Fig.6E). Module 2 is expressed at the early
stage, and the GO enrichment results indicate the xylo-
glucan metabolic process, wax biosynthetic process,
cell wall biogenesis, and cell–cell signaling (Fig.6E and
TableS7). Modules 1 and 4 include genes expressed at
the mid-term, and the GO enrichment results indicate
photosynthesis, reductive pentose-phosphate cycle,
and metal iron transport (Fig.6E and TableS7). Genes
of module 3 are expressed at the later stage, and the
GO enrichment results reveal cell redox homeostasis,
nucleosome assembly, agglutination involved in conju-
gation, nucleosome positioning, and chromosome con-
densation (Fig.6E and TableS7). Module 5 is expressed
at both early and late stages, and the GO enrichment
results indicate the methionine biosynthetic process
(Fig.6E and TableS7).
Salt stress promotes thedevelopment ofsalt bladders
inquinoa
GO enrichment analyses showed significant enrichment
of terms responsive to salt stress in SC and EBC clusters.
In addition, we identified several marker genes in stalk
and salt bladder cell clusters in response to salt stress,
such as bHLH112-like (LOC110693480), LOC110707708,
LOC11069094, and LOC110705347. Among them, it was
reported that AtbHLH112 belongs to subfamily F and
responds to abiotic adversity through increased proline
levels and enhances ROS scavenging thus enhancing tol-
erance to abiotic stress (Liu etal. 2015). e observation
that these genes related to salt stress are highly expressed
in SC and EBC cells suggests that salt stress has a poten-
tial role in influencing the development and function of
salt bladders.
To characterize the effect of salt treatment on the
development of PC, GC, and EBC, we treated one-week-
old quinoa seedlings with 150 mM NaCl for two weeks
(Fig.7A). It was observed that the growth rate of quinoa
seedlings treated with salt was slightly lower compared
to the control (Figs.7A and 9A). Image J (Version 1.2.4,
RRID:SCR_003070) software was employed to count
the number and size of cells. Statistical analysis showed
that the EBC density on the leaf surface increases signifi-
cantly with salt treatment, especially in the lower epider-
mis (Fig.7B, C). e density of PC and GC in the upper
epidermis significantly increased after salt treatment
(Fig.7D). ese results imply that Na+ stimulates the for-
mation of salt bladders.
Nitrogen promotes thedevelopment ofsalt bladders
KEGG pathway enrichment analysis revealed that nitro-
gen metabolism is significantly enriched in PC cell clus-
ters, and sulfur metabolism is significantly enriched in
both PCs and EBC (Fig. 4B). ese results suggest that
nitrogen metabolism and sulfur metabolism may have
potential roles in influencing the development of salt
bladders.
To analyze the effects of nitrogen, and sulfur on the
development of PC, GC, and EBC, we grew quinoa under
Fig. 6 The development trajectory of EBC from MMC. A-B Monocle 2 was used to simulate the developmental trajectory of EBC over pseudotime,
with each dot representing a single cell. C The distribution of different EBC-associated cell types along these pseudotime trajectories is shown,
with each dot color-coded by cell type. D The expression profiles of the two most variable genes along pseudotime were identified using Monocle
2’s differential Gene Test function (q-value < 0.01). The black line represents the overall expression trend, with each dot indicating a single cell. E
A heatmap displays genes with highly variable expression across five gene modules along pseudotime. Additionally, a dot plot presents the GO
enrichment analysis results for these highly variable genes
(See figure on next page.)
Page 12 of 23
Liuetal. Stress Biology (2024) 4:47
Fig. 6 (See legend on previous page.)
Page 13 of 23
Liuetal. Stress Biology (2024) 4:47
nitrogen/sulfur deficiency for two weeks. Growth of qui-
noa was significantly inhibited, especially in the case of
nitrogen deficiency (Fig.8A). e number of salt bladders
on the surface of quinoa increased in the absence of sul-
fur and decreased in the absence of nitrogen (Fig.8A, B,
C). is indicates that sulfur plays an inhibitory role in
salt bladder formation while nitrogen positively regulates
salt bladder formation. Statistical analysis of the num-
ber of PCs and GCs in the upper epidermis showed that
nitrogen deficiency significantly decreases the number of
PCs and GCs (Fig.8B, D).
ATML1 and PDF2 are known to regulate epidermal
cell development (Abe etal. 2003). erefore, we exam-
ined the expression of the homologs of ATML1 and PDF2
corresponding to PDF2-LIKE and PDF1-LIKE, respec-
tively, under nitrogen deprivation. e results showed
that both the ATML1 and PDF2 homologs which regu-
late the development of quinoa leaves, are repressed
under nitrogen deficiency conditions (Fig. 8F). is
repression leads to a disruption in the normal growth
pattern, culminating in the abnormal enlargement of
provascular cells (PCs) (Fig. 8E). Under conditions of
sulfur deficiency, a pronounced increase in the density
of PCs within the quinoa upper epidermis was observed
(Fig. 8D). Additionally, there was a significant reduc-
tion in the individual area of PCs (Fig.8E). In stark con-
trast, the sulfur-deficient treatment induces a significant
increase in the GC count (Fig.8D). ese observations
Fig. 7 Effect of salt stress on the development of salt bladders and PCs in quinoa leaves. A The growth phenotype of quinoa under control
conditions and after 2 weeks of 150 mM NaCl treatment; Scale bar: 1 cm. B SEM images illustrating the impact of salt stress on the density of salt
bladders on the upper and lower epidermis of quinoa leaves; Scale bar: 100 μm. C-D The density of salt bladder, PC, and GC on the surface of quinoa
under control and salt stress conditions (** p < 0.01, t-test)
Fig. 8 Effects of nitrogen and sulfur deficiency on the development of salt bladders and PCs in quinoa leaves. A The growth phenotypes
of quinoa after two weeks of nitrogen and sulfur deficiency; Scale bar: 1 cm. B SEM images illustrating the impact of nitrogen and sulfur deficiency
on the density of salt bladders on the upper and lower epidermis of quinoa leaves; Scale bar: 100 μm. C-E Changes in density of salt bladder cells,
PC, GC, and size of PC under nitrogen deficiency and sulfur deficiency treatment (* p < 0.05, ** p < 0.01, *** p < 0.001, t-test). F The expression level
of PDF1-like and PDF2-like decreases under nitrogen deficiency (*** p < 0.001, t-test)
(See figure on next page.)
Page 14 of 23
Liuetal. Stress Biology (2024) 4:47
Fig. 8 (See legend on previous page.)
Page 15 of 23
Liuetal. Stress Biology (2024) 4:47
suggest a potential facilitative effect of nitrogen on and
inhibitory role of sulfur in the developmental process of
quinoa salt bladders.
Salt stress‑induced salt bladder development
isindependent ofnitrogen signaling
By observing salt bladders after salt stress, we found that
high salt concentrations promote the development of
salt bladders in quinoa leaves (Fig.7B). To test whether
high concentrations of Na+ could act as a signal to pro-
mote salt bladder development, we grew quinoa seed-
lings under salt stress and nitrogen deficiency. e results
showed that the development and height of plants sub-
jected to this combined treatment were significantly
reduced compared with the control and plants subjected
only to salt stress (Fig.9A, C). SEM observation of leaves
showed that the number of salt bladders was significantly
increased by combined nitrogen deficiency and salt stress
compared with nitrogen deprivation (Fig.9A, B, D), sug-
gesting that salt stress promotes the development of salt
bladders under nitrogen deficiency and that the develop-
ment of salt bladders induced by high concentrations of
Na+ is independent of the nitrogen signals. In addition,
statistical analyses of PCs revealed that the area of PCs
Fig. 9 Effect of nitrogen deficiency and salt stress co-treatment on the development of salt bladders and PCs in quinoa leaves. A The growth
phenotypes of quinoa after two weeks under 150 mM NaCl or nitrogen deficiency treatments; Scale bar: 1 cm. B SEM images illustrating the impact
of 150 mM NaCl or nitrogen deficiency on the density of salt bladders on the upper epidermis of quinoa leaves; Scale bar: 100 μm. C Effects
of nitrogen deficiency and salt stress co-treatment on quinoa plant height (*** p < 0.001, t-test); D-E Changes of density of salt bladder, PCs, and GCs
after nitrogen deficiency with salt stress co-treatment
Page 16 of 23
Liuetal. Stress Biology (2024) 4:47
in quinoa leaves co-treated with nitrogen deficiency and
salt stress slightly decreased compared with the nitrogen
deficiency treatment, partially restoring the phenotype of
abnormally enlarged PCs induced by nitrogen deficiency
(Fig. 9E). ese results suggest that salt stress slightly
restores the inhibitory effect on phototrophic growth
caused by nitrogen deficiency. is may be a mechanism
of adaptation to adverse growth conditions such as salin-
ity and nitrogen deficiency which occurred during the
evolution of quinoa.
Construction ofaregulatory network oftranscription
factors ofpigment gland morphogenesis
To elucidate the regulatory mechanisms of EBC devel-
opment, we conducted a comprehensive analysis of the
transcription factors of the different cell clusters. We
searched for transcription factors among all differen-
tially expressed genes and generated a diagram that sys-
tematically classified the transcription factors enriched
in each cell cluster. Two transcription factors were
retrieved in SC-7, but not exclusively expressed in SC-7
(Fig.10A and TableS8). ere are 33 transcription fac-
tors were retrieved in SC-11, of which 13 were specifi-
cally expressed in SC-7 (Fig. 10A and TableS8). ere
are 73 transcription factors were retrieved in pre-SC,
of which one was specifically expressed only in pre-SC
(Fig.10A and TableS8). ere are 142 transcription fac-
tors were retrieved in the EBC, of which 24 were specifi-
cally expressed in the EBC (Fig.10A and TableS8). We
used STRING (https:// cn. string- db. org/) to predict the
interactions between the above transcription factors and
visualize the interaction network via cystoscope (Fig.10B
and TableS9). Based on functional annotations of tran-
scription factors specifically expressed in pre-SC, SC, and
EBC, 6 candidate transcription factors were screened
for possible involvement in salt bladder development
(Fig. 10C). TCP5 directly promotes the transcrip-
tion of KNAT3 and indirectly activates the expression
of SAW1. Earlier studies also showed that TCP5 regu-
latesKNAT3andSAW1in a temporal- and spatial-spe-
cific manner during the formation of serrations (Yu etal.
2021). e YABBY gene family, predominantly expressed
in lateral organs with polarity, is essential for leaves to
initiate as dorsiventral structures and subsequently acti-
vate the developmental pathways crucial for lamina
formation (Sarojam etal. 2010). In particular, the failure
to establish a marginal leaf domain prevents the initia-
tion of CINCINATTA class TCP genes (CIN-TCPs) and
leads to the reactivation of shoot apical meristem (SAM)
specific developmental programs (Sarojam et al. 2010).
Transcription of PI4Kγ5 is relatively high at the early
stage and decreases along with the leaf development,
and is finally restricted at the leaf margin, especially at
the serration tips of mature leaves, which is consistent
with PI4Kγ5 regulating cell division at the leaf margin by
regulating auxin synthesis. It is hypothesized that PI4Kγ5
interacts with membrane-bound ANAC078 to promote
its proteolytic processing, possibly through phosphoryla-
tion, to maintain the normal auxin concentration, and
hence regulate the final leaf shape with weak serrations.
Deficiency of PI4Kγ5 results in the defective interaction
and loss of ANAC078 cleavage, resulting in enhanced
auxin synthesis and promoting cell proliferation at leaf
teeth with highly deep serrations (Tang etal. 2016). e
repression of BRON (BRONTOSAURUS), encoding a
C2H2-like zinc finger transcription factor, lifts its repres-
sion on the cyclins CYCD3;1 and CYCP4;1, leading to
BR-induced cell division (Clark et al. 2021). efoliar
applicationof GA3increased the expression ofGT-3band
salt tolerance (Wang etal. 2020), trichome developmen-
tal selectors GLABRA3(GL3) and GLABRA1(GL1),
encoding basic helix-loop-helix (bHLH) and MYB tran-
scription factors. Several of the GL3/GL1 direct targets
are expressed early during trichome formation, includ-
ing the transcription factors MYC1 (bHLH), and SCL8
(GRAS),associated with trichome formation (Morohashi
& Grotewold 2009). Since the application of NaCl pro-
motes the development of EBCs and nitrogen deficiency
inhibits their development, we examined the expression
of candidate transcription factors under different treat-
ments. e expression of transcription factors TCP5,
YAB5, NAC078, and T1P17.40 was significantly increased
with 150 mM NaCl, and conversely, their expression
was significantly decreased under nitrogen deficiency
(Fig.10D). YABBY family transcription factors promote
the formation of pre-SC while GRAS, NAC, Trihelix, and
C2H2 family transcription factors promote the forma-
tion of EBC. TCP family transcription factors promote
the formation of SC (Fig. 10E). ese results suggest
that transcription factors TCP5, YAB5, NAC078, SCL8,
Fig. 10 Transcription factors regulatory network of EBC development in quinoa. A Venn diagram that systematically categorizes the transcription
factors enriched in each cell cluster. B Transcription factors regulatory network of EBC development inferred from pre-SC, SC, and EBC. C The violin
plot provides a visual representation of the expression patterns for candidate transcription factors. D Gene expression of candidate transcription
factors from pre-SC, SC, and EBCs after nitrogen deficiency and salt stress. E Proposed model of transcription factors for the regulation of EBC
development
(See figure on next page.)
Page 17 of 23
Liuetal. Stress Biology (2024) 4:47
Fig. 10 (See legend on previous page.)
Page 18 of 23
Liuetal. Stress Biology (2024) 4:47
GT-3B, and T1P17.40 may play an important role in the
regulation of EBC development.
Discussion
Single‑cell analysis ofsalt bladder development inquinoa
e differentiation of salt bladders is irreversible and
accompanied by dramatic changes in organelles and cel-
lular morphology. Using SEM and TEM, we observed
dynamic changes in the shape of intracellular subcel-
lular organelles during salt bladder development. Using
single-cell transcriptomic techniques, we could study the
interplay of quinoa salt bladder-specific developmental
regulatory programs and the impact of external envi-
ronmental factors on the fate decisions of different cell
types from a single-cell perspective. e combination of
marker genes identified in past studies and GO analysis
enable us to reliably classify and define cell types. Fur-
thermore, a series of novel marker genes were identified
across various cell types. Additionally, we investigated
the impact of minerals and NaCl on the development of
salt bladder cells by examining their development pat-
terns in quinoa leaves under conditions of nutrient defi-
ciency and salt treatment.
By SEM of true leaves of quinoa seedlings (Fig.1), we
observed that the development of salt bladders undergoes
a special differentiation process, which leads to the grad-
ual expansion of their apical vesicles and the accumula-
tion of salt. We used transmission electron microscopy to
observe salt bladders at different stages of development
(Fig.2) and found that during salt bladder development,
the primary epidermal cells undergo pendulous pericy-
cle divisions to form SCs and EBCs. e EBCs, through
plasmodesmata, continuously absorb vesicles and trans-
port vesicles from the periphery of the epidermal cells,
so that their vesicles increase in number continuously,
and eventually almost fill up the whole salt bladder cells.
As a result of this process, organelles such as Golgi and
mitochondria are squeezed to the edge of the salt blad-
der cells. e nucleus in the stalk cells, together with
the surrounding endoplasmic reticulum and Golgi, pro-
motes the transport of substances in the epidermal cells
and salt bladders. e epidermal cells are dense, with a
large number of chloroplasts, mitochondria, endoplasmic
reticulum, and vesicles, which provide the energy and
carriers for the transport of substances.
e development of salt bladder cells in quinoa is
influenced by various critical factors including salinity,
temperature, and metabolites (Xie et al. 2022). Previ-
ous studies, mostly focused on the whole plant, could
not clearly distinguish the specific functions of these
factors in different cell types. For example, low salinity
promotes quinoa growth whereas high salinity inhibits
quinoa growth, suggesting that quinoa has a very efficient
osmoregulatory system to adapt to sudden increases
in salt stress (Hariadi etal. 2011). Utilizing DEGs from
distinct cell clusters combined with GO analysis, we
identified genes potentially involved in regulating the
development of salt bladder cells. For example, GO heat-
map analysis showed that genes preferentially expressed
in SC and EBC were mainly involved in S-adenosylme-
thionine metabolic processes, copper ion binding, and
oxidative phosphorylation (Fig. 4A). Notably, genes
highly expressed in SC were involved in the formation
of cytoplasmic ribosomes. Stalk cells lack vesicles, but
are dense with an abundant endoplasmic reticulum and
Golgi apparatus to provide carriers for material transport
(Otterbach etal. 2021). is suggests that genes expressed
in SCs may be involved in protein synthesis during sol-
ute transport. GO heatmap analysis also showed that
MPC_1, MPC_2, and MPC_5 participated in relatively
similar GO processes (Fig.4A). Primarily, these processes
involve precursor metabolite synthesis and energy pro-
duction, which implies an intricate interplay among these
cell types regarding gene expression and their respective
cellular functions. e analysis of transcription factor
interactions within specific cell types is crucial for under-
standing the regulatory mechanisms that govern cellu-
lar differentiation and function. Analysis of interactions
of transcription factors specifically expressed in pre-
SC, SC, and EBCs suggests that TCP5, YAB5, NAC078,
SCL8, GT-3B, and T1P17.40 may play important roles in
the developmental regulation of EBCs (Fig.10E). TCP5,
a member of the TCP family of transcription factors, is
known for its role in regulating gene expression related
to cell cycle progression and organ development (Yu
et al. 2021). During petal development, the process of
cell division operates as a built-in timing mechanism that
propels the transition from cell division to cell enlarge-
ment. And transcription factor TCP5 is central to regu-
lating this transition (Huang & Irish 2015). TCP5, a key
regulatory factor in plant developmental processes,
potentially plays a crucial role in modulating the devel-
opment of stalk cells in quinoa’s salt bladders (Huang &
Irish 2024). Interestingly, within the context of stalk cells,
the presence of TCP5 potentially curbs additional cell
division and expansion. is implies that TCP5’s expres-
sion could act as a developmental switch, transitioning
cells from a phase of growth to one of maturation. e
precise regulation of TCP5 is thus likely pivotal for the
proper development of both petals and stalk cells, high-
lighting the complexity of gene expression patterns in
orchestrating plant organogenesis. eYABBYgene fam-
ily plays a crucial role in plant development, particularly
in the specification of abaxial cell fate.YABBY genes play
a pivotal role in the regulation of plant development by
repressing the expression of knotted1-like homeobox
Page 19 of 23
Liuetal. Stress Biology (2024) 4:47
(KNOX) homeobox genes, which in turn limits mer-
istem formation and maintains distinct growth zones
within the plant’s structural organization (Kumaran etal.
2002). A recent study reveals the molecular mechanism
by which KNOX1 regulates node-internode morpho-
genesis in stems through antagonistic and synergistic
interactions with the YABBY gene (Tsuda etal. 2024).
YAB5, a member of the YABBY family, is implicated in
the regulation of cell polarity and differentiation which
are critical for the establishment of cell identity and tis-
sue patterning (Sarojam etal. 2010). e overexpression
of YABBY1 in both tobacco and Arabidopsis plants led
to a marked increase in trichome density on the epider-
mis, indicating that AaYABBY1 may play a crucial role in
trichome development and regulation of Artemisia argyi
(Cui etal. 2024). NAC078, a member of the NAC family
of transcription factors, is associated with various devel-
opmental processes, including the regulation of cell dif-
ferentiation and the response to environmental stimuli
(Tang etal. 2016). GT-3B is involved in the modulation of
gene expression related to cell growth and differentiation
(Wang etal. 2020). SCL8 is essential for the maintenance
of stalk cell identity and the regulation of asymmetric
cell division (Morohashi & Grotewold 2009). Our find-
ings underscore the significance of these transcription
factors in the developmental trajectory of EBCs. Further
research is warranted to elucidate the precise molecular
mechanisms by which these factors interact and to deter-
mine their individual and collective contributions to the
regulation of cell fate decisions. is knowledge will be
instrumental in advancing our understanding of plant
development and may have implications for the manip-
ulation of plant growth and regeneration processes for
agricultural and biotechnological applications.
Salt stress promotes thedevelopment ofsalt bladder
andPC inquinoa
After the salt bladder matures or is stimulated by exter-
nal forces such as wind or rain, the bladder -like cells
rupture, excreting salt out of the body, thus reducing the
damage of salt to plants (Shabala etal. 2014). Mesembry-
anthemum crystallinum L. is a model plant for the study
of salt bladders. It has been shown that Mesembryanthe-
mum crystallinum L. increases significantly the Na+ con-
centration in its salt bladders after treatment with NaCl
(Adams etal. 2005). Treatment of mutants lacking salt
bladders with 400 mM NaCl for two weeks resulted in a
decrease in Na+ and Cl− content within the aboveground
tissues and their salt tolerance, demonstrating that salt
bladders accumulate excess salt in the body and help to
improve its salt tolerance (Agarie etal. 2007). Our results
show that the number of salt bladders of quinoa increases
after treatment with 150 mM NaCl, and that quinoa
grows better than under normal conditions (Fig. 7A).
In addition, we found that the number of PCs of quinoa
increases under salt stress. ese results suggest that salt
treatment promotes the development of salt bladders,
which in turn enhances salt-secretion and salt-tolerance
of quinoa and ultimately improves the growth of quinoa
under salt stress. EBCs on the leaf surface of Mesembry-
anthemum crystallinum L. contribute to plant growth
by acting as reservoirs and favor salt tolerance by main-
taining ionic segregation and stability within active tis-
sues (Agarie etal. 2007). Similarly, it has been shown that
EBCs are also present on the surface of quinoa, which
increases the seed’s capacity to swell to complete cell
division and differentiation under low salt stress (Böhm
et al. 2018). ese results suggest that the presence of
EBCs in quinoa increases growth and development and
epidermal cell number under salt stress as compared to
seedlings grown under control conditions. By subjecting
quinoa seedlings to low salt treatment and nutrient dep-
rivation, we found that the low concentration of salt may
act as a driving force to promote quinoa development
and salt bladder formation (Fig.7B-C).
Nitrogen regulates salt bladder andPC development
inquinoa
Nitrogen is an essential element in plant growth and
development, and nitrogen can increase crop yield and
improve crop quality. It has been shown that nitrogen
supply strongly affects the relationship between photo-
synthesis and yield, with higher yields in high-nitrogen
than in low-nitrogen grown plants (Bascuñán-Godoy
etal. 2018). GO analysis of DEGs in different cell types
showed that genes expressed in salt bladders are involved
in nitrogen and sulfur metabolism (Fig.4A). Under sulfur
deprivation, the growth of quinoa was inhibited. How-
ever, the development of salt bladders was significantly
promoted (Fig.8A). Under conditions of nitrogen defi-
ciency, both the growth of quinoa leaves and the develop-
ment of salt bladders were significantly inhibited (Fig.8A,
C). Notably, significant changes in quinoa leaf develop-
ment occurred under nitrogen deprivation, with a signifi-
cant decrease in the number of salt bladders on the leaf
surface and a significant increase in PC size (Fig.8C). We
found that the expression of ATML1 and PDF2 homologs,
which affect PC development, was significantly reduced,
consistent with the results of abnormal PC development
(Fig.8F). Further studies on salt stress treatment of qui-
noa seedlings under nitrogen deprivation showed that
although NaCl treatment stimulates the development of
salt bladders under these conditions, it did not improve
the growth of quinoa (Fig.9A). Nitrogen deficiency is a
crucial factor affecting plant development and response
to various stresses. During later stages of leaf growth,
Page 20 of 23
Liuetal. Stress Biology (2024) 4:47
the density of glandular trichomes may become more
nitrogen-limited (Bilkova etal. 2016). is nitrogen limi-
tation can directly impair the production and function-
ality of these protective structures, weakening the plant’s
overall stress response capabilities. In the case of quinoa,
nitrogen is essential for its growth and the proper devel-
opment of its distinctive salt bladders. ese epidermal
structures are instrumental in managing salt stress, and
their development is significantly influenced by the lev-
els of nitrogen present in the soil. is study suggests that
while salt treatments can initiate the growth of salt blad-
ders, their enhancement is dependent on the presence of
sufficient nitrogen (Fig.8A). Inadequate nitrogen levels
may hinder the development of these bladders. Moreo-
ver, nitrogen deficiency can impact the expression of
genes (Corteggiani Carpinelli etal. 2014) and regulatory
pathways that dictate the development of salt bladders.
ese results suggest that nitrogen is very important for
quinoa growth and that salt treatment can only enhance
the growth of quinoa by promoting the development of
salt bladder bladders if sufficient nitrogen is available.
Materials andmethods
Plant material andgrowth conditions
Plant material was selected from fully matured and
healthy quinoa seeds. Initially, the seeds were rinsed with
water to eliminate impurities, followed by a 3-min soak
in a 0.5% sodium hypochlorite solution for sterilization.
Subsequently, the seeds were rinsed with distilled water
to remove any remaining sodium hypochlorite. e qui-
noa seeds were sown in pots with a diameter of 15 cm,
each filled with vermiculite. Ten seeds were planted per
pot, and after germination, four uniformly growing plants
were retained per pot, with aberrantly small or large
plants being removed. e incubation conditions were
maintained at 25°C with a photoperiod of 16 h of light
and 8 h of darkness. Each treatment consisted of at least
four pots, with four plants per pot. e control group was
irrigated with 1/2 MS medium, while the salt treatment
group received 1/2 MS medium supplemented with 150
mM NaCl. e nitrogen deprivation group was watered
with 1/2 MS medium devoid of nitrogen, and the sulfur
deficiency group was watered with 1/2 MS medium lack-
ing sulfur. Each experiment was replicated three times to
ensure accuracy and reliability.
cDNA synthesis andRT‑qPCR tomeasure relative gene
expression
RNA was extracted from 0.5 mg of quinoa leaves using
FastPure Plant Total RNA Isolation Kit (Vazyme,
China) 0.1 μg of RNA was taken and synthesized using
a 4 × gDNA wiper in the HiScript II 1st Strand cDNA
Synthesis Kit (Vazyme) before the reverse transcription
step to remove genomic contamination. Anchored Oligo
(dT) 23 VN was designed for binding site anchoring to
synthesize full-length cDNA for cloning and performing
RT-qPCR (Reverse transcription quantitative PCR). Spe-
cific primers were designed based on the cDNA sequence
information of quinoa in the GenBank nucleic acid
sequence database (TableS10). RT-qPCR was performed
on a qTower real-time PCR System (Analytik Jena, Ger-
many) and the enzyme reagent was NovoStart®SYBR
qPCR SuperMix plus (novoprotein, China). e PCR sys-
tem was configured and run according to the instructions
provided by the manufacturer, and relative gene expres-
sion was calculated using the 2-ΔΔct method.
Preparation ofprotoplasts
e true leaves of two-week-old quinoa seedlings were
cut into strips that were 1–2mm in length with a razor
blade and immersed in a freshly prepared enzymatic
solution (refer to the system in the pre-publication arti-
cle from our lab (Liu etal. 2020)) and subjected to a vac-
uum for 10 min. e material was placed in the dark and
shaken at 40 rpm on a thermostatic shaker at 28°C for 2
h. Isolated protoplasts were washed three times using 8%
mannitol buffer and then filtered through a 40 μm cell
sieve. e activity of the cells was detected using Taipan
blue staining and the cell concentration was counted with
a hematocrit plate.
cDNA library construction andsequencing
e protoplast suspension concentration was adjusted
to 1000 / µL and GEMs were generated according to the
10 × Chromium Single-Cell 3 ‘GEM (Gel Beads in-Emul-
sion) Library & Gel Bead Kit v3.1 instructions. GEMs
wereconducted with reverse transcription for 45 min at
53°C and 5 min at 85°C to extract cDNA with a 10 × bar-
code and UMI information. High-quality cDNA librar-
ies were chosen for high-throughput sequencing on the
Illumina Nova 6000 Pe15 platform after the cDNA was
amplified and examined.
Single‑cell transcriptome data analysis
Raw reads were compared to the quinoa reference
genome using Cell Ranger 3.0.0, the official software of
10 × genomics, to obtain quality control results such as
high-quality cell counts, gene counts, and genome com-
parison rates of the raw data. e initial quality control
results from Cell Ranger analyses were rigorously re-
evaluated using the Seurat R package to meticulously
exclude data associated with multiple cells, doublets,
or unbound cells. eoretically, the number of genes
(nGene), the number of UMIs (nUMI), and the propor-
tion of mitochondrial genes (percent_mito) expressed in
most of the cells will be concentrated in a certain region,
Page 21 of 23
Liuetal. Stress Biology (2024) 4:47
and based on their distribution characteristics a distribu-
tion model can be fitted, which can be used to find out-
of-domain values in them and to exclude abnormal data.
e quality control criterion was to retain cells with gene
counts and UMI counts within ± 2 times the standard
deviation of the mean and with mitochondrial gene ratios
below 100% as high-quality cells for downstream analy-
sis. Based on the dimensionality reduction results of PCA
(Principal Components Analysis), cell cluster clustering
was visualized by UMAP (Uniform Manifold Approxima-
tion and Projection), and the clustering algorithm SNN
(Shared nearest neighbor). e differences between the
specified cell clusters and all the remaining cell clusters
were tested using the bimodal test to filter the specific
marker genes for each cell cluster. e differential gene
expression was determined by Wilcoxon rank-sum test
available in Seurat, alongside the Benjamini–Hochberg
method to correct for multiple testing. Genes with an
adjusted p-value below the threshold of 0.05 were identi-
fied as marker genes. ese markers were subsequently
sorted by their log-fold change in expression specific to
the cluster of interest, as opposed to their expression in
other clusters. DEGs were identified by applying strin-
gent criteria: a p-value threshold of less than 0.05 and a
fold-change magnitude of at least 2.
Pseudotime analysis
Using the monocle 2 R package (Qiu et al. 2017), we
screened out the genes with a large degree of variation in
gene expression between cells performed spatial dimen-
sionality reduction based on their expression profiles, and
constructed a minimum spanning tree (MST). Accord-
ing to the MST branches, the longest paths were found
to represent the differentiation trajectories of cells with
similar transcriptional profiles. Firstly, extract the data
from the Seurat object and create the CellDataSet(CDS)
using the newCellDataSet function. e size factor and
dispersion were assessed by estimateSizeFactors and esti-
mateDispersions, which helped us standardize mRNA
differences between cells and conduct subsequent dif-
ference analysis. e genes were filtered by setting the
parameters min_expr = 0.1 and num_cells_expressed ≥ 10
to obtain the genes needed for subsequent operations.
Seurat was used to select highly variable genes, and
setOrderingFilter was used to embed genes into CDS
object. e reduceDimension (CDS, max_components 2,
method ‘DDRTree’) was used to reduce the dimension of
the data.
Drawing ofTF network diagram
To identify differentially expressed TFs specifically
enriched in pre-stalk cells (pre-SC), stalk cells (SC),
and epidermal bladder cells (EBC), we conducted a
comprehensive screening analysis. We performed a pro-
tein–protein interaction (PPI) network analysis using the
STRING database (version 11.0, RRID: SCR_005223).
e outcome of the PPI analysis was downloaded from
the STRING database in tab-separated values (TSV)
format. Finally, the visualization of the protein–protein
interaction network was achieved using Cytoscape (ver-
sion 3.4.0, RRID: SCR_003032).
Abbreviations
DEGs Differentially expressed genes
EBC Epidermal bladder cells
GC Guard cell
GO Gene Ontology
KEGG Kyoto Encyclopedia of Genes and Genomes
LN Low nitrogen
LS Low sulfur
MST Minimum spanning tree
PCA Principal Components Analysis
pre-SC pre-stalk cells
RNA-seq RNA sequencing
ROS Reactive oxygen species
RT-qPCR Reverse transcription quantitative PCR
SC Stalk cells
scRNA-seq single cell RNA sequencing
SEM Scanning electron microscopy
SNN Shared nearest neighbor
TEM Transmission electron microscopy
TF Transcription factor
UMAP Uniform Manifold Approximation and Projection
UMI Unique molecular identifiers
UV Ultraviolet
Supplementary Information
The online version contains supplementary material available at https:// doi.
org/ 10. 1007/ s44154- 024- 00189-3.
Additional file 1: Fig. S1. Observation of plasmodesmata between initial
bladder cells and neighboring epidermal cells by transmission electron
microscopy. Fig. S2. Observation and analysis of the ultrastructure of
mature salt bladders and adjacent epidermal cells. Fig. S3. Observation
and analysis of the ultrastructure of mature stalk cells. Fig. S4. Top 57 GO
terms of all clusters and expression features of L0C110682269. Fig. S5. New
marker genes of MMC, pre-SC, SC, and EBC for feature map presentation.
Additional file 2: Table S1. Sequencing summary statistics showing the
total number of cells before and after trimming and quality filtering.
Table S2. All differentially expressed genes in each cell cluster. Table S3.
Top 10 differentially expressed genes in each cell cluster. Table S4. The GO
analysis of all differentially expressed genes in each cell cluster. Table S5.
The KEGG analysis of all differentially expressed genes in each cell cluster.
Table S6. GO enrichment analysis of the genes with highly variable
expression of the three identified gene modules. Table S7. GO enrichment
analysis of the genes with highly variable expression of the five identified
gene modules. Table S8. Differently expressed TFs in each cluster. Table S9.
String protein–protein interaction network analysis data. Table S10. The
primers for RT-qPCR.
Acknowledgements
We are deeply grateful to Prof. Jean-David Rochaix for his insightful comments
and invaluable feedback during the preparation of this manuscript.
Authors’ contributions
Conceptualization of the project: X.S. and Z.L. Experimental design: X.S. and H.L.
Performance of experiments: Y.Z., Y.L., C.L, P.G., Q.Z., X.S., M.L., L.K., Y.X., L.Y., E.G. and Data
analysis: H.L. and Y.L. M anuscript drafting: H.L. and Y.L Manuscript revise: X.S., and A.Q.
All authors have read and agreed with the published version of the manuscript.
Page 22 of 23
Liuetal. Stress Biology (2024) 4:47
Funding
This research was supported by the National Key Research and Development
Program of China (No.2022YFD1200300).
Data availability
The raw sequence data reported in this paper have been deposited in the
Genome Sequence Archive (Genomics, Proteomics & Bioinformatics 2021)
in National Genomics Data Center (Nucleic Acids Res 2022), China National
Center for Bioinformation / Beijing Institute of Genomics, Chinese Academy of
Sciences (GSA: CRA016880) that are publicly accessible at https:// ngdc. cncb.
ac. cn/ gsa.
Declarations
Ethics approval and consent to participate
All authors consent to participate.
Consent for publication
All authors consent for publication.
Competing interests
The authors declare that they have no competing interests.
Received: 18 July 2024 Accepted: 3 September 2024
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