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Phytosulfokine promotes fruit ripening and quality
via phosphorylation of transcription factor DREB2F
in tomato
Hanmo Fang ,
1,†
Jinhua Zuo ,
2,†
Qiaomei Ma ,
1
Xuanbo Zhang ,
1
Yuanrui Xu,
1
Shuting Ding ,
1
Jiao Wang ,
1
Qian Luo,
1
Yimei Li,
1
Changqi Wu ,
1
Jianrong Lv ,
1
Jingquan Yu
1
and Kai Shi
1,
*
1 Department of Horticulture, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou 310058, China
2 Institute of Agro-Products Processing and Food Nutrition, Beijing Academy of Agriculture and Forestry Sciences, Beijing 100097, China
*Author for correspondence: kaishi@zju.edu.cn
†
These authors are co-first authors.
The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the
Instructions for Authors (https://academic.oup.com/plphys/pages/General-Instructions) is Kai Shi.
Abstract
Phytosulfokine (PSK), a plant peptide hormone with a wide range of biological functions, is recognized by its receptor
PHYTOSULFOKINE RECEPTOR 1 (PSKR1). Previous studies have reported that PSK plays important roles in plant growth, de-
velopment, and stress responses. However, the involvement of PSK in fruit development and quality formation remains largely
unknown. Here, using tomato (Solanum lycopersicum) as a research model, we show that exogenous application of PSK pro-
motes the initiation of fruit ripening and quality formation, while these processes are delayed in pskr1 mutant fruits.
Transcriptomic profiling revealed that molecular events and metabolic pathways associated with fruit ripening and quality
formation are affected in pskr1 mutant lines and transcription factors are involved in PSKR1-mediated ripening. Yeast screening
further identified that DEHYDRATION-RESPONSIVE ELEMENT BINDING PROTEIN 2F (DREB2F) interacts with PSKR1.
Silencing of DREB2F delayed the initiation of fruit ripening and inhibited the promoting effect of PSK on fruit ripening.
Moreover, the interaction between PSKR1 and DREB2F led to phosphorylation of DREB2F. PSK improved the efficiency of
DREB2F phosphorylation by PSKR1 at the tyrosine-30 site, and the phosphorylation of this site increased the transcription level
of potential target genes related to the ripening process and functioned in promoting fruit ripening and quality formation.
These findings shed light on the involvement of PSK and its downstream signaling molecule DREB2F in controlling climacteric
fruit ripening, offering insights into the regulatory mechanisms governing ripening processes in fleshy fruits.
Research article
Received September 28, 2023. Accepted December 16, 2023. Advance access publication January 12, 2024
© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com
Introduction
Fleshy fruits serve as an important nutritional source for hu-
mans and animals, as they contain abundant beneficial nutri-
ents, including carbohydrates, lipids, proteins, vitamins, and
minerals (Seymour et al. 2013). Fruit ripening exerts a profound
influence on the ultimate quality and economic value of the
fruit. This complex, dynamic, and programmed process encom-
passes a series of physiological and biochemical changes, result-
ing in the development of characteristic color, texture, flavor,
and aroma (Giovannoni et al. 2017). Comprehensive insights
into the mechanisms of fruit ripening constitute a vital theor-
etical foundation and practical tools for controlling ripening
processes, enhancing fruit quality traits, and extending posthar-
vest shelf life.
The initiation and coordination of fruit ripening are tightly
regulated by multiple phytohormones (Kumar et al. 2014; Li
et al. 2021). Ethylene promotes fruit ripening and is essential
for the ripening of climacteric fruits, which undergo a burst
of respiration and ethylene production during ripening.
(Fenn and Giovannoni 2021). Other hormones such as auxin,
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abscisic acid (ABA), jasmonic acid, and brassinosteroids (BR)
also influence fruit ripening (Zhu et al. 2015; Mou et al. 2016;
Li et al. 2017b; Yue et al. 2020). Plant hormones usually work
with downstream transcription factors to regulate the
expression of ripening-related genes, which further coordi-
nates the synthesis and accumulation of pigments, metabol-
ism of sugars, evolution of aroma volatiles, and changes
to cell wall structure, ultimately influencing fruit ripening
and quality. For example, ethylene inhibits the expression
of ETHYLENE RESPONSE FACTOR. F12 (ERF. F12),
preventing ERF.F12 from suppressing the transcription of
POLYGALACTURONASE 2a (PG2A) and PECTATE LYASE
(PL), the genes related to fruit softening (Deng et al. 2022).
BR promotes the expression of the key component of BR sig-
nal transduction, BRASSINAZOLE RESISTANT1 (BZR1), and
BZR1 can directly transcribe and activate PHYTOENE
SYNTHASE1 (PSY1) to promote carotenoid synthesis (Sang
et al. 2022). Recently, however, it has become very clear
that peptide hormones, derived from precursors with amino
acid backbones, have emerged as greater than anticipated vi-
tal signaling molecules in plants (Wang et al. 2015). Despite
extensive research on the functions of traditional hormones
in fruit ripening, the contribution of plant peptide hormones
to this process was largely unknown.
Phytosulfokine (PSK) is a disulfated pentapeptide
[Tyr(SO
3
H)-Ile-Tyr(SO
3
H)-Thr-Gln] that is produced from
precursors of ∼80 to 110 amino acids encoded by the PSK
gene family, which appears to be ubiquitous in angiosperms
(Sauter 2015). PSK has a regulatory role in fruit development
and quality formation during cold storage. The application of
PSK delays senescence and improves the accumulation of
antioxidant nutrients such as melatonin, phenols, flavonoids,
and anthocyanins in strawberry (Fragaria × ananassa)
fruit during storage at 4 °C (Aghdam et al. 2021). The
PSK1-treated loquat (Eriobotrya japonica) fruit exhibited sig-
nificantly lower activities of cell wall–degrading enzymes and
a delayed increase of firmness under refrigerated storage con-
ditions. PSK increases levels of polyamine, proline, and
γ-aminobutyric acid, substances that are beneficial to human
health, to alleviate cold-induced browning of banana
(Musa sp.) (Wang et al. 2022). The molecular mechanisms
underlying the regulatory roles of PSK in biological processes,
such as root growth, drought-induced flower abscission, and
disease resistance, have been clarified to some extent
through genetic studies (Yamakawa et al. 1998; Mosher
et al. 2013; Han et al. 2014; Stuhrwohldt et al. 2015;
Reichardt et al. 2020; Ding et al. 2023). In contrast, compar-
able research on the involvement of PSK in regulating fruit
ripening and quality formation has lagged far behind.
The perceptions of PSK by its receptors and subsequent
signal transduction are essential prerequisites for PSK to elicit
downstream molecular and physiological responses. PSK is
perceived by 2 membrane-bound PSK receptors (PSKRs),
PSKR1 and PSKR2, in Arabidopsis (Arabidopsis thaliana)
and tomato (Solanum lycopersicum), classified as leucine-rich
repeat receptor-like kinases (LRR-RLKs) (Matsubayashi et al.
2002; Kutschmar et al. 2009; Zhang et al. 2018). The recogni-
tion primarily occurs through PSKR1, which plays a major
role in PSK-mediated root growth in Arabidopsis and resist-
ance to Botrytis cinerea in tomato, while PSKR2 acts as an al-
ternative receptor with lower PSK binding affinity (Amano
et al. 2007; Zhang et al. 2018). Furthermore, transcription fac-
tors are involved in PSK signal transduction and modulate
the expression of downstream components. In loquat,
PSK1 plays an inhibitory role in lignin biosynthesis by repres-
sing the expression of MYB DOMAIN PROTEIN 1 (MYB1) and,
its target gene, 4-COUMARATE COENZYME LIGASE (4CL), the
key lignin biosynthetic genes (Xu et al. 2014; Song et al. 2017).
The C-REPEAT BINDING FACTOR 6 (CBF6) participates in
PSK-retarded cold-induced internal browning by directly
suppressing the expression of LIPOXYGENASE 5 (LOX5) in
peach (Prunus persica L.) fruit. Additionally, a microarray ap-
proach has been utilized to identify multiple PSK-responsive
genes in Arabidopsis. It was found that PSK signals nonhair
cell fate by inducing the expression of WEREWOLF (WER),
its paralog MYB DOMAIN PROTEIN 23 (MYB23), and
At1g66800, which participate in the control of root hair for-
mation (Lee and Schiefelbein 2001; Kaufmann et al. 2021).
Therefore, we hypothesize that transcription factors may
serve as an integral component in PSK signaling across di-
verse physiological processes, including fruit ripening, which
is well worth investigating.
Tomato is known as one of the foremost vegetable crops
globally, possessing substantial commercial value. Tomato
also serves as a research model for climacteric fruits due
to its availability of genome sequences and amenability to
genetic modification. Thus, tomato was selected as the
model system to investigate PSK-mediated fruit ripening.
Here, we demonstrated that the PSK-PSKR1 signaling
positively regulates the onset of fruit ripening and the tran-
scription factor DEHYDRATION-RESPONSIVE ELEMENT
BINDING PROTEIN 2F (DREB2F) is an important compo-
nent in this process. PSKR1 phosphorylates DREB2F, and
this phosphorylation is involved in PSK-mediated ripening.
This study reveals the role and mode of PSK and its down-
stream components in fruit ripening and sheds light on the
regulatory mechanism of climacteric fruit ripening and
quality formation.
Results
PSK promotes the initiation of ripening and
carotenoid accumulation of tomato fruits
To explore the putative role of PSK in tomato fruits, the
spatiotemporal expression patterns of genes related to PSK
biosynthesis were assessed by qPCR. The PSK precursor genes
displayed differential expression across the tissues tested,
including roots, leaves, flowers, and fruits, indicating tissue-
specific roles of PSKs (PSK1, PSK2, PSK3, PSK3L, PSK4, PSK5,
PSK6, PSK7) (Fig. 1A). Notably, PSK5 exhibited extremely
high expression levels in fruits and was increasingly expressed
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during fruit growth, with a peak expression at the breaker
(Br) stage (Fig. 1A). The expression pattern suggests en-
hanced PSK signaling is involved in the unripe-to-ripe phase
transition of fruit. Exogenous PSK was used to treat mature
green fruits, and color transition, hardness, and ethylene pro-
duction of fruits were measured. The results showed PSK
treatment accelerated color transition, with PSK-treated
fruits turning orange 3 d posttreatment (DPT) while control
fruits remained yellow (Fig. 1, B and C). Consistent with the
color transition of fruit pericarp in advance, PSK-treated
fruits also displayed an earlier softening and increase in
ethylene production (Fig. 1, D and E), indicating PSK accel-
erates the onset of ripening. Since the accumulation of car-
otenoids is not only a hallmark of fruit ripening but also
impacts nutritional composition of fruits, major carotenoids
(lycopene, β-carotene, lutein) in tomato were examined at 3
d intervals. Lycopene and β-carotene levels were higher in
the PSK-treated fruits compared with the control fruits
from 3 to 9 d after treatment (Fig. 1, F and G), and lutein
was only elevated at 9 d after treatment (Fig. 1H). These
Figure 1. PSK promotes the transition to fruit ripening in tomato. A) Relative PSKs transcript levels in different tissues, as assessed by qPCR. DPA,
d postanthesis; MG, mature green; Br, breaker; Br + 5, 5 d after the Br stage. B) Effect of exogenous PSK treatment on ripening progression of tomato
fruit. WT tomato fruits were picked at mature green stage and treated. Fruits in horizontal rows are biological replicates. DPT, d posttreatment. Bar,
2 cm. C) to E) Effect of exogenous PSK treatment on color (C), firmness (D), and ethylene production (E) of tomato fruit at different ripening stages.
The different colored panels in D) and E) signify the same treatments as indicated in C). F) to H) Effect of exogenous PSK treatment on accumulation
of lycopene (F), β-carotene (G), and lutein (H) in tomato fruit at different ripening stages. The different colored panels in G) and H) signify the same
treatments as indicated in F). Data are presented in A), C) to E), and F) to H) as the means of 3 biological replicates (±SD, n = 3), and asterisks
indicate statistical significance using Tukey's t test, P < 0.05.
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data demonstrate that PSK promotes the transition to
ripening and causes a corresponding change in quality
attributes.
The onset of fruit ripening is delayed in tomato
pskr1 mutants
PSKRs are indispensable in mediating the effects of PSK. To
explore the roles of PSKRs in fruit ripening, the expression
patterns of PSKR1 and PSKR2 were examined in different or-
gans, including roots, leaves, flowers, and fruits, by qPCR
(Supplementary Fig. S1). PSKR1 and PSKR2 were ubiquitously
expressed in all assayed tissues. Critically, the transcript levels
of PSKR1 were higher than PSKR2 in each tissue tested. The
expression profile of PSKR1 in fruits closely mirrored that
of PSK5, with both peaking at the Br stage, while PSKR2 ex-
pression gradually increased over ripening progression. To
further confirm the role of PSKR1 during fruit ripening, we
utilized 2 homozygous lines of PSKR1 knockout mutants
pskr1#1 and pskr1#2 generated previously (Hu et al. 2023).
Remarkably, pskr1 mutants delayed the onset of fruit ripen-
ing (Fig. 2A). Both pskr1 mutants reached the Br stage 2 to 3
d later than the wild type (WT) (Fig. 2B). Moreover, pskr1
fruits exhibited later color transition, softening, and ethylene
emission (Fig. 2, C to E). The levels of lycopene and
β-carotene in pskr1 mutant fruits were lower than those in
WT from 45 to 49 d postanthesis (DPA), with lutein levels
being lower at 49 DPA (Fig. 2, F to H). Consistent with the
delayed ripening in pskr1 mutant lines, key ripening-related
genes were repressed in pskr1 fruits at 45 DPA, such as lyco-
pene biosynthesis genes (PSY1, PHYTOENE DESATURASE
[PDS]), fruit softening genes (EXPANSIN 1 [EXP1], PG2a),
ethylene biosynthetic and responsive genes (1-AMINOCYC
LOPROPANE-1-CARBOXYLATE SYNTHASE 2 [ACS2], 1-AMIN
OCYCLOPROPANE-1-CARBOXYLATE OXIDASE 1 [ACO1],
E4), and ripening regulator genes (RIPENING INHIBITOR
[RIN], FRUITFULL 1 [FUL1], APETALA2a [AP2a]) (Fig. 2,
I to L). Together, these data support that blocking
the endogenous PSK signal pathway delays the initiation
of fruit ripening and affects fruit quality, which is consistent
with the promoting effect of exogenous PSK on fruit
ripening.
Changes in metabolic processes related to fruit
ripening and transcription factors in tomato pskr1
mutants
PSKR1 is essential for PSK to trigger the downstream signal-
ing cascade and elicit the appropriate cellular responses.
To gain a deeper molecular insight into the changes
throughout the ripening process in pskr1 mutants, we
performed RNA-seq analysis of pskr1#1 mutant and WT
fruits harvested at 43, 45, 47, and 49 DPA. First, we examined
coexpressed gene clusters (modules) that showed a high
association with PSKR1-mediated ripening using weighted
gene coexpression network analysis (WGCNA). As a result,
7,730 genes (FPKM ≥ 0.1, coefficient of variation [CV] ≥
0.5) were divided into 9 modules (Fig. 3A). Notably, the
brown module exhibited the clearest positive correlation
with carotenoids and ethylene while showing the most nega-
tive correlation with firmness (Fig. 3A). This suggests that the
genes in the brown module were highly responsive to
PSKR1-mediated ripening. The heat map depicting changes
in fold expression of 293 genes within the brown module in-
dicated a global increase in expression levels during ripening
in the WT fruits, compared with the pskr1 mutant (Fig. 3B,
Supplementary Table S1). Gene Ontology (GO) enrichment
analysis revealed disruption of PSKR1 expression affects bio-
logical processes related to protein–chromophore linkage,
cell wall organization, lipid transport, hormone response,
and primary metabolism during fruit ripening (Fig. 3C,
Supplementary Table S2), demonstrating the molecular
changes underlying PSKR1 regulation of physiological pro-
cesses in fruit ripening from a transcriptome perspective.
Furthermore, given that the PSK-PSKR1 signaling is closely
associated with the onset of ripening, we identified 8,654 and
6,341 genes expressed differentially between 43 and 45 DPA
in WT and pskr1 mutant (fold change ≥ 1.5 and P < 0.05), re-
spectively (Fig. 3D, Supplementary Table S3). Among these
differentially expressed genes (DEGs), 3,828 were upregu-
lated, and 4,826 were downregulated in the WT during fruit
ripening. As for the pskr1 mutant, 2,867 genes were upregu-
lated, and 3,474 genes were downregulated as the fruit
ripened. Excluding the 2,245 upregulated and the 2,830
downregulated genes in both WT and pskr1 mutant, we
then focused on the remaining 4,784 DEGs that exhibited
inconsistent expression trends between WT and pskr1 mu-
tant from 43 to 45 DPA (Supplementary Table S4). Kyoto
Encyclopedia of Genes and Genomes (KEGG) analysis re-
vealed that multiple metabolic pathways, such as carbon,
starch and sucrose, fatty acid, α-linolenic acid, riboflavin,
and nitrogen metabolism, as well as terpenoid backbone,
steroid, and carotenoid biosynthesis, are affected in pskr1
mutant (Fig. 3E, Supplementary Table S5). These data suggest
that PSKR1 participates in the control of the metabolism of
various nutrients in fruits during ripening. Additionally, ex-
pression of genes involved in plant hormone signal transduc-
tion and transcription factors, 2 major regulators of fruit
ripening, was also altered in the pskr1 mutant, indicating
a possible connection between PSKR1, plant hormone, and
transcription factors within the regulatory network that
governs fruit ripening (Fig. 3E).
Transcription factor DREB2F interacts with PSKR1
To further explore the mechanisms of the PSK-PSKR1-
mediated fruit ripening, a yeast 2-hybrid (Y2H) screen was
performed. The cDNA of the inner juxtamembrane domain
and the kinase domain of PSKR1 (referred to as PSKR1JK)
was fused in-frame to the GAL4 DNA-binding domain
(PSKR1JK-BD) and used as the bait to screen a Y2H library
containing tomato fruit cDNAs. Of 123 candidate PSKR1-
interacting proteins (Supplementary Table S6), 1 clone en-
coding transcription factor DREB2F was verified to strongly
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interact with PSKR1 by performing Y2H assay (Fig. 4A).
DREBs belong to the AP2/ERF transcription factor family
and play diverse roles in plant physiology. DREBs are
implicated in the regulation of various attributes of fruit ri-
pening and quality, including firmness, flavor, and aroma
(Nishawy et al. 2015; Kuang et al. 2017; Gupta et al. 2022).
Figure 2. PSKR1 deficiency represses the transition to fruit ripening. A) Different ripening stages in WT and pskr1 mutant lines (pskr1#1 and
pskr1#2). DPA, d postanthesis. Bar, 1 cm. B) Time from anthesis to the breaker stage in WT and pskr1 mutant lines. C) to E) Fruit coloration
(C), firmness (D), and ethylene production (E) in WT and pskr1 mutant fruits at different ripening stages. The different colored panels in D)
and E) signify the same treatments as indicated in C). F) to H) Accumulation of lycopene (F), β-carotene (G), and lutein (H) in WT and pskr1 mutant
fruits at different ripening stages. The different colored panels in G) and H) signify the same treatments as indicated in F). I) to L) Relative expression
of carotenoid synthesis genes (I) PSY1 and PDS; fruit softening-related genes (J) EXP1 and PG2a; ethylene biosynthesis and responsive genes (K) ACS2,
ACO1, and E4; and ripening-related transcription factors (L) RIN, FUL1, and AP2a in WT and pskr1 mutant lines at 45 DPA. Data in B) are presented as
the means ± SD of 10 biological replicates (n = 10). Data in C) to L) are presented as the means ± SD of 3 biological replicates (n = 3). Asterisks in-
dicate statistical significance using Tukey's t test, P < 0.05.
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They are also integral components of multiple phytohor-
mone signaling cascades (Niu et al. 2002; Shi et al. 2012; Li
et al. 2017a; Xie et al. 2019). We thus hypothesized that
DREB2F may be a downstream target of PSKR1 signaling.
To further confirm whether PSKR1 directly interacts with
DREB2F, we performed an in vitro glutathione S-transferase
(GST) pull-down assay. Consistently, MBP-PSKR1JK could
be specifically pulled down by the glutathione beads immo-
bilizing with GST-DREB2F (Fig. 4B). Moreover, the interaction
between PSKR1 and DREB2F in planta was further confirmed
by split-luciferase (LUC) and co-immunoprecipitation
(Co-IP) assays (Fig. 4, C and D).
Figure 3. RNA-seq analysis revealed biological processes and molecular components associated with PSKR1-regulated fruit ripening. A) Module–
trait relationships between modules and carotenoids, firmness, and ethylene production. Each row represents a module eigengene; each column
represents a fruit ripening trait. Each cell contains the corresponding correlation coefficient and P-value. The table is color-coded by correlation
according to the color legend. B) Heat map depicting the transcript levels of the genes in the brown module in WT and pskr1 fruits at different
ripening stages. DPA, d postanthesis. The original transcript levels were subjected to data adjustment by normalization and hierarchical clustering
using an R package. The list of the brown genes is shown in Supplementary Table S1. C) Enriched GO biological process categories of the genes in
the brown module. The detailed list is given in Supplementary Table S2. D) Venn diagram showing the overlap between genes upregulated or down-
regulated at 45 DPA compared with 43 DPA in WT and pskr1 fruits, respectively. The detailed list is given in Supplementary Table S3. E) KEGG
pathway analysis of the genes with an inconsistent expression pattern between WT and pskr1 fruits from 43 to 45 DPA. The detailed list is given
in Supplementary Tables S4 and S5. The stars indicate important KEGG pathways related to the regulation of fruit ripening.
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DREB2F is required for the PSK-PSKR1
signaling-mediated fruit ripening
To determine whether DREB2F participates in PSK-regulated ri-
pening, efficient gene silencing of DREB2F was achieved using a
virus-induced gene silencing (VIGS) approach in WT fruits
(Supplementary Fig. S2) (Fu et al. 2005). The empty TRV
vector was transformed into fruits as a negative control.
Consistent with the phenotype of the pskr1 mutants,
silencing of DREB2F also delayed fruit ripening by 2 to 3 d
(Supplementary Fig. S3). Furthermore, treating detached green
DREB2F-silenced fruits with exogenous PSK revealed that de-
creased DREB2F expression significantly inhibited the promot-
ing effect of PSK on fruit ripening (Fig. 5). Specifically, the onset
of ripening in DREB2F-silenced fruits was still delayed despite
PSK treatment (Fig. 5A). TRV-0 and TRV-DREB2F turned yellow
at 3 and 6 DPT, respectively. When the total carotenoid
contents (including lycopene, β-carotene, and lutein) were
comparable between TRV-0 and TRV-DREB2F, the total carot-
enoid contents in PSK-treated TRV-DREB2F fruits at 6 DPT were
48% lower compared with PSK-treated TRV-0 fruits at 3 DPT
(Fig. 5B). The levels of fruit coloration, firmness, and ethylene
production all exhibited consistent trends (Fig. 5, C to E). To
predict the ripening-related genes regulated by DREB2F, we
used the JASPAR website (https://jaspar.genereg.net/) and iden-
tified E4, E8, EXP1, ZDS, and AP2a as potential target genes of
AtDREB2F, the Arabidopsis homolog of SlDREB2F. qPCR ana-
lysis showed that PSK increased the expression of these poten-
tial target genes, but the inductive effect of PSK was inhibited in
TRV-DREB2F fruits at 3 DPT (Fig. 5F). These results indicate that
DREB2F lies in the PSK signaling pathway and plays an import-
ant role in regulating fruit ripening.
PSK enhances phosphorylation of DREB2F by PSKR1
to promote the transition to fruit ripening
The above findings suggest that PSKR1 and DREB2F pro-
teins cooperate in the control of fruit ripening. PSKR1 is a
receptor kinase possessing both autophosphorylation and
transphosphorylation activities (Kaufmann et al. 2017).
Figure 4. Tomato DREB2F protein interacts with PSKR1. A) Y2H assay. The interactions between the GAL4 DNA activation domain (AD) fusion of
DREB2F and GAL4 DNA-binding domain (BD) fusion of PSKR1JK was detected. The transformed yeast cells were cultured on synthetic dropout
medium lacking Trp and Leu (SD-T-L), followed by further selection on medium lacking Trp-Leu- Ade-His (SD-T-L-A-H). PSKR1JK, consisting of
the inner juxtamembrane domain and the protein kinase domain of PSKR1 protein. B) In vitro pull-down assays. GST or GST- DREB2F fusion pro-
teins coupled with glutathione beads were used to pull down (PD) MBP-PSKR1JK. The input fusion proteins were indicated by immunoblots with
anti-MBP antibody and anti-GST antibody, respectively. Asterisks indicate the specific bands of GST and DREB2F-GST. C) Split-LUC imaging assays.
N. benthamiana leaves were coinfiltrated with Agrobacterium expressing N-LUC and C-LUC fusion proteins. EV and ERF12-cLUC served as negative
controls. Bar, 1 cm. The pseudocolor bar indicates the range of luminescence intensity. D) Co-IP assays. PSKR1-HA was coexpressed with
DREB2F-GFP or GFP in N. benthamiana leaves. The protein was precipitated using GFP-Trap. Both immunoprecipitated and input samples were
subjected to western blotting using an anti-HA or anti-GFP antibody.
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The impairment of kinase activity resulted in a compro-
mised growth-promoting ability of PSKR1 in the root and
shoot of Arabidopsis (Hartmann et al. 2014), indicating
the essential role of PSKR1 kinase activity in PSK signal
transduction. Thus, it is reasonable to hypothesize that
the kinase activity of PSKR1 may be involved in the regula-
tion of fruit by PSK and investigate whether the interaction
between PSKR1 and DREB2F results in the phosphorylation
of DREB2F. To test this hypothesis, PSKR1 and DREB2F were
coexpressed in Nicotiana benthamiana leaves. Since PSKR1
can phosphorylate serine, threonine, and tyrosine residues
(Muleya et al. 2016), immunoblotting analysis in planta was
performed using antibodies that recognizes the phosphory-
lated serine and threonine residues (anti-pSer/pThr) or tyrosine
residues (anti-pTyr) on DREB2F. Interestingly, the results
showed that PSKR1 increased the phosphorylation state of
DREB2F protein at tyrosine residues but not at serine or threo-
nine residues (Fig. 6A). Furthermore, the level of DREB2F phos-
phorylation was further enhanced in the presence of PSK
(Fig. 6A).
To identify the PSKR1-mediated phosphorylation sites on
DREB2F, the phosphorylated recombinant DREB2F, pro-
duced by PSKR1 in vitro, was subjected to liquid chromatog-
raphy–tandem MC (LC–MS/MS). Two sites on DREB2F
protein at tyrosine-30 (Y30) and tyrosine-69 (Y69) were found
to be phosphorylated by PSKR1 (Fig. 6, B and C). Both sites are
located in the AP2 domain (Fig. 6B). Next, we substituted the
tyrosine (Y) residues with nonphosphorylatable phenylalanine
Figure 5. Silencing of DREB2F delays fruit ripening and inhibits the promoting effect of PSK on ripening. A) Effect of exogenous PSK treatment on
ripening progression of TRV-0 and TRV-DREB2F fruits harvested at the mature green stage. CK, control; DPT, d posttreatment. The images were
digitally extracted for comparison. Bar, 2 cm. B) Effect of exogenous PSK treatment on accumulation of carotenoids in TRV-0 and TRV-DREB2F fruits.
Carotenoid content was measured in TRV-0 fruits at 3 DPT and in TRV-DREB2F fruits at 6 DPT. C) to E) Effect of exogenous PSK treatment on color
(C), firmness (D), and ethylene production (E) of TRV-0 and TRV-DREB2F fruits at different ripening stages. F) Relative expression of potential target
genes of DREB2F in TRV-0 and TRV-DREB2F fruits at 3 DPT. Data in B) to F) are presented as the means ± SD of 3 biological replicates (n = 3), and
different letters indicate significant differences (P < 0.05) according to Tukey's test.
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Figure 6. PSK enhances PSKR1-mediated phosphorylation of DREB2F at the Y30 residue, and phosphorylation of DREB2F at Y30 functions in the
initiation of fruit ripening. A) PSK promotes DREB2F phosphorylation by PSKR1 in vivo. DREB2F-GFP and PSKR1-HA (EV as a negative control) were
transiently coexpressed in N. benthamiana. After 48 h, leaves were treated with 10 μM PSK or water control, and samples were collected 2 h later.
Anti-pSer/pThr or anti-pTyr antibody was used to detect DREB2F phosphorylation after immunoprecipitation with anti-GFP agarose beads.
B) Schematic diagram showing the phosphorylation sites of DREB2F identified by MS. The blue bar indicates the AP2 domain of DREB2F. C) In vitro
mass spectrometric analysis of DREB2F phosphorylation site by PSKR1. Phosphorylation of DREB2F by PSKR1 at the Y30 site (upper panel) and Y69
site (lower panel). Phosphorylated tyrosine residues in DREB2F are highlighted in red. D) Ripening phenotype produced by transient overexpression
of DREB2F, DREB2F
Y30F
, and DREB2F
Y69F
in WT fruits. The photograph was taken 5 d after Agrobacterium-mediated infiltration. EV-GFP was used as
negative control. The images were digitally extracted for comparison. Bar, 1 cm. E) Relative transcript levels of DREB2F, E4, E8, EXP1, ZDS, and AP2a in
fruits transiently expressing either GFP, DREB2F, DREB2F
Y30F
, or DREB2F
Y69F
5 d after Agrobacterium-mediated infiltration. F) The Y30 site of
DREB2F is crucial for PSK-induced phosphorylation by PSKR1. Proteins were collected from N. benthamiana leaves after treatment with either
10 μM PSK or water (control) for 2 h. They were then subjected to immunoprecipitation using anti-GFP agarose beads. G) Tentative model of
the role of PSK in promoting ripening initiation. PSK5 expression reaches maximal level at the transition of fruit ripening. At this stage, the percep-
tion of PSK by PSKR1 stimulates PSKR1-mediated DREB2F phosphorylation. This subsequently leads to transcriptional activation of
ripening-associated putative target genes of DREB2F to promote the initiation of fruit ripening. The solid lines represent direct evidence, and
the dashed lines represent potential pathways that currently lack evidence in this system. The blue rectangle represents the DREB2F protein,
and the yellow circle represents phosphorylation modification. Data in E) are presented as the means ± SD of 3 biological replicates (n = 3), and
different letters indicate significant differences (P < 0.05) according to Tukey's test.
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(F) to generate DREB2F
Y30F
and DREB2F
Y69F
variants. To fur-
ther investigate the biological function of DREB2F tyrosine
phosphorylation in fruit ripening, the DREB2F variants were
transiently expressed in MicroTom fruits at the mature green
stage due to its well-established transient overexpression sys-
tem. Notably, the Y30F mutation, but not Y69F, compromised
the DREB2F-promoted transition to ripening (Fig. 6D).
Consistently, E4, E8, EXP1, ZDS, and AP2a transcript levels
were reduced in DREB2F
Y30F
-overexpressing fruits compared
with DREB2F- and DREB2F
Y69F
-overexpressing fruits at 5 d
after infiltration (Fig. 6E). In addition, the Y30F mutation
largely suppressed PSK-induced DREB2F phosphorylation by
PSKR1 (Fig. 6F). In summary, these results indicated that the
phosphorylation of DREB2F at Y30 is involved in the
PSK-mediated fruit ripening.
Discussion
The plant peptide hormone PSK plays important roles in
growth, development, and stress responses in plants. PSK
has been shown to participate in regulating cell elongation,
root meristem, quiescent center cell development, pollen
tube growth, plant immunity, and response to abiotic stress
(van den Berg et al. 1995; Kutschmar et al. 2009; Stuhrwohldt
et al. 2011; Reichardt et al. 2020; Ding et al. 2023). However,
PSK's involvement in the regulation of fruit development and
quality formation as well as the underlying mechanism re-
main poorly understood. Here, we demonstrate that PSK, a
peptide perceived by PSKR1, promoted the initiation of fruit
ripening through enhancing DREB2F phosphorylation in to-
mato (Fig. 6G). Several lines of evidence presented below sup-
port this conclusion.
First, the PSK-PSKR1 signaling accelerates the onset of fruit
ripening and quality formation. qPCR analysis showed that
PSK5 and PSKR1 are predominantly expressed in fruits, with
their expression peaking at the Br stage during fruit develop-
ment (Fig. 1A and Supplementary Fig. S1). The Tomato
Expression Atlas (http://tea.solgenomics.net) database showed
a similar expression pattern. Furthermore, exogenous PSK
treatment promoted the initiation of fruit ripening, while
the pskr1 mutants exhibited a delayed onset of ripening and
significant changes in the expression of genes related to fruit
ripening and quality (Figs. 1, 2, and 3E). Based on these results,
we provide both molecular and genetic evidence for the pro-
moting effect of PSK signaling on fruit ripening and quality for-
mation, such as carotenoid accumulation. There are 2 PSKRs in
tomato (Zhang et al. 2018). PSKR1 exhibited markedly higher
expression than PSKR2 during fruit ripening and displayed
the ripening-associated expression pattern (Supplementary
Fig. S1). Our previous work found that PSKR1 showed a stron-
ger binding affinity for PSK than PSKR2 did (Zhang et al. 2018).
These findings imply a predominant role for PSKR1 in fruit ri-
pening. However, as an alternative PSKR, the roles and under-
lying mechanisms of PSKR2 in the ripening process deserve
further investigation. As previously reported, PSK treatment
delayed senescence and increased the levels of antioxidant
nutrients during cold storage (Song et al. 2017; Aghdam and
Alikhani-Koupaei 2021; Jiao 2022; Wang et al. 2022). These
findings demonstrate that PSK possesses diverse functional
roles during fruit development, being able to modulate both
naturally developing and postharvest cold-stored fruits
in different ways. Additionally, various plant peptides are im-
plicated in modulating fruit developmental processes.
CLAVATA3 (CLV3) binds to its receptor CLV1 and then initi-
ates a signaling cascade that suppresses expression of the tran-
scription factor WUSCHEL (WUS), leading to increased locule
number in tomato fruits. (Xu et al. 2015). Moreover,
RAPID ALKALINIZATION FACTOR 3 (ScRALF3)-RNAi plants
have smaller fruits with fewer seeds owing to improper
embryo sac development (Chevalier et al. 2013). Regarding ri-
pening, NUCLEAR LOCALIZATION SIGNAL OCTAPEPTIDE 1
(NOP-1) delays tomato fruit ripening by blocking ethylene sig-
naling through preventing the formation of the ETHYLENE
INSENSITIVE 2-ETHYLENE RECEPTOR 1 (EIN2-ETR1) complex,
a classic module in ethylene signaling. (Bisson et al. 2016). The
expression of the GOLVEN (GLV) genes PpGLV4 and PpGLV8/
CTG134 exhibits a ripening-regulated pattern, and PpGLV4
peptide treatment dramatically induces the expression of
PpACS1 in peach callus (Busatto et al. 2017; Wang et al.
2019). These findings highlight the importance of incorporat-
ing peptides into the regulatory framework that controls fruit
development. Our data further strengthen this conclusion by
rigorously demonstrating the role of PSK peptides in regulat-
ing the transition of fruit ripening.
Second, PSK signaling regulates fruit ripening through the
transcription factor DREB2F. The RNA-seq analysis suggested
the involvement of transcription factors and plant hormones
in PSKR1-mediated fruit ripening (Fig. 3E). Moreover, the Y2H
screening, together with the protein–protein interaction study
presented here, demonstrated an interaction between PSKR1
and the transcription factor DREB2F (Fig. 4). The TRV-DREB2F
fruits exhibited similar delayed ripening phenotypes with
pskr1 mutant lines (Supplementary Fig. S3). Furthermore,
the promotive effect of PSK on fruit ripening was compro-
mised in DREB-silenced fruits (Fig. 5). DREBs, like ERFs, which
play a crucial role in fruit ripening, belong to the AP2/ERF
transcription factor family. Recently, increasing studies
have proven that DREBs are involved in the control of fruit
development. MaDREB2 directly regulates genes for cell
wall modification and aroma production, such as MaEXP1,
XYLOGLUCAN ENDOTRANSGLYCOSYLASE 7 (MaXET7),
ALCOHOL DEHYDROGENASE1 (MaADH1), and PYRUVATE
DECARBOXYLASE (MaPDC) (Kuang et al. 2017). In addition,
the overexpression of CgDREB increases the levels of organic
acids and sugars in tomato (Nishawy et al. 2015). Moreover,
SlDREB3 overexpression represses the onset of ripening and ac-
tivation of multiple ripening-related genes that govern ethyl-
ene, carotenoids, and softening (Gupta et al. 2022). Our
study revealed that silencing of DREB2F delayed fruit ripening
and the expression of its potential target genes, which are as-
sociated with ripening (Fig. 5). In summary, our findings sug-
gest that transcription factor DREB2F appears to be a critical
2748 |PLANT PHYSIOLOGY 2024: 194; 2739–2754 Fang et al.
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component for the functions of PSK signaling and demon-
strated the importance of DREB2F in PSK-regulated fruit ri-
pening in tomato. Interestingly, aside from functioning in
PSK signaling, DREBs have also been demonstrated to be asso-
ciated with various phytohormones. For instance, ABA
INSENSITIVE 4 (ABI4), a member of the DREB-A3 group, is re-
quired for ABA signaling (Finkelstein et al. 1998; Niu et al.
2002). Additionally, the cold-responsive DREB1a/b/c are im-
portant elements of the ethylene and BR signaling pathways
that modulate cold tolerance in plants (Shi et al. 2012;
Li et al. 2017a). The involvement of phytohormones in
PSKR1-mediated ripening, as well as the close association be-
tween DREBs and multiple phytohormones, implies that PSK
may crosstalk with other plant hormones to modulate fruit ri-
pening and quality attribute development through DREB2F.
This possibility deserves further exploration.
Third, the interaction between PSKR1 and DREB2F leads to
phosphorylation of DREB2F, which functions in fruit ripening
and can be enhanced by PSK. PSKR1 used to be classified as
serine/threonine protein kinases, but recent research indi-
cates that recombinant cytoplasmic domains of PSKR1 also
phosphorylate on tyrosine residues (Hartmann et al. 2015;
Muleya et al. 2016). Phosphorylation assays showed that
PSK improves the efficiency of DREB2F phosphorylation by
PSKR1 at the Y30 site and the phosphorylation of this site
contributes to promoting the onset of fruit ripening
(Fig. 6). Kinase activity is essential for PSKR1-mediated
growth (Hartmann et al. 2014). Introducing kinase-impaired
PSKR1 (K762E) into the pskr knockout line was insufficient to
recover normal root or shoot growth. Our study demon-
strates the involvement of PSKR1 kinase activity in the regu-
lation of fruit ripening, and we identified DREB2F as a
phosphorylation substrate of PSKR1, providing a theoretical
basis for the transcriptional regulation downstream of the
PSK signaling pathway in regulating fruit ripening.
Phosphorylation has been implicated as posttranslational
regulators influencing the activity of DREB2-type proteins
(Liu et al. 1998; Agarwal et al. 2007). In both animals and
plants, the phosphorylation of transcription factors is an im-
portant mechanism that affects their translocation into the
nucleus (Schindler et al. 1992; Yang et al. 2022). Given that
PSKR1 is mainly localized on the membrane, it is highly plaus-
ible that DREB2F is phosphorylated by PSKR1 prior to enter-
ing the nucleus to perform its transcriptional functions. Our
results showed that the phosphorylation at Y30 within the
AP2 domain of DREB2F promoted the expression of potential
downstream target genes and advanced the initiation of fruit
ripening in tomato (Fig. 6, D and E). DREB2-type proteins
have a conserved region known as the PEST sequence
(RSDASEVTSTSSQSEVCTVETPGCV), which is rich in serine/
threonine residues and contains potential phosphorylation
sites (Lata and Prasad 2011). Phosphorylation of this region
leads to the rapid degradation of DREB2-type proteins
(Rogers et al. 1986). Deletion of this region in DREB2-type
proteins, such as AtDREB2A, GmDREB2A, and MaDREB20,
enables them to activate target genes and enhance abiotic
stress tolerance in Arabidopsis, soybean (Glycine max), and
banana, respectively (Sakuma et al. 2006; Mizoi et al. 2013;
Chaudhari et al. 2023). In contrast, we found that DREB2F
does not contain any PEST sequences and its phosphoryl-
ation mediated by PSKR1 occurred at a tyrosine residue
(Fig. 6B). These features may account for the distinct mode
of action of DREB2F compared with the aforementioned
DREB2-type proteins. Furthermore, previous research has
found that phosphorylation of PgDREB2A reduced its bind-
ing affinity for DRE/CRT elements (Agarwal et al. 2007).
Taken together, the potential regulation of DREB2F activity
and nuclear import through PSKR1-mediated phosphoryl-
ation is worth further in-depth investigation.
In summary, we demonstrate the role of the peptide hor-
mone PSK in promoting the initiation of fruit ripening and
quality formation and reveal the integral involvement of
DREB2F in PSK signaling. We find that PSK enhances
PSKR1-mediated DREB2F phosphorylation, which also func-
tions in fruit ripening. This study expands our knowledge
of the regulatory network governing climacteric fruit ripen-
ing and sheds light on PSK signaling in modulating the ultim-
ate phases of fleshy fruit development, which determines
fruit quality.
Materials and methods
Plant material and growth conditions
Tomato (S. lycopersicum) variety “Condine Red” was used as
the WT in this study. The pskr1 mutant lines were previously
generated by CRISPR/Cas9 and have already been published
in Hu et al. (Hu et al. 2023). The homozygosity of these mu-
tants was confirmed by PCR amplification and sequencing.
All the primers used are listed in Supplementary Table S7.
Tomato plants were grown at 22/20 °C and 80% relative
humidity with a 14/10 h (day/night). Lighting was at 400
μmol m
−2
s
−1
. For the measurement of time to ripen, flowers
were tagged at anthesis. Fruits at 43, 45, 47, and 49 DPA were
harvested to analyze a* value, firmness, ethylene production,
carotenoid contents, and gene expressions. All fruit stages
were harvested in at least 3 biological replicates. Upon har-
vesting, the pericarps were frozen immediately in liquid ni-
trogen and stored at −80 °C until used.
Vector construction
For Y2H analysis, the fragment of PSKR1JK (inner juxtamem-
brane domain and the protein kinase domain) were cloned
into the pGBKT7, and DREB2F was cloned into pGADT7.
To generate plasmids for the luciferase complementation
(split-LUC) assay, the corresponding genes were amplified
from cDNA and cloned into the pDONR-ZEO intermediate
vector. After sequencing confirmation, the fragment from the
pDONR-ZEO vector was assembled into pCAMBIA1300-35S-
cLUC-RBS or pCAMBIA1300-35S-HA-nLUC-RBS destination
vector. For pull-down assay and in vitro phosphor site identifi-
cation, the fragment of PSKR1JK was cloned into pMAL-c2x,
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and SlDREB2F was cloned into pGEX-4T-1. To generate
PSKR1-HA and SlDREB2F-GFP, the coding regions of PSKR1 and
SlDREB2F were amplified and inserted into pFGC1008-35S-
HA-C and pAC402–35S-eGFP-C, respectively. The point-mutant
plasmids were generated by site-directed mutagenesis (NEB). All
the primers used are listed in Supplementary Table S7.
VIGS in tomato fruits
VIGS in tomato fruits was performed as described previously
(Fu et al. 2005). cDNA fragment of SlDREB2F was amplified
using the primers listed in Supplementary Table S7 and
cloned into pTRV2 to construct TRV-SlDREB2F. The con-
struct was introduced in Agrobacterium tumefaciens strain
GV3101. Bacteria containing TRV- SlDREB2F or empty vec-
tors (EVs) were mixed in equal volume with those containing
the pTRV1 plasmid and then infiltrated into immature green
fruits. Gene silencing was verified by qPCR using primers in
Supplementary Table S7. Each infiltration involved 3 repli-
cates with 6 plants each time.
Agrobacterium-mediated transient gene expression in
tomato fruits
The SlDREB2F coding sequence was cloned into the
pAC402–35S-eGFP vector to generate an overexpression
construct. The plasmids were introduced into A. tumefa-
ciens strain GV3101. Agrobacterium-mediated transient
expression was performed as described previously
(Orzaez et al. 2006). Tomato fruits at the mature green
stage were detached and then infiltrated. Completely infil-
trated fruits were placed at room temperature for 5 d and
collected.
Chemical treatments
WT tomato fruits at the mature green stage were treated by
injecting them with 10 nmol PSK (Iris Biotech, Germany) or
an equal volume of double distilled water and then placed at
room temperature. The pericarp tissues of the tomato fruits
were collected at 3, 6, 9, and 12 DPT, frozen in liquid nitrogen,
and stored at −80 °C.
Measurement of color, firmness, and ethylene
production of fruit
Fruit pericarp coloration was quantified using a HunterLab
MiniScan XE Plus colorimeter (Hunter Associates
Laboratory Inc., United States) with the CIE L*a*b color sys-
tem (Komatsu et al. 2016). The a* value denotes the extent
of red-to-green coloration. Firmness of the pericarp was
assayed using the TA-XT2i texture analyzer (Stable Micro
Systems Ltd, United Kingdom). The rate of penetration was
1 mm s
−1
with a final penetration depth of 2 mm. For the
measurement of ethylene production, each fruit was en-
closed in a 330 mL sealed container and then incubated at
20 °C for 2 h. Then, 1 mL gas sample was analyzed using
the 6890N GC system (Agilent, United States) equipped
with a flame ionization detector, as previously described
(Li et al. 2020).
Measurement of carotenoids
Carotenoid extraction was performed as described
previously (Xu et al. 2023). The extracts were dried under
N
2
flow, resuspended in 100 μL of ethyl acetate, and injected
for ultraperformance liquid chromatography analysis
(Thermo Fisher Scientific).
Protein–protein interaction assays
The Y2H screening assay was conducted as previously de-
scribed (Ding et al. 2023). pGBKT7-PSKR1JK was used as
the bait. The tomato cDNA library construction and Y2H
screening were performed according to the manufacturer’s
protocol (Clontech). To test for protein–protein interac-
tions, pGADT7-DREB2F was cotransformed with pGBKT7-
PSKR1JK into yeast strain AH109 and cultured on synthetic
dropout medium lacking Trp and Leu (SD-T-L) at 28 °C,
followed by further selection on medium lacking Trp-Leu-
Ade-His (SD-T-L-A-H).
The pull-down assays were performed as described previ-
ously (Ding et al. 2023). The GST- and MBP-fused recombin-
ant proteins were purified from Escherichia coli BL21. GST or
GST-tagged proteins coupled to glutathione beads were in-
cubated with MBP-tagged proteins in pull-down buffer
(20 mM Tris–HCl pH 7.5, 100 mM NaCl, 0.1 mM EDTA, 0.2%
Triton-X100) at 4 °C for 1 h with rotation. The beads were
collected and washed 5 times with washing buffer (20 mM
Tris–HCl, pH 7.5, 300 mM NaCl, 0.1 mM EDTA, 0.5% Triton-
X100). Protein interactions were detected by immunoblot-
ting using an anti-MBP antibody (Cell Signaling
Technology, United States).
The split-LUC assays were performed as described
previously (Ma et al. 2022). The corresponding plasmids
were transiently expressed in N. benthamiana leaves by
A. tumefaciens GV3101 strains. Images were captured
10 min after spraying the abaxial surface of leaves with
1 mM luciferin solution using an HRPCS5 camera (Photek,
Britain).
The Co-IP assays were performed as described previously
(Ding et al. 2023). Two d after A. tumefaciens infiltration,
N. benthamiana leaves were harvested and extracted
with IP buffer. The clarified supernatant was incubated
with GFP-Trap Magnetic Agarose (Chromotek, Germany)
for 2 h at 4 °C. The beads were washed 5 times with
wash buffer and once with 50 mM Tris–HCl (pH 7.5).
Immunoprecipitated proteins were detected by immuno-
blotting with anti-GFP and anti-HA antibodies (Thermo
Fisher Scientific, United States).
Phosphorylation assay
The in vitro phosphor site identification with LC–MS/MS
analysis was performed as described (Ding et al. 2023). In
vitro phosphorylation assays were performed by incubating
purified MBP-PSKR1JK and GST-DREB2F in kinase buffer at
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room temperature for 3 h. The reaction was terminated by
adding SDS loading buffer. GST-DREB2F was separated by
SDS–PAGE and digested with trypsin overnight for further
MS analysis. Phosphopeptides were analyzed by LC-MS/MS
according to a previous study (Zhou et al. 2018). The phos-
phopeptides were analyzed with LTQ Orbitrap Elite
(Thermo Fisher, United States), according to the previous
study (Zhou et al. 2018). For in vivo phosphorylation assays,
the corresponding vectors were coexpressed in N. benthami-
ana leaves for 2 d and infiltrated with or without 10 μM PSK.
The leaves were homogenized in IP buffer and incubated
with GFP-Trap Magnetic Agarose (Chromotek, Germany).
After rotating for 2 h at 4 °C, the proteins were subjected
to western blotting using anti-pSer/pThr antibody
(ECM Biosciences, United States) or anti-pTyr antibody
(GenScript, United States).
RNA extraction and qPCR analysis
RNA was isolated from different tissues/organs/stages of to-
mato plants using the RNAsimple Total RNA Kit (Tiangen,
China) for roots, leaves, and flowers and the E.Z.N.A. Plant
RNA Kit (Omega Bio-tek, United States) for pericarp of fruits.
Reverse transcription was performed according to the man-
ufacturer's instructions (Toyobo, Japan). qPCR assays were
conducted using the SYBR Green PCR Master Mix Kit
(Takara, Japan) on a LightCycler 480 II system (Roche,
Germany). The relative mRNA levels of the genes were nor-
malized using the Actin gene as an internal control.
Gene-specific primers are listed in Supplementary Table S7.
RNA-seq analysis
Total RNA was extracted from fruits of WT and pskr1#1 lines
at 43, 45, 47, and 49 DPA, with 3 biological replicates each.
RNA-seq library preparation and paired-end sequencing
were conducted by Biomarker Technologies (Beijing, China)
using an Illumina NovaSeq 6000. The data were analyzed using
the online BMKCloud bioinformatics platform (www.
biocloud.net). R with WGCNA package was used to build a co-
expression network; the parameters of WGCNA used default
settings, except that the power was 6, TOMType was unsigned,
minModuleSize was 30, and mergeCutHeight was 0.25
(Langfelder and Horvath 2008).
Statistical analysis
At least 3 independent biological replicates were used for
each experiment. Data were analyzed using SAS version 8, fol-
lowed by Tukey's test at the 5% level. Bar graphs were gener-
ated in GraphPad Prism 8.
Accession numbers
Sequence data from this article can be found in the Sol
Genomics Network (https://solgenomics.net/) database un-
der the following accession numbers: PSKR1 (Solyc01g
008140), PSKR2 (Solyc07g063000), PSK1 (Solyc09g009130),
PSK2 (Solyc11g066880), PSK3 (Solyc02g092110), PSK3L (Solyc
02g092120), PSK4 (Solyc01g106830), PSK5 (Solyc10g083580),
PSK6 (Solyc06g074540), PSK7 (Solyc04g077580), DREB2F
(Solyc10g080310), ERF12 (Solyc11g012980), PSY1 (Solyc03
g031860), PDS (Solyc03g123760), ZDS (Solyc01g097810),
ACS2 (Solyc01g095080), ACO1 (Solyc07g049530), E4 (Solyc
03g111720), E8 (Solyc09g089580), EXP1 (Solyc06g051800),
PG2a (Solyc07g049530), RIN (Solyc05g012020), FUL1 (Solyc
06g069430), AP2a (Solyc03g044300), and Actin (Solyc
03g078400).
Acknowledgments
We would like to thank Prof. Xian Li (Zhejiang University) for
assistance with the measurement of fruit color, firmness, and
ethylene production.
Author contributions
K.S. conceived the research; H.F. and K.S. designed the experi-
ments; H.F., Q.M., X.Z., Y.X., S.D., J.W., Q.L., Y.L., C.W., J.L., and
K.S. performed the experiments; J.Y. discussed interpretations
with K.S.; H.F. and K.S. analyzed the data; J.Z. provided tech-
nical/intellectual support; H.F. and K.S. wrote the paper with
contributions from other authors.
Supplementary data
The following materials are available in the online version of
this article.
Supplementary Figure S1. Relative PSKRs transcript levels
in different tissues assessed by qPCR.
Supplementary Figure S2. DREB2F gene silencing effi-
ciency in VIGS tomato fruits.
Supplementary Figure S3. Time from anthesis to the
breaker stage in TRV-0 and TRV-DREB2F fruits.
Supplementary Table S1. Transcript levels of genes of the
brown module in WT and pskr1 fruits at different ripening
stages.
Supplementary Table S2. Selected GO categories en-
riched among genes in the brown module.
Supplementary Table S3. DEGs between 43 and 45 DPA
in WT and pskr1 mutant fruits.
Supplementary Table S4. Genes exhibited inconsistent
expression trends between WT and pskr1 mutant from 43
to 45 DPA.
Supplementary Table S5. Selected KEGG pathways en-
riched among genes exhibited inconsistent expression trends
between WT and pskr1 mutant from 43 to 45 DPA.
Supplementary Table S6. PSKR1-interacting candidates
identified from Y2H screening.
Supplementary Table S7. Primers used in this study.
Funding
This work was supported by the National Natural Science
Foundation of China (32172650), the Key Research and
Development Program of Zhejiang Province (2021C02040),
Phytosulfokine signaling promotes fruit ripening PLANT PHYSIOLOGY 2024: 194; 2739–2754 |2751
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and the Starry Night Science Fund of the Zhejiang University
Shanghai Institute for Advanced Study (SN-ZJU-SIAS-0011).
Conflict of interest statement. None declared.
Data availability
The data used to support the findings of this study are in-
cluded within the article and the supporting information file.
References
Agarwal P, Agarwal PK, Nair S, Sopory SK, Reddy MK.
Stress-inducible DREB2A transcription factor from Pennisetum glau-
cum is a phosphoprotein and its phosphorylation negatively regu-
lates its DNA-binding activity. Mol Genet Genomics. 2007:277(2):
189–198. https://doi.org/10.1007/s00438-006-0183-z
Aghdam MS, Alikhani-Koupaei M. Exogenous phytosulfokine α
(PSKα) applying delays senescence and relief decay in strawberry
fruits during cold storage by sufficient intracellular ATP and
NADPH availability. Food Chem. 2021:336:127685. https://doi.org/
10.1016/j.foodchem.2020.127685
Aghdam MS, Sayyari M, Luo Z. Exogenous phytosulfokine α applica-
tion delays senescence and promotes antioxidant nutrient accumu-
lation in strawberry fruit during cold storage by triggering
endogenous phytosulfokine α signaling. Postharvest Biol Tec.
2021:175:111473. https://doi.org/10.1016/j.postharvbio.2021.111473
Amano Y, Tsubouchi H, Shinohara H, Ogawa M, Matsubayashi Y.
Tyrosine-sulfated glycopeptide involved in cellular proliferation
and expansion in Arabidopsis. Proc Natl Acad Sci. 2007:104(46):
18333–18338. https://doi.org/10.1073/pnas.0706403104
Bisson MM, Kessenbrock M, Muller L, Hofmann A, Schmitz F,
Cristescu SM, Groth G. Peptides interfering with protein-protein in-
teractions in the ethylene signaling pathway delay tomato fruit ri-
pening. Sci Rep. 2016:6(1):30634. https://doi.org/10.1038/srep30634
Busatto N, Salvagnin U, Resentini F, Quaresimin S, Navazio L, Marin
O, Pellegrini M, Costa F, Mierke DF, Trainotti L. The peach RGF/
GLV signaling peptide pCTG134 is involved in a regulatory circuit
that sustains auxin and ethylene actions. Front. Plant Sci. 2017:8:
1711. https://doi.org/10.3389/fpls.2017.01711
Chaudhari RS, Jangale BL, Krishna B, Sane PV. Improved abiotic stress
tolerance in Arabidopsis by constitutive active form of a banana
DREB2 type transcription factor, MaDREB20. CA, than its native
form, MaDREB20. Protoplasma. 2023:260(3):671–690. https://doi.
org/10.1007/s00709-022-01805-7
Chevalier E, Loubert-Hudon A, Matton DP. ScRALF3, a secreted
RALF-like peptide involved in cell–cell communication between
the sporophyte and the female gametophyte in a solanaceous spe-
cies. Plant J. 2013:73(6):1019–1033. https://doi.org/10.1111/tpj.12096
Deng H, Chen Y, Liu Z, Liu Z, Shu P, Wang R, Hao Y, Su D, Pirrello J,
Liu Y, et al. SlERF.F12 modulates the transition to ripening in tomato
fruit by recruiting the co-repressor TOPLESS and histone deacety-
lases to repress key ripening genes. Plant Cell. 2022:34(4):
1250–1272. https://doi.org/10.1093/plcell/koac025
Ding ST, Lv JR, Hu ZJ, Wang J, Wang P, Yu JQ, Foyer CH, Shi K.
Phytosulfokine peptide optimizes plant growth and defense via glu-
tamine synthetase GS2 phosphorylation in tomato. Embo J.
2023:42(6):e111858. https://doi.org/10.15252/embj.2022111858
Fenn MA, Giovannoni JJ. Phytohormones in fruit development and
maturation. Plant J. 2021:105(2):446–458. https://doi.org/10.1111/
tpj.15112
Finkelstein RR, Li Wang M, Lynch TJ, Rao S, Goodman HM. The
Arabidopsis abscisic acid response locus ABI4 encodes an
APETALA2 domain protein. Plant Cell. 1998:10(6):1043–1054.
https://doi.org/10.1105/tpc.10.6.1043
Fu DQ, Zhu BZ, Zhu HL, Jiang WB, Luo YB. Virus-induced gene silen-
cing in tomato fruit. Plant J. 2005:43(2):299–308. https://doi.org/10.
1111/j.1365-313X.2005.02441.x
Giovannoni J, Cuong N, Ampofo B, Zhong S, Fei Z. The epigenome
and transcriptional dynamics of fruit ripening. Annu Rev Plant Biol.
2017:68(1):61–84. https://doi.org/10.1146/annurev-arplant-042916-
040906
Gupta A, Upadhyay RK, Prabhakar R, Tiwari N, Garg R, Sane VA, Sane
AP. SlDREB3, a negative regulator of ABA responses, controls seed ger-
mination, fruit size and the onset of ripening in tomato. Plant Sci.
2022:319:111249. https://doi.org/10.1016/j.plantsci.2022.111249
Han J, Tan JF, Tu LL, Zhang XL. A peptide hormone gene, GhPSK pro-
motes fibre elongation and contributes to longer and finer cotton fi-
bre. Plant Biotechnol J. 2014:12(7):861–871. https://doi.org/10.1111/
pbi.12187
Hartmann J, Fischer C, Dietrich P, Sauter M. Kinase activity and cal-
modulin binding are essential for growth signaling by the phytosul-
fokine receptor PSKR1. Plant J. 2014:78(2):192–202. https://doi.org/
10.1111/tpj.12460
Hartmann J, Linke D, Bonniger C, Tholey A, Sauter M. Conserved
phosphorylation sites in the activation loop of the Arabidopsis phy-
tosulfokine receptor PSKR1 differentially affect kinase and receptor
activity. Biochem J. 2015:472(3):379–391. https://doi.org/10.1042/
BJ20150147
Hu Z, Fang H, Zhu C, Gu S, Ding S, Yu J, Shi K. Ubiquitylation of
PHYTOSULFOKINE RECEPTOR 1 modulates the defense response
in tomato. Plant Physiol. 2023:192(3):2507–2522. https://doi.org/10.
1093/plphys/kiad188
Jiao C. PpCBF6 is involved in phytosulfokine α-retarded chilling injury
by suppressing the expression of PpLOX5 in peach fruit. Front Plant
Sci. 2022:13:874338. https://doi.org/10.3389/fpls.2022.874338
Kaufmann C, Motzkus M, Sauter M. Phosphorylation of the phytosul-
fokine peptide receptor PSKR1 controls receptor activity. J Exp Bot.
2017:68(7):1411–1423. https://doi.org/10.1093/jxb/erx030
Kaufmann C, Stuhrwohldt N, Sauter M. Tyrosylprotein
sulfotransferase-dependent and -independent regulation of root de-
velopment and signaling by PSK LRR receptor kinases in Arabidopsis.
J Exp Bot. 2021:72(15):5508–5521. https://doi.org/10.1093/jxb/erab233
Komatsu T, Mohammadi S, Busa LS, Maeki M, Ishida A, Tani H,
Tokeshi M. Image analysis for a microfluidic paper-based analytical
device using the CIE L*a*b* color system. Analyst. 2016:141(24):
6507–6509. https://doi.org/10.1039/C6AN01409G
Kuang JF, Chen JY, Liu XC, Han YC, Xiao YY, Shan W, Tang Y, Wu
KQ, He JX, Lu WJ. The transcriptional regulatory network mediated
by banana (Musa acuminata) dehydration-responsive element bind-
ing (MaDREB) transcription factors in fruit ripening. New Phytol.
2017:214(2):762–781. https://doi.org/10.1111/nph.14389
Kumar R, Khurana A, Sharma AK. Role of plant hormones and their
interplay in development and ripening of fleshy fruits. J Exp Bot.
2014:65(16):4561–4575. https://doi.org/10.1093/jxb/eru277
Kutschmar A, Rzewuski G, Stuhrwohldt N, Beemster GTS, Inze D,
Sauter M. PSK-α promotes root growth in Arabidopsis. New
Phytol. 2009:181(4):820–831. https://doi.org/10.1111/j.1469-8137.
2008.02710.x
Langfelder P, Horvath S. WGCNA: an R package for weighted correl-
ation network analysis. BMC Bioinformatics. 2008:9(1):559. https://
doi.org/10.1186/1471-2105-9-559
Lata C, Prasad M. Role of DREBs in regulation of abiotic stress re-
sponses in plants. J Exp Bot. 2011:62(14):4731–4748. https://doi.
org/10.1093/jxb/err210
Lee MM, Schiefelbein J. Developmentally distinct MYB genes encode
functionally equivalent proteins in Arabidopsis. Development.
2001:128(9):1539–1546. https://doi.org/10.1242/dev.128.9.1539
Li H, Ye K, Shi Y, Cheng J, Zhang X, Yang S. BZR1 positively regulates
freezing tolerance via CBF-dependent and CBF-independent path-
ways in Arabidopsis. Mol Plant. 2017a:10(4):545–559. https://doi.
org/10.1016/j.molp.2017.01.004
2752 |PLANT PHYSIOLOGY 2024: 194; 2739–2754 Fang et al.
Downloaded from https://academic.oup.com/plphys/article/194/4/2739/7517350 by guest on 16 April 2024
Li S, Chen K, Grierson D. Molecular and hormonal mechanisms regu-
lating fleshy fruit ripening. Cells. 2021:10(5):1136. https://doi.org/10.
3390/cells10051136
Li S, Zhu B, Pirrello J, Xu C, Zhang B, Bouzayen M, Chen K, Grierson
D. Roles of RIN and ethylene in tomato fruit ripening and
ripening-associated traits. New Phytol. 2020:226(2):460–475.
https://doi.org/10.1111/nph.16362
Li T, Xu Y, Zhang L, Ji Y, Tan D, Yuan H, Wang A. The
jasmonate-activated transcription factor MdMYC2 regulates
ETHYLENE RESPONSE FACTOR and ethylene biosynthetic genes to
promote ethylene biosynthesis during apple fruit ripening. Plant
Cell. 2017b:29(6):1316–1334. https://doi.org/10.1105/tpc.17.00349
Liu Q, Kasuga M, Sakuma Y, Abe H, Miura S, Yamaguchi-Shinozaki
K, Shinozaki K. Two transcription factors, DREB1 and DREB2, with
an EREBP/AP2 DNA binding domain separate two cellular signal
transduction pathways in drought-and low-temperature-responsive
gene expression, respectively, in Arabidopsis. Plant Cell. 1998:10(8):
1391–1406. https://doi.org/10.1105/tpc.10.8.1391
Ma Q, Hu Z, Mao Z, Mei Y, Feng S, Shi K. The novel leucine-rich re-
peat receptor-like kinase MRK1 regulates resistance to multiple
stresses in tomato. Hortic Res. 2022:9:uhab088. https://doi.org/10.
1093/hr/uhab088
Matsubayashi Y, Ogawa M, Morita A, Sakagami Y. An LRR receptor
kinase involved in perception of a peptide plant hormone, phytosul-
fokine. Science. 2002:296(5572):1470–1472. https://doi.org/10.1126/
science.1069607
Mizoi J, Ohori T, Moriwaki T, Kidokoro S, Todaka D, Maruyama K,
Kusakabe K, Osakabe Y, Shinozaki K, Yamaguchi-Shinozaki K.
GmDREB2A; 2, a canonical DEHYDRATION-RESPONSIVE ELEMENT-
BINDING PROTEIN2-type transcription factor in soybean, is post-
translationally regulated and mediates dehydration-responsive
element-dependent gene expression. Plant Physiol. 2013:161(1):
346–361. https://doi.org/10.1104/pp.112.204875
Mosher S, Seybold H, Rodriguez P, Stahl M, Davies KA, Dayaratne S,
Morillo SA, Wierzba M, Favery B, Keller H, et al. The tyrosine-
sulfated peptide receptors PSKR1 and PSY1R modify the immunity
of Arabidopsis to biotrophic and necrotrophic pathogens in an an-
tagonistic manner. Plant J. 2013:73(3):469–482. https://doi.org/10.
1111/tpj.12050
Mou W, Li D, Bu J, Jiang Y, Khan ZU, Luo Z, Mao L, Ying T.
Comprehensive analysis of ABA effects on ethylene biosynthesis
and signaling during tomato fruit ripening. PLoS One. 2016:11(4):
e0154072. https://doi.org/10.1371/journal.pone.0154072
Muleya V, Marondedze C, Wheeler JI, Thomas L, Mok YF, Griffin
MD, Manallack DT, Kwezi L, Lilley KS, Gehring C, et al.
Phosphorylation of the dimeric cytoplasmic domain of the phytosul-
fokine receptor, PSKR1. Biochem J. 2016:473(19):3081–3098. https://
doi.org/10.1042/BCJ20160593
Nishawy E, Sun X, Ewas M, Ziaf K, Xu R, Wang D, Amar M, Zeng Y,
Cheng Y. Overexpression of Citrus grandis DREB gene in tomato af-
fects fruit size and accumulation of primary metabolites. Sci Hortic.
2015:192:460–467. https://doi.org/10.1016/j.scienta.2015.06.035
Niu X, Helentjaris T, Bate NJ. Maize ABI4 binds coupling element1 in
abscisic acid and sugar response genes. Plant Cell. 2002:14(10):
2565–2575. https://doi.org/10.1105/tpc.003400
Orzaez D, Mirabel S, Wieland WH, Granell A. Agroinjection of to-
mato fruits. A tool for rapid functional analysis of transgenes directly
in fruit. Plant Physiol. 2006:140(1):3–11. https://doi.org/10.1104/pp.
105.068221
Reichardt S, Piepho HP, Stintzi A, Schaller A. Peptide signaling for
drought-induced tomato flower drop. Science. 2020:367(6485):
1482–1485. https://doi.org/10.1126/science.aaz5641
Rogers S, Wells R, Rechsteiner M. Amino acid sequences common to
rapidly degraded proteins: the PEST hypothesis. Science.
1986:234(4774):364–368. https://doi.org/10.1126/science.2876518
Sakuma Y, Maruyama K, Osakabe Y, Qin F, Seki M, Shinozaki K,
Yamaguchi-Shinozaki K. Functional analysis of an Arabidopsis
transcription factor, DREB2A, involved in drought-responsive gene
expression. Plant Cell. 2006:18(5):1292–1309. https://doi.org/10.
1105/tpc.105.035881
Sang K, Li J, Qian X, Yu J, Zhou Y, Xia X. The APETALA2a/DWARF/
BRASSINAZOLE-RESISTANT 1 module contributes to carotenoid
synthesis in tomato fruits. Plant J. 2022:112(5):1238–1251. https://
doi.org/10.1111/tpj.16009
Sauter M. Phytosulfokine peptide signalling. J Exp Bot. 2015:66(17):
5160–5168. https://doi.org/10.1093/jxb/erv071
Schindler C, Shuai K, Prezioso VR, Darnell JE Jr. Interferon-dependent
tyrosine phosphorylation of a latent cytoplasmic transcription factor.
Science. 1992:257(5071):809–813. https://doi.org/10.1126/science.
1496401
Seymour GB, Østergaard L, Chapman NH, Knapp S, Martin C. Fruit
development and ripening. Annu Rev Plant Biol. 2013:64(1):219–241.
https://doi.org/10.1146/annurev-arplant-050312-120057
Shi Y, Tian S, Hou L, Huang X, Zhang X, Guo H, Yang S. Ethylene sig-
naling negatively regulates freezing tolerance by repressing expres-
sion of CBF and type-A ARR genes in Arabidopsis. Plant Cell.
2012:24(6):2578–2595. https://doi.org/10.1105/tpc.112.098640
Song H, Wang X, Hu W, Yang X, Diao E, Shen T, Qiang Q. A
cold-induced phytosulfokine peptide is related to the improvement
of loquat fruit chilling tolerance. Food Chem. 2017:232:434–442.
https://doi.org/10.1016/j.foodchem.2017.04.045
Stuhrwohldt N, Dahlke RI, Kutschmar A, Peng X, Sun MX, Sauter M.
Phytosulfokine peptide signaling controls pollen tube growth and fu-
nicular pollen tube guidance in Arabidopsis thaliana. Physiol Plant.
2015:153(4):643–653. https://doi.org/10.1111/ppl.12270
Stuhrwohldt N, Dahlke RI, Steffens B, Johnson A, Sauter M.
Phytosulfokine-α controls hypocotyl length and cell expansion in
Arabidopsis thaliana through phytosulfokine receptor 1. PLoS One.
2011:6(6):e21054. https://doi.org/10.1371/journal.pone.0021054
van den Berg C, Willemsen V, Hage W, Weisbeek P, Scheres B. Cell fate
in the Arabidopsis root meristem determined by directional signalling.
Nature. 1995:378(6552):62–65. https://doi.org/10.1038/378062a0
Wang D, Huang H, Jiang Y, Duan X, Lin X, Soleimani Aghdam M, Luo
Z. Exogenous phytosulfokine α (PSKα) alleviates chilling injury of ba-
nana by modulating metabolisms of nitric oxide, polyamine, proline,
and gamma-aminobutyric acid. Food Chem. 2022:380:132179.
https://doi.org/10.1016/j.foodchem.2022.132179
Wang J, Li H, Han Z, Zhang H, Wang T, Lin G, Chang J, Yang W, Chai J.
Allosteric receptor activation by the plant peptide hormone phyto-
sulfokine. Nature. 2015:525(7568):265–268. https://doi.org/10.1038/
nature14858
Wang Y, Ding YF, Wang XB, Chen HJ, Cao HB, Niu L, Pan L, Lu ZH, Cui
GC, Zeng WF, et al. Analysis of PpGLV gene family suggests that
PpGLV4 peptide coordinates auxin and ethylene signaling in peach.
Sci Hortic. 2019:246:12–20. https://doi.org/10.1016/j.scienta.2018.10.026
Xie Z, Nolan TM, Jiang H, Yin Y. AP2/ERF transcription factor regula-
tory networks in hormone and abiotic stress responses in
Arabidopsis. Front Plant Sci. 2019:10:228. https://doi.org/10.3389/
fpls.2019.00228
Xu C, Liberatore KL, MacAlister CA, Huang Z, Chu Y-H, Jiang K,
Brooks C, Ogawa-Ohnishi M, Xiong G, Pauly M. A cascade of ara-
binosyltransferases controls shoot meristem size in tomato. Nat
Genet. 2015:47(7):784–792. https://doi.org/10.1038/ng.3309
Xu J, Liu S, Cai L, Wang L, Dong Y, Qi Z, Yu J, Zhou Y. SPINDLY inter-
acts with EIN2 to facilitate ethylene signalling-mediated fruit ripen-
ing in tomato. Plant Biotechnol J. 2023:21(1):219–231. https://doi.
org/10.1111/pbi.13939
Xu Q, Yin XR, Zeng JK, Ge H, Song M, Xu CJ, Li X, Ferguson IB, Chen
KS. Activator- and repressor-type MYB transcription factors are in-
volved in chilling injury induced flesh lignification in loquat via their
interactions with the phenylpropanoid pathway. J Exp Bot.
2014:65(15):4349–4359. https://doi.org/10.1093/jxb/eru208
Yamakawa S, Sakuta C, Matsubayashi Y, Sakagami Y, Kamada H,
Satoh S. The promotive effects of a peptidyl plant growth factor,
Phytosulfokine signaling promotes fruit ripening PLANT PHYSIOLOGY 2024: 194; 2739–2754 |2753
Downloaded from https://academic.oup.com/plphys/article/194/4/2739/7517350 by guest on 16 April 2024
phytosulfokine-α, on the formation of adventitious roots and ex-
pression of a gene for a root-specific cystatin in cucumber hypoco-
tyls. J Plant Res. 1998:111(3):453–458. https://doi.org/10.1007/
BF02507810
Yang S, Cai W, Shen L, Cao J, Liu C, Hu J, Guan D, He S. A
CaCDPK29–CaWRKY27b module promotes CaWRKY40-mediated
thermotolerance and immunity to Ralstonia solanacearum in pep-
per. New Phytol. 2022:233(4):1843–1863. https://doi.org/10.1111/
nph.17891
Yue P, Lu Q, Liu Z, Lv T, Li X, Bu H, Liu W, Xu Y, Yuan H, Wang A.
Auxin-activated MdARF5 induces the expression of ethylene biosyn-
thetic genes to initiate apple fruit ripening. New Phytol. 2020:226(6):
1781–1795. https://doi.org/10.1111/nph.16500
Zhang H, Hu Z, Lei C, Zheng C, Wang J, Shao S, Li X, Xia X, Cai X,
Zhou J, et al. A plant phytosulfokine peptide initiates auxin-
dependent immunity through cytosolic Ca
2+
signaling in tomato.
Plant Cell. 2018:30(3):652–667. https://doi.org/10.1105/tpc.17.00537
Zhou J, Liu D, Wang P, Ma X, Lin W, Chen S, Mishev K, Lu D, Kumar
R, Vanhoutte I. Regulation of Arabidopsis brassinosteroid receptor
BRI1 endocytosis and degradation by plant U-box PUB12/
PUB13-mediated ubiquitination. Proc Natl Acad Sci USA.
2018:115(8):E1906–E1915. https://doi.org/10.1073/pnas.1712251115
Zhu T, Tan W-R, Deng X-G, Zheng T, Zhang D-W, Lin H-H. Effects of
brassinosteroids on quality attributes and ethylene synthesis in post-
harvest tomato fruit. Postharvest Biol Tec. 2015:100:196–204. https://
doi.org/10.1016/j.postharvbio.2014.09.016
2754 |PLANT PHYSIOLOGY 2024: 194; 2739–2754 Fang et al.
Downloaded from https://academic.oup.com/plphys/article/194/4/2739/7517350 by guest on 16 April 2024
