Erf G3

Plant Biotechnology Journal (2023), pp. 1–16 doi: 10.1111/pbi.
14177
SlERF.G3-Like mediates a hierarchical transcriptional

cascade to regulate ripening and metabolic changes in
tomato fruit
Shengjie You1, Yu Wu1, Wen Li1, Xiaofeng Liu1, Qinlan Tang1, Fengkun Huang2,3, Yan Li2,3, Hsihua Wang1,
Mingchun Liu1 and Yang Zhang1,*
1
Key Laboratory of Bio-resource and Eco-environment of Ministry of Education, College of Life Sciences, Sichuan University, Chengdu, Sichuan, People’s Republic of
China
2
Sanya Nanfan Research Institute of Hainan University, Hainan Yazhou Bay Seed Laboratory, Sanya, China
3
College of Tropical Crops, Hainan University, Haikou, China
Received 14 March 2023; Summary

revised 25 July 2023; The tomato ripening process contains complex changes, including ethylene signalling, cell wall
accepted 2 September 2023. softening and numerous metabolic changes. So far, much is still unknown about how tomato
*Correspondence (Tel +86 28 85470795;
plants precisely coordinate fruit maturation and metabolic regulation. In this paper, the ERF
fax +86 82 85412571; email yang.zhang@
family transcription factor SlERF.G3-Like in tomato was found to be involved in the regulation of
scu.edu.cn)
ethylene synthesis, cell wall degradation and the flavonoid pathway. We show that the master
ripening regulator SlRIN was found to directly bind to the promoter region of SlERF.G3-Like to
activate its expression. In addition, we managed to increase the production of resveratrol
derivatives from ~1.44 mg/g DW in E8:VvStSy line to ~2.43 mg/g DW by crossing p35S:
SlERF.G3-Like with the E8:VvStSy line. Our data provide direct evidence that SlERF.G3-Like, a
Keywords: tomato, ERF, hierarchical transcriptional factor, can directly manipulate pathways in which tomatoes can
phenylpropanoid, ripening, metabolic coordinate fruit maturation and metabolic changes. We also attest that SlERF.G3-Like can be
engineering. used as an effective tool for phenylpropanoid metabolic engineering.
factors in the regulation of ethylene biosyntheses, such as

Introduction
RIPENING-INHIBITOR (RIN) (Vrebalov et al., 2002), COLORLESS
The ripening of tomatoes, the representative climacteric fruit, is NONRIPENING (CNR)(Manning et al., 2006) and NON-RIPENING
controlled by the plant hormone ethylene, a range of transcrip- (NOR) (Giovannoni, 2004). There are certain differences between
tion factors and other developmental cues (Bapat et al., 2010). SlRIN mutants induced by CRISPR/Cas9 and natural mutant rin,
During tomato fruit ripening, ethylene production and signalling, but SlRIN is still essential for the maturation process (Ito et al.,
fruit softening and metabolic changes take place under the tight 2017; Li et al., 2018a, 2019; Vrebalov et al., 2002). SlRIN
control of a regulatory network (Giovannoni, 2004; Seymour significantly impacts genes involved in maturation within this
et al., 2013). This regulatory network of transcription factors regulatory network. Master regulator modulation often involves
collectively determines the expression of downstream ripening- direct regulation, interactions and feedback regulation. A better
related genes (Li et al., 2019). understanding of the function of master transcription factors in
During tomato ripening, various genes are differentially tomato is gained through the study of hierarchical transcriptional
expressed and the fruit undergoes several physiological and cascades.
biochemical changes, including hardness, flavour, colour Fruit softening is also a prominent feature during the ripening
and aroma (Giovannoni, 2004; Karlova et al., 2014; Qin of climacteric fruit (Karlova et al., 2014; Shi et al., 2022).
et al., 2016). At the onset of ripening, like other climatic fruits, Hemicellulose and pectin in the cell wall are solubilized and
tomato fruit experiences a burst of respiration and a surge in depolymerized by various cell wall-related enzymes, leading to
ethylene production (Alexander and Grierson, 2002). The softening fruits (Karlova et al., 2014; Wang et al., 2018a). The
ethylene biosynthetic pathway has been well characterized and early report showed that polygalacturonase (PG) activity in
involves the action of two key enzymes, ACC synthase (ACS) and tomatoes could be reduced by antisense RNA (Smith, 1988).
ACC oxidase (ACO) (Hoffman, 1984). Researchers demonstrated After silencing SlPG, no difference in the firmness of tomato fruit
that SlACS2 and SlACS4 were consistently expressed in increasing was detected by vertical pressing compared with controls,
amounts during ripening and were the central genes expressed although the inhibition of the expression of SlPG did improve
during tomato ripening and that, among the six ACOs, SlACO1 its storage life (Schuch et al., 1991). However, simultaneous
and SlACO4 appeared to be more important (Liu et al., 2015). To inhibition of LePG and LeEXP1 expression caused significantly
date, a rich literature confirms the involvement of transcription firmer tomato fruit (Powell et al., 2003). Also, silencing SlPL
Please cite this article as: You, S., Wu, Y., Li, W., Liu, X., Tang, Q., Huang, F., Li, Y., Wang, H., Liu, M. and Zhang, Y. (2023) SlERF.G3-Like mediates a hierarchical
transcriptional cascade to regulate ripening and metabolic changes in tomato fruit. Plant Biotechnol. J., https://doi.org/10.1111/pbi.14177.
ª 2023 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd. 1
This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use,
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2 Shengjie You et al.
enhanced fruit firmness (Yang et al., 2017). These studies suggest expression of ripening-related and cell wall-related genes. Our
that softening is a complex process involving the synergistic study complements the link between the ripening process and
action of multiple enzymes. flavonoid metabolism. Furthermore, our findings show that SlRIN,
In addition, the ripening process involves many metabolic a master regulator during tomato fruit ripening, can bind to two
changes that affect not only the texture of the fruit but also its CArG motifs upstream of the promoter of SlERF.G3-Like and
taste, colour and aroma (Gao et al., 2022; Qin et al., 2016; Shi induce its expression. SlERF.G3-Like is a key TF responding to
et al., 2021). The change in carotenoids has been thoroughly master regulator RIN and controls multiple ripening-related
studied, and it significantly increases during the maturation processes. In addition, co-expression of SlERF.G3-Like with the
process (Klee and Giovannoni, 2011; Seymour et al., 2013). In resveratrol biosynthetic gene VvStSy can significantly enhance
addition to carotenoids, phenylpropanoids, such as flavonoids, resveratrol production in tomato fruit. Altogether, this study
are also involved in the process of colour change in tomato fruit reveals the coordinated maturation and metabolic model of
(Zhang et al., 2015a), but little has been reported on the SlERF.G3-Like as a hierarchical transcriptional factor in fruit
relationship between the ripening process and flavonoid ripening that can be applied to future breeding.
metabolism.
Numerous studies have reported transcription factors involved Results
in the regulation of ripening. To comprehend the process of fruit
SlERF.G3-Like positively regulates tomato fruit ripening
ripening regulation, it is crucial to investigate the connections
between higher level regulatory genes, intermediate regulators We previously screened SlERF.G3-Like as a potential flavonoid
and downstream effectors. Ethylene Responsive Factor (ERF) is biosynthesis regulator in tomato fruit (Li et al., 2020b). To further
conservatively widespread in plants. It is involved in the control of investigate the function of SlERF.G3-Like in tomatoes we
plant growth and developmental programmes, response to generated slerf.g3-like knockout mutants in the MicroTom
abiotic stress and plays a dynamic role in primary and secondary background using CRISPR/Cas9, as well as overexpression lines
metabolic regulation (Bailey-Serres et al., 2012; De Boer et al., driven by the cauliflower mosaic virus (CaMV) 35S promoter.
2011; Licausi et al., 2013; Yang et al., 2005). The crucial role We genotyped 14 gene-edited seedlings by PCR amplification
played by ERF TFs in the ripening of tomato fruit was revisited and and sequencing over the edited site in the T0 generation. In total,
defined (Julien Pirrello et al., 2012). Increasing numbers of ERF TFs we obtained two homozygous lines named CR-slerf.g3-like-3
have been characterized by fruit ripening regulators and are (CR-3) and CR-slerf.g3-like-8 (CR-8), respectively. CR-3 harboured
involved in regulating a specific aspect of fruit ripening (Cao a 4-bp deletion, while CR-8 carried an 8-bp deletion (Figure S1).
et al., 2020; Deng et al., 2022; Lee et al., 2012; Liu et al., 2013, Both lines have premature stop codons compared with the WT.
2014). We grew the CR-3 and CR-8 into the T2 generation. Individual
Due to its high yield and high contents of various metabolites, plants containing the homozygous mutation but no T-DNA
tomato is an ideal chassis for metabolic engineering (Fan insertions were selected for subsequent analyses (Figure S1d).
et al., 2021; Li et al., 2018b, 2022). In addition to introducing On the other hand, we obtained 16 independent 35S:
biosynthetic genes, the use of transcription factors can signifi- SlERF.G3-Like-OE lines in the T0 generation (Figure S2a). Two
cantly enhance the production of valuable compounds in tomato lines, OE-SlERF.G3-Like-K (OE-K) and OE-SlERF.G3-Like-S (OE-S),
fruit (Fu et al., 2018; Jian et al., 2019; Sun et al., 2020). For with representative phenotypes and expression levels, were
example, overexpression of CYP76AD6 in the background of selected for further phenotypic and molecular analyses
AtMYB12 results in further accumulation of L-DOPA (Breitel (Figures 1a,b and S2).
et al., 2021). And simultaneously, overexpression of SlWRKY35 To analyse SlERF.G3-Like related phenotypes, knockout
and SlLCYE further enhances lutein production from 290 to mutants, overexpression lines and WT were sown and grown
422.6 lg g1 DW in tomato (Yuan et al., 2022). Resveratrol together. There was no significant difference between CR and
(3,40 ,5-trihydroxystilbene) is a non-flavonoid polyphenolic com- WT in the period of vegetative growth (Figure S3c). However,
pound naturally synthesized in only a few plant species (Vannozzi significant differences were observed during the fruit ripening
et al., 2012). Its potential value in the nutraceutical, medicinal and process (Figure 1a). Compared with the WT fruits, which reached
cosmeceutical fields is extensively characterized (Valletta et al., the breaker stage at 39 days post anthesis (DPA), the CR-slerf.g3-
2021). The master transcription factor AtMYB12, which regulates like fruits displayed a significant delay in ripening (breaker stage
the flavonoid pathway, was reported to obtain tomatoes enriched at 41 DPA), while the OE fruits were significantly earlier (breaker
with high resveratrol by crossing with the transgenic line stage at 36 DPA) (Figure 1a,c).
overexpressing VvStSy (stilbene synthase) (Zhang et al., 2015a). In addition, we assessed the fruit hue angle, which indicates
Given the high demand for the production of valuable colour saturation. Compared with WT, the fruit hue angle was
compounds, new metabolic engineering tools are needed. significantly increased in OE-K and OE-S fruits, which eventually
In this study, we comprehensively characterize the roles of show an orange phenotype in OE lines (Figures 1d and S3a,b).
SlERF.G3-Like in regulating tomato ripening and metabolism. The fruit hue angle was not significantly different between CR
SlERF.G3-Like is expressed explicitly during tomato fruit ripening, and WT fruits at the Br + 15 stage (Figures 1d and S3a). All these
which also predicts its vital role in the ripening process. Stable results indicate that SlERF.G3-Like is a positive regulator of
knockout mutants of slerf.g3-like by CRISPR/Cas9 and tomato fruit ripening.
stable overexpressing transgenic lines under a constitutive
Alternation of SlERF.G3-Like expression levels
promoter of 35S were generated. Phenotypical characterization
significantly affects the expression of ripening-related
indicated that SlERF.G3-Like also participates in regulating the
and metabolic genes
ripening process in tomatoes. Comprehensive transcriptomic and
metabolic analyses showed that SlERF.G3-Like could promote To elucidate how SlERF.G3-Like affected the ripening process of
fruit ripening as well as affect fruit softening by regulating the tomato fruits, we analysed global gene expression profiling by
ª 2023 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd., 1–16
Roles of SlERF.G3-Like in tomato fruit 3
Figure 1 Alternation of SlERF.G3-Like expression affects tomato fruit ripening. (a) Fruit ripening processes of CR, OE and WT lines. The WT fruit reached
the breaker stage at 39 DPA. While the OE lines arrived at the breaker at 36 DPA, and the CR lines were at 41 DPA. The black arrow indicates RNA-seq
sampling time. (b) Statistics analysis of ripening time by days from anthesis to the breaker stages in CR, OE and WT lines. Asterisks indicate statistical
significance compared with WT fruit using Student’s t-test, *P < 0.05; **P < 0.01, n > 25. (c)The hug angle was significantly increased in the OE lines.
Fruits were measured at the Br + 15 stage. Asterisks indicate statistically significant differences compared with WT using Student’s t-test, *P < 0.05;
**P < 0.01, n > 25. NS, no significance.
RNA-seq of CR-slerf.g3-like, OE-SlERF.G3-Like and WT fruits at 39 SlERF.G3-Like (Figure 2b). A series of vital genes involved in
DPA (Figure 1a). A principal component analysis (PCA) showed ethylene biosynthesis and signalling, the cell wall, the carotenoid
that OE-K and OE-S lines shared a similar transcriptome quite pathway and some TFs relating to the ripening of fruit were
distinct from the control (Figure S4a). affected (Figure 2c). Gene ontology (GO) analysis also indicated
To determine differentially expressed genes (DEGs), we that loss of SlERF.G3-Like function affected multiple metabolic
selected only genes with a false discovery rate P-value correction pathways, including the secondary metabolic process, cell wall
≤0.05 for each line. In total, we detected 1383 DEGs, of which organization or biogenesis, phenylpropanoid metabolic process,
678 were upregulated and 705 were downregulated between flavonoid metabolic process, pigment metabolic process, poly-
the CR-slerf.g3-like and WT lines (Figure S4b). In addition, we saccharide metabolic process and terpenoid biosynthetic process
identified 1667 upregulated genes and 2982 downregulated (Figure S4d). In summary, SlERF.G3-Like plays a vital role in
genes in OE-SlERF.G3-Like lines compared with the WT tomato fruit ripening by affecting the transcription of ripening-
(Figure S4c). Among all the DEGs, we screened out 488 DEGs associated genes as well as metabolic genes. It seems like
that displayed an opposite expression pattern in CR-slerf.g3-like SlERF.G3-Like can significantly regulate the ripening and meta-
and OE-SlERF.G3-Like fruits. Among these 488 DEGs, 210 genes bolic networks.
were co-upregulated in OE lines but co-downregulated in CR lines
SlERF.G3-Like affects ethylene production by regulating
and 288 genes were co-downregulated in OE lines but co-
the expression of SlACO1 and SlACS2
upregulated in CR lines (Figure 2a).
The Kyoto Encyclopedia of Genes and Genomes (KEGG) Ethylene plays a crucial role in regulating the climacteric
annotation of these 488 DEGs indicated that multiple metabolic fruit ripening process. According to the currently accepted
pathways are significantly affected by the expression of model, ethylene biosynthesis comprises two steps catalysed by
Figure 2 RNA-seq profiling of CR-slerf.g3-like and OE-SlERF.G3-Like fruits. (a) Venn diagram of differently expressed genes in CR and OE lines. (b) KEGG
enrichment of 488 DEGs showed an opposite expression pattern between the OE and CR lines. (c) Comparison of ripening-related gene expression patterns
in RNA-seq data. (d) Comparison of metabolic-related gene expression patterns in RNA-seq data.
1-aminocyclopropane-1-carboxylic acid synthase (ACS) and 1- polymerase chain reaction (RT-qPCR) analysis proved the consis-
aminocyclopropane-1-carboxylic acid oxidase (ACO) (Yang and tent results (Figure S8).
Hoffman, 1984). The previous studies confirmed that ACS2 and Ethylene production was investigated in CR-slerf.g3-like,
ACS4 are the primary family members expressed during ripening OE-SlERF.G3-Like and WT fruit by gas chromatography (GC).
while pointing to ACO1 and ACO4 as the central ACO genes We monitored ethylene production from 35 to 51 DPA. The peak
supporting ripening-associated ethylene production (Liu et al., of climacteric ethylene production shifted from 41 DPA in the WT
2015; Nakatsuka et al., 1998; Theologis et al., 1992). In our RNA- to 43 DPA in CR-slerf.g3-like fruits (Figure 3b) and occurred 1 day
seq data, a series of important ethylene biosynthesis and signal earlier in OE-SlERF.G3-Like fruits than in the WT. Compared with
transduction pathway genes were significantly affected in the WT fruit, the amount of ethylene produced was significantly
transgenic lines. We examined the expression level of 14 ACS increased in the OE-SlERF.G3-Like lines but decreased in the CR-
genes and 6 ACO genes that had previously been reported (Liu slerf.g3-like lines (Figure 3b).
et al., 2015). Only ACS2, ACO1 and ACO3 are differentially A dual-luciferase assay was performed to understand whether
expressed in CR and OE lines. Other fruit ripening-related SlERF.G3-Like directly regulates the transcription of SlACS2 and
ethylene biosynthesis genes, such as SlACS4 and SlACO4, were SlACO1 during tomato fruit ripening. The relative LUC/REN ratio
not present in the DEGs. Two ethylene biosynthesis genes, ACO1 in tobacco leaves is co-transformed with CaMV35S-SlERF.G3-Like
and ACS2, attracted our attention because they are required for and CaMV35S-REN/pSlACS2-LUC or CaMV35S-REN/pSlACO1-
tomato fruit ripening. They all showed decreased transcript LUC were significantly higher than in leaves co-transformed with
abundance in CR-slerf.g3-like fruits compared with the WT and CaMV35S-Empty and CaMV35S-REN/pSlACS2-LUC or CaMV35S-
displayed an opposite expression pattern in OE-SlERF.G3-Like REN/pSlACO1-LUC (Figure 3c-e), indicating that SlERF.G3-Like
(Figure 3a). Supplementary reverse transcription quantitative could activate the promoter activity of SlACS2 and SlACO1 in
Figure 3 SlERF.G3-Like influences ethylene production by directly regulating the expression of SlACS2 and SlACO1. (a) The expression levels of SlACO1
and SlACS2 were significantly upregulated in the OE lines but significantly decreased in the CR lines. Bars represent standard deviation (SD) of three
independent replicates. Asterisks indicate statistical significance using the Student’s t-test, *P < 0.05; **P < 0.01. (b) Ethylene production in CR, OE and
WT fruits at different ripening stages. (c) Schematic representation of transient expression assay design for SlERF.G3-Like activation of the SlACS2 and
SlACO1 promoters. (d,e) SlERF.G3-Like induces the activities of proSlACS2/ proSlACO1 in the dual-Luc assay. Each value represents the means of three
biological replicates. Asterisks indicate statistical significance using the Student’s t-test, *P < 0.05; **P < 0.01.
vivo. Further, we explored the sites where SlERF.G3-Like binds to Then, we performed further truncation, and the assay results
the SlACO1 promoter. The SlACO1 promoter contains a GCC-like showed that the activation activity of SlERF.G3-Like on the
motif, and we mutated it. In addition, we truncated the promoter SlACO1 promoter decreased substantially at 129 bp to 1
fragment (Figure S5a). The results of the Dual-Luc experiments fragment of the proSlACO1 (Figure S5e,f). The yeast one-hybrid
showed no significant differences among these fragments assay indicated SlERF.G3-Like directly interacts with the pro-
(Figure S5b-d). This indicated that the actual binding site might SlACO1 from 547 bp to 305 bpfragment (Figure S5g). Given
be present on the 547 bp to 1 fragment of the proSlACO1. all these data, SlERF.G3-Like can activate the promoters of
SlACS2 and SlACO1, in which SlERF.G3-Like can specifically bind bind to the promoters of SlPG2a and SlPL in vivo and activate
to a fragment of from 547 bp to 305 bp on the SlACO1 their expression (Figure 4h). Furthermore, a total of 14 cell wall-
promoter region. related genes, including endo-glucanase 2 (SlCEL2), expansin1
We further investigated whether the expression of SlERF.G3- (SlEXP1), b-galactosidases (SlTBG), xyloglucan endotransglycosy-
Like is under the control of ethylene. RT-qPCR results indicated lase/hydrolase (SlXTH), endo-b-mannanase (SlMAN), xylosidase
that the expression of SlERF.G3-Like was significantly induced in (SlXY) and xylanase (SlXyn) were downregulated in both CR-K
mature green (MG) fruits treated with exogenous ethylene (Figure and CR-S fruits (Figure 2c). Collectively, these results imply that
S6a). On the other hand, SlERF.G3-Like expression level was SlERF.G3-Like positively regulates the cell wall by targeting
significantly inhibited under the treatment of 1- SlPG2a and SlPL.
methylcyclopropen (1-MCP), an ethylene perception inhibitor
SlERF.G3-Like positively regulates the phenylpropanoid
(Figure S6b). The results indicated that SlERF.G3-Like showed
pathway
some response to exogenous ethylene and increased its
expression upon ethylene treatment. The phenylpropanoid metabolic pathway is an essential second-
To investigate whether the change in ethylene production in ary metabolic pathway in plants and is synthesized from
the fruits of OE-SlERF.G3-Like plants persisted in their non-fruit phenylalanine produced by the shikimate pathway as a substrate
tissues, OE-SlERF.G3-Like and WT tomato seedlings were treated (Jacobowitz and Weng, 2020). Many important phenylpropa-
with exogenous ethylene. However, upon treatment with exoge- noids, such as coumarins, anthocyanins, flavonoids and lignins,
nous ethylene, hypocotyl elongation was significantly decreased in have vital roles in plant growth and development, disease and
OE seedings, and root elongation showed no apparent difference antioxidant capacity and pigment formation (Dong and
between WT and OE, probably suggesting a higher sensitivity to Lin, 2021; Jacobowitz and Weng, 2020; Zhang et al., 2015b).
the hormone for the OE lines (Figure S6c-f). Taken together, these The GO terms of the secondary metabolic process were
results demonstrated that SlERF.G3-Like positively regulate ethyl- dominantly enriched in the biological process group, especially
ene biosynthesis in tomato fruit by directly targeting the promoters the phenylpropanoid metabolic process. Our previous study
of SlACS2 and SlACO1. suggests that SlERF.G3-Like can directly regulate the expression
of SlCHS1 (chalcone synthase 1), SlFLS (flavonol synthase) and
SlERF.G3-Like directly regulates the softening of tomato
SlF3H (flavanone-3-hydroxylase) (Li et al., 2020b). SlCHS1 is
fruit by regulating the expression of SlPG2a and SlPL
responsible for the first committed step in the flavonoid
The softening of tomato fruit is a significant physiological change pathway, and most of the downstream genes were upregulated
during the ripening process (Giovannoni, 2004; Seymour in OE lines and downregulated in CR lines, suggesting enhanced
et al., 2013). To evaluate the effect of the SlERF.G3-Like on activity of this branch of the pathway (Figure 2d). On the other
tomato fruit softening, the firmness of CR, OE and WT fruits at hand, overexpression of SlERF.G3-Like in tomatoes increased
the Br + 15 stage was measured by a texture analyser. CR-K and transcript levels of genes involved in primary metabolism.
CR-S mutant fruits were significantly firmer than the WT at the Compared with WT fruit, nearly all of the genes encoding
Br + 15 stage examined. In contrast, the overexpressed lines enzymes of glycolysis and the shikimate pathways were
showed the opposite trend (Figure 4c). Furthermore, histological expressed more highly in p35S: SlERF.G3-Like tomato fruit than
sectioning from the fruit epicarp indicated compact cells and control fruit. And both primary and secondary metabolisms
increased cell numbers in the exocarp of CR fruit compared with promote flavonoid biosynthesis in tomatoes (Figure 2d).
WT fruit. In comparison, the exocarp cells are more loosely Lignin is a major component of the secondary cell wall and can
arranged in OE fruit (Figure 4a,e,f). On the other hand, the cuticle provide mechanical strength for plants. Formation, movement and
thickness of the OE lines was also significantly thinner than the polymerization of monolignols are all aspects of lignification
WT lines (Figure 4b,d). and this process results from the coordinated expression of several
Softening is one of the irreversible features of ripening fruits. enzymes (Boerjan et al., 2003; Boudet et al., 2003). Monolignols
Extensive studies on softening during fruit ripening in tomatoes are derived from the general phenylpropanoid pathway, and the
showed that softening is a complex process with multiple related enzymes have been well-characterized in Arabidopsis and
enzymes working consecutively and synergistically, contributing poplar (Shi et al., 2022; Vanholme et al., 2019). It is worth
to altering fruit texture (Wang et al., 2018a). Silencing SlPL, the mentioning that p-coumaric acid acts as the common precursor for
only gene confirmed in tomatoes, was shown to increase fruit both flavonoid and monolignol biosynthesis (Figure 5). By sequence
firmness and improve fruit shelf life (Yang et al., 2017). GO alignment of key enzymes with verified functions, it was
enrichment analysis of the DEGs showed that SlERF.G3-Like found in our data that cinnamyl CoA reductase (CCR), caffeic acid
regulates cell wall organization or biogenesis pathways O-methyltransferase (COMT) and cinnamyl alcohol dehydrogenase
(Figure S3d). To investigate the molecular mechanism of (CAD) showed significant upregulation in CR lines and down-
SlERF.G3-Like in regulating tomato fruit softening, transcriptome regulation in OE lines. The last major steps in lignin synthesis are
analysis was performed. Functional annotation of the DEGs catalysed by laccase (LAC) or peroxidase (PRX). Putative genes,
indicates that the expression levels of SlPG2a and SlPL were including 10 PRXs and two LACs, showed the same trend as other
downregulated in CR-slerf.g3-like fruits but upregulated in monolignol biosynthesis genes. And all of them offer an opposite
OE-SlERF.G3-Like lines (Figure 4g). To confirm the regulatory role trend to the key genes of the flavonoid pathway. SlERF.G3-Like is
of SlERF.G3-Like in the expression of cell wall-related genes, we involved in regulating the flavonoid pathway, and increased
assessed their transcript levels in CR, OE and WT fruits by RT- flavonoid biosynthesis reduces the availability of these precursors,
qPCR (Figure S7). thereby directing a change in the direction of metabolic flow and
A dual-luciferase assay was performed to detect whether thus reducing lignin biosynthesis (Figure 5). The redistributed
SlERF.G3-Like could directly regulate the promoter activity of metabolic flow also contributes to the softening phenotype of
SlPG2a and SlPL. The results showed that SlERF.G3-Like could p35S:SlERF.G3-Like fruits.
Figure 4 SlERF.G3-Like influences the cell wall by regulating the expression of SlPG2a and SlPL. (a) Paraffin transverse sections of pericarp tissues at the Br
stage in CR, OE and WT lines. Bars = 200 lm. (b) Micrographs of fruit epicarp transverse sections at Br stage in CR, OE and WT lines to visualize the cuticle.
Bars = 25 lm. (c) The firmness of tomato fruit at the Br + 10 stage. Red asterisks indicate statistical significance using the Student’s t-test, *P < 0.05;
**P < 0.01, n = 10. (d-f) Statistical analysis regarding cuticle thickness (n = 23), cell size (average value in one vision, n = 6) and cell number in one image
(n = 6). Red asterisks indicate statistical significance using the Student’s t-test, *P < 0.05; **P < 0.01. (g) The expression levels of cell wall-related genes.
Bars represent standard deviation (SD) of three independent replicates. Asterisks indicate statistical significance using the Student’s t-test, *P < 0.05;
**P < 0.01. (h) SlERF.G3-Like induces the activities of proSlPG2a / proSlPL in the Dual-Luc assay. The effectors were SlERF.G3-Like or SlRIN-driven by
CaMV35S. Each value represents the mean of three biological replicates. The lowercase indicates a significant difference (one-way ANOVA, Tukey’s post-
hoc test, P < 0.05).
WT (Zhong et al., 2013) (Figure S8c). RT-qPCR data showed the

SlRIN directly activates the expression of SlERF.G3-Like
same result (Figure S8d). Based on these results, SlRIN is a
during tomato ripening
potential upstream factor of SlERF.G3-Like.
Our data indicate SlERF.G3-Like can regulate multiple ripening- To verify this hypothesis, we tested whether SlRIN can directly
related pathways. Interestingly, the expression level of SlERF.G3- activate transcription from the SlERF.G3-Like promoter with
Like is significantly co-expressed with the master ripening transient expression assays in N. benthamiana leaves. Luciferase
regulator RIN in the Tomato Expression Atlas (TEA; http://tea. (LUC) activity derived from proSlERF.G3-Like: LUC was approxi-
solgenomics.net/) database (Figure S8a). Previously, analysis data mately 25–30 fold higher than the control (Figure 6b). Although
revealed that MADS-box proteins could bind to the specific DNA SlRIN appears to promote SlERF.G3-Like transcription, the specific
sequences known as CArG-box, including C(C/T)(A/T)6(A/G)G, C binding site is unknown. Since there are five CArG motifs within
(A/T)8G or C(C/T)(A/T)G(A/T)4(A/G)G (Fujisawa et al., 2013; Ito 1425 bp, and we also predicted two possible binding sites at
et al., 2008). As shown in Figure 6a, the 1.5 kb promoter of 30 bp and 280 bp by data analysis, we truncated the
SlERF.G3-Like contains five CArG-box motifs, which are putative promoter region to confirm the actual binding site further. We
binding motifs of MADS proteins. A ChIP-seq dataset that divided the SlERF.G3-Like promoter into five fragments, desig-
predicted direct RIN targets showed significant fragment enrich- nated P1 to P5, with the P2 fragment containing the three CArG-
ment at the promoter of SlERF.G3-Like (Zhong et al., 2013) box motifs: CArG-box2, located 1033-1023 bp, CArG-box3,
(Figure S8b). Furthermore, the expression level of SlERF.G3-Like located 948-938 bp and CArG-box4, located 913-903 bp
was markedly reduced by 98% in the rin mutant compared with upstream of the translational start codon (the first nucleotide of
Figure 5 SlERF.G3-Like controls the expression of genes involved in the phenylpropanoid pathway. The schematic diagram of phenylpropanoid,
flavonoid and lignin biosynthetic pathways. SlERF.G3-Like directly regulated genes are shown by yellow arrows. Colours signify the average of three
technical duplications of the expression level from RNA-seq data for the respective genes. Red and blue show upregulation and downregulation. Each of
the squares represents a different line, as shown in the top left of the figure. PAL, phenylalanine ammonia-lyase; C4H, cinnamic acid 4-hydroxylase;
4CL, 4-coumaroyl-CoA ligase; CHS, chalcone synthase; CHI, chalcone isomerase; F3H, flavanone 3-hydroxylase; F3’H, flavanone 30 -hydroxylase;
F3’5’H, flavanone 30 ,50 -hydroxylase; FLS, flavonol synthase; HCT, hydroxycinnamoyl-coenzyme A: quinate/shikimate hydroxycinnamoyl transferase;
C3H, p-coumarate 3-hydroxylase; COMT, caffeic acid O-methyltransferase; CCR, cinnamoyl-CoA reductase; CAD, cinnamyl alcohol dehydrogenase;
LAC, laccase; PRX, peroxidase.
the ATG start codon being designated as 1), respectively expression, and all of the CArG motifs, the CArG-box2 and CArG-
(Figure 6a). The rest of the fragment information is shown in box5 motifs, were critical binding sites.
Figure 6a. From the results (Figure 6b), the truncated P2 and P3
Application of SlERF.G3-Like in metabolic engineering
fragments still have a strong activation, and the P2 fragment has
a more significant activation than the full-length fragment. This Previous studies indicate that increasing the demands of
result indicated that the CArG-box1 motif might be invalid. particular metabolic pathways would redirect the metabolic flux
Next, an electrophoretic mobility shift assay (EMSA) revealed towards the pathways of interest (Morandini, 2013). In addition,
that the GST–SlRIN fusion protein directly bound to SlERF.G3-Like the expression levels of genes involved in the glycolysis pathway
CArG-box2 and CArG-box5 motifs (Figure 6g,h and S9a-d). (SlENO et al.), the shikimate pathway (SlDAHPS, SlEPSPS2 et al.)
Moreover, yeast one-hybrid (Y1H) analyses indicated that SlRIN and the phenylpropanoid pathway (SlPAL, Sl4CL et al.) are
binds to the two fragments containing the 3* CArG-box2 and significantly induced in the p35S:SlERF.G3-Like lines (Figure 2d).
CArG-box5 elements (Figure 6e,f). The data showed that over- These genes dominate in guiding metabolic flux into the
expression of SlRIN strongly activated the promoter of SlERF.G3- phenylpropanoid pathway (Ying et al., 2020; Zhang et al.,
Like, whereas mutation of the CArG-box2 and CArG-box5 motifs 2015a). Therefore, we hypothesize that SlERF.G3-Like can
in the SlERF.G3-Like promoter significantly reduced its activity significantly increase the demands of flavonol biosynthesis to
(Figure 6c,d). Notably, the activation capacity of SlRIN was not guide the metabolic flux into phenylpropanoid biosynthesis. And
wholly lost, implying that there may be other remaining binding it might be a new tool for engineering phenylpropanoid compounds
sites. The results indicated that SlRIN could bind directly to the in tomato fruit. We then attempted to use SlERF.G3-Like to redirect
CArG motif upstream of the SlERF.G3-Like promoter to induce its metabolism to produce specialized metabolites. Resveratrol is a
Figure 6 SlRIN directly regulates the expression of SlERF.G3-Like during tomato ripening. (a) Schematic diagram of CArG motifs in proSlERF.G3-Like. Dark
blue squares indicate the positions of the five CArG elements, and light blue squares represent predicted binding sites. The reporter vector contained
different promoter fragments of proSlERF.G3-Like fused to LUC. (b) Dual-luciferase assay showing relative SlRIN activation of the different promoter
fragments of proSlERF.G3-Like. Each value represents the mean of three biological replicates. Asterisks indicate statistical significance using the Student’s
t-test, *P < 0.05; **P < 0.01. (c,d) SlRIN directly binds to the CArG motif in proSlERF.G3-Like to induce its expression. A mutated version
(mproSlERF.G3-Like) was designed as a negative control. Each value represents the mean of three biological replicates. The lowercase indicates a
significant difference (one-way ANOVA, Tukey’s post-hoc test, P < 0.05). (e,f) Y1H assay showing the binding of SlRIN to the proSlERF.G3-Like. The
SlERF.G3-Like promoter sequence from 688 to 589 contains the one CArG motif fused to the HIS2 reporter gene. The SlERF.G3-Like promoter from
1214 to 1184 was repeated three times and fused to the HIS2 reporter gene in the Y1H assay. pDEST22-EV, empty vector used as the negative
control; pDEST22-SlRIN, prey vector containing SlRIN. (g,h) EMSA shows the binding of SlRIN to the proSlERF.G3-Like. + and indicate the presence
and absence of the probe or protein. (i) The regulatory model of the comprehensive roles of SlERF.G3-Like.
natural polyphenolic antioxidant widely known for its potential hybrid tomato plants improved the production of resveratrol
health benefits (Vestergaard and Ingmer, 2019). Therefore, it is a derivatives from ~1.44 mg/g DW in E8:VvStSy tomato fruits to
promising target for metabolic engineering using plant chassis. ~2.31 mg/g DW in E8: VvStSy x p35S: SlERF.G3-Like line and
Previous data showed that the p35S: VvStSy tomato, although ~2.43 mg/g DW in p35S: SlERF.G3-Like x E8:VvStSy line (Figure 7c;
producing resveratrol compounds in its fruit, showed poor fertility Figures S12,S13; Table S3). Further enhanced resveratrol produc-
and stress tolerance. This was probably due to the side effects of tion indicates the potential of SlERF.G3-Like as an efficient tool
resveratrol overexpression in vegetative tissues (Ingrosso et al., with potential for metabolic engineering
2011). To engineer new tomato lines with resveratrol production
without yield penalties, we overexpressed VvStSy, the key
Discussion
resveratrol biosynthetic gene, under the fruit-specific E8 promoter
and selected line#9 to cross with p35S: SlERF.G3-Like tomato SlERF.G3-Like was initially identified as a positive regulator for the
(Figures S10a–d and S11a–c). Both the F1 generation hybrids and flavonoid pathway in tomato fruit (Li et al., 2020b). Changes in
the p35S: SlERF.G3-Like showed orange phenotype compared with carotenoids are widely characterized during maturation (Klee and
WT fruits at the Br + 10 stage (Figure 7b). We further analysed the Giovannoni, 2011; Seymour et al., 2013), while several studies
contents of resveratrol and its derivatives, including resveratrol, have reported the regulation of the flavonoid pathway during the
piceid (resveratrol glycosides, Res-Glc), piceid isoform, methylated tomato ripening process (Adato et al., 2009; Li et al., 2020b;
resveratrol-glycosides (MeRes-Glc) and resveratrol-di-glycosides Zhang et al., 2015a). In tomatoes, naringenin chalcone was
(Res-Glc-Glc) (Zhang et al., 2015a). LC–MS analysis indicated significantly lower in fruits of FUL1/FUL2-suppressed lines than in
simultaneous overexpression of VvStSy and SlERF.G3-Like in F1 wild-type fruits (Fujisawa et al., 2014). The previous study
Figure 7 Application of SlERF.G3-Like in resveratrol metabolic engineering. (a) Schematic of the regulatory network of SlERF.G3-Like to the resveratrol
metabolic engineering. (b) Fruit phenotypes of MicroTom at the Br + 10 stage. (c) LC–MS analysis of the total resveratrol contents, including resveratrol,
piceid (Res-Glc), piceid isoform, methylated resveratrol-glycosides (MeRes-Glc) and resveratrol-di-glycosides (Res-Glc-Glc) in different transgenic and hybrid
tomato lines. ND, not detected.
reported the regulation of SlERF.G3-Like in the flavonoid pathway increasing carotenoid contents in CR fruit could be due to
(Li et al., 2020b), and in this research, new evidence is provided the reduction of flux into flavonoid biosynthesis. The previous
for the accumulation of flavonoids during the process of data indicate that the induction of carotenoid biosynthesis by
maturation. Notably, a yeast two-hybrid assay observed an SlWRKY35 could lead to decreased flavonoids, suggesting that
interaction between FUL1 and SlERF.G3-Like proteins both pathways could compete for substrates (Yuan et al., 2022).
(Figure S14). These results predict that, in addition to directly Although carotenoid accumulation was present in the final fruits
activating the expression of key structural genes of the flavonoid of CR lines, unlike direct targeting of SlPSY1 and thus affecting
pathway, SlERF.G3-Like may be involved in the regulation of the carotenoid content, this could be a cascade change resulting from
flavonoid pathway by interacting directly or indirectly with FUL1 the redistribution of metabolic flow caused by the induction of
in fruit ripening. flavonoid biosynthetic genes by SlERF.G3-Like (Fu et al., 2018).
SlERF.G3-Like is also found to be involved in the regulation of We previously analysed the expression profile of carotenoid
ethylene synthesis and the hardness of cell walls during tomato biosynthesis genes in transcriptomic data of E8:SlERF.G3-Like
fruit ripening. Overexpression of SlERF.G3-Like under constitutive lines, and no significant changes were observed among those
promoter 35S activated the expression of SlACO1 and SlACS2, lines (Li et al., 2020b). In addition, the Dual-Luc assay verified that
which play a significant function in the ethylene synthesis SlERF.G3-Like does not activate the promoters of SlPSY1 and
pathway (Liu et al., 2015), resulting in increased ethylene SlPDS (Figure S16). All these data suggest that SlERF.G3-Like does
production. The cell wall phenotype and DEGs suggest that not directly affect carotenoid biosynthetic genes.
SlERF.G3-Like directly affects cell wall texture. The promoter of As a climacteric fruit, the ripening process of tomato fruit
SlPL, which by far has the most determined impact on the involves complex changes in morphology and metabolites,
hardness of the cell wall, is positively regulated by SlERF.G3-Like triggered by the strong ethylene production during the ripening
and can be activated in vitro (Yang et al., 2017). SlPG2a is process (Seymour et al., 2013). Several master regulators have been
another gene involved in the cell wall that SlERF.G3-Like can identified, such as RIN, a MADS-box transcription factor identified
directly activate. Other reported cell wall-associated enzymes, and characterized by an originally cloned rin tomato mutation
such as SlEXP1, SlTBG, SlMAN, SlXY and SlXyn are significantly (Vrebalov et al., 2002). The rin mutant led to a ripening-inhibition
increased in OE lines while showing the opposite trend in CR phenotype, indicating the central role of RIN during tomato
lines. Whether these genes are direct targets of SlERF.G3-Like ripening (Fujisawa et al., 2011, 2013; Vrebalov et al., 2002).
needs further confirmation. SlLOB1 was reported to target SlEXP1 Recent studies showed that rin is a gain-of-function mutant, and
in tomato directly, and the transcript and protein levels of there are differences between the phenotypes of the RIN knockout
SlEXP1 were strongly repressed in RNAi lines (Shi et al., 2021). and rin mutant (Ito et al., 2017; Li et al., 2018a). However, there is
SlLOB1 was a DEG in RNA-seq data, with decreased expression in no doubt that RIN is required for the progression of ripening and
CR lines and increased expression in OE lines. In addition, these plays a central role in tomato fruit ripening (Li et al., 2020a).
two genes have different tissue expression profiles and functions. Considering the central role of RIN during ripening, there
SlLOB1 was initially induced at the mature green (MG) stage, is should be a hierarchical transcriptional cascade for the accurate
highly expressed during the BR stage and has the highest non- regulation of various downstream morphologic and metabolic
fruit expression in stems (Shi et al., 2021). SlERF.G3-Like changes. Similar regulatory networks have been established in
expression occurs specifically at the fruit ripening stages. Unlike other plant hormone signalling pathways. For example,
SlERF.G3-Like, SlLOB1 did not affect other tomato ripening OsDREB1C, a transcriptional regulator belonging to the AP2/ERF
phenotypes, including the maximum ethylene production and the family, can be involved in the regulation of both photosynthesis
onset of ripening. According to our findings, p35S:SlERF.G3-Like and nitrogen utilization (Wei et al., 2022). In the JA signalling
has a significantly shorter time to induce ethylene production and pathway, the master regulator MYC2 and its downstream MYC2-
a higher maximum ethylene production than WT. This result targeted transcription factors (MTFs) form various MYC2-MTF
suggests that SlLOB1 has more specific functions for cell wall modules, which initiate and amplify specific aspects of the JA-
changes, while SlERF.G3-Like has a broader pleiotropic effect on signalling transcriptional output: the MYC2-JA2L module medi-
the maturation process. ates the wound response while the MYC2-ERF.C3 module
In the wild-type tomato fruit, the peel is orange predominantly regulates the pathogen response (Du et al., 2017).
due to flavonoids, while the flesh is red mainly due to carotenoids Transcriptomic data and phenotypes suggest that SlERF.G3-Like
(Adato et al., 2009; Fernandez-Moreno et al., 2016; Wang is an intermediate regulator influencing the most physiological
et al., 2018b). However, when TFs regulating flavonoid biosyn- subdomain of the maturation transition. Combining Dual-Luc, Y1H
thesis were overexpressed throughout the fruit, the tomato fruit and EMSA assays, we verified that SlRIN is an upstream regulator of
pericarps were enriched with flavonoids and turned yellow (Luo SlERF.G3-Like. SlERF.G3-Like is an essential intermediate compo-
et al., 2008; Ying et al., 2020). Our data support the idea that nent of the SlRIN regulatory network. Systematic elucidation of
overexpression of SlERF.G3-Like affects the onset of ripening. regulatory networks at all levels contributes to further understand-
Eventually, OE fruits show an orange colour mainly due to the ing of the molecular mechanisms of master regulators.
enrichment of flavonoids. We examined the carotenoid content Notably, many known intermediate TFs in the RIN hierarchical
(mainly lycopene, lutein, zeaxanthin, a-carotene and b-carotene) transcriptional cascade regulate a single aspect of ripening. For
in fruits at the Br + 10 stage, and there was no change in the instance, our previous studies indicate that SlRIN can directly
total carotenoid content in OE and WT lines (Figure S15). In activate the expression of SlWRKY35, an inducer of SlDXS1, to
contrast, there was a tendency for the total carotenoid content to guide carbon flux towards the MEP pathway and enhance
increase in the CR line compared with WT, but the changes were downstream carotenoid biosynthesis (Yuan et al., 2022). In the
different for each compound specifically. For example, lycopene meantime, SlRIN can also inhibit the expression of SlGCR, an
was elevated in CR fruits relative to WT fruits, but b-carotene was inducer of SlLCYE, to suppress the production of lutein from the
decreased in CR lines and increased in OE lines (Table S2). The main carotenoid pathway (Ren et al., 2022). Together, the SlRIN-
WRKY35 and SlRIN-SlGCR modules accurately regulate the Agrobacterium-mediated tomato plant transformation and were
significant induction of carotenoid biosynthesis during tomato introduced into Agrobacterium tumefaciens strain EHA105 (Ying
ripening. The current SlRIN-SlERF.G3-Like module, however, is at et al., 2020).
least associated with ethylene production, cell wall degradation
RNA extraction and RT-qPCR analysis
and phenylpropanoid metabolism. The hierarchical transcriptional
cascade of plant hormone signalling seems more complicated Total RNA was extracted from tomato fruit using the Fastpure Plant
than expected. More intermediate TFs are yet to be identified. Total RNA Isolation Kit (Vazyme Nanjing, China; RC401-01)
Due to their medicinal functions, tomato fruit enriched with following the instructions. First-strand cDNA was reverse tran-
specialized metabolites is suitable for processing purposes rather scribed from 1 lg of total RNA using the PrimeScriptTM RT reagent
than fresh consumption (Li et al., 2018b). Therefore, normal, kit (AK4201; Takara Bio, Kusatsu, Japan) according to the
even fast, softening is an ideal trait for such types of varieties. manufacturer’s instructions. RT-qPCR was performed using AceQ
However, when TFs such as AtMYB12 and Del/Ros1 were used as Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China; Q511-
metabolic engineering tools, fruit ripening and over-ripening 02) on a Bio-Rad CFX384 Real-Time PCR System (Initial incubation
were significantly delayed due to the increased antioxidant at 95 °C for 3 min, followed by 40 cycles of 95 °C for 10 s and 60 °C
capacity by flavonoid enrichment (Zhang et al., 2013, 2015b). for 30 s, and added melting curve analysis during the 65–95 °C).
Our study indicates SlERF.G3-Like can positively regulate tomato The primer sequences used in this study are listed in Table S1.
ripening by activating the expression of genes involved in
Transcriptome profiling
ethylene synthesis, cell wall degradation and phenylpropanoid
pathway. Therefore, it can be an ideal tool for engineering high Total RNA was extracted from the fruits at 39 DPA for WT,
antioxidant compounds in tomato fruit while maintaining the overexpression and gene-edited lines. RNA-Seq library construc-
ordinary ripening and over-ripening processes. tion and high-throughput sequencing were conducted by
To summarize, our results suggest that SlERF.G3-Like drives Novogene (Beijing, China). The library preparations were
functionally diverse transcriptional programmes that regulate sequenced on an Illumina Novaseq 6000 platform, and 150 bp
ethylene synthesis, cell wall degradation and the phenylpropanoid paired-end reads were generated. Transcriptome analysis was
pathway during tomato ripening. And SlRIN, which plays a global carried out as previously described (Yuan et al., 2022). DEGs were
regulatory role in tomato fruit ripening, can control its expression. found using a significance threshold of a log2 fold change of 1.
The conservative role of the SlERF.G3-Like remains to be further RNA seq data reported in this paper have been deposited in the
explored. Overall, our data revealed the comprehensive roles of Genome Sequence Archive (Chen et al., 2021) in National
SlERF.G3-Like, and, in particular, provided new evidence linking Genomics Data Center, China National Center for Bioinforma-
the ripening and the phenylpropanoid pathway during the tion/Beijing Institute of Genomics, Chinese Academy of Sciences
maturation of tomato fruits. This work offers new insights into (GSA: CRA009040) and are publicly accessible at https://ngdc.
how plants control intricate biological processes despite limited cncb.ac.cn/gsa.
genomic information. Since targeting global regulators like SlRIN
Ethylene production measurements
causes many phenotypic changes, including some adverse effects
such as flavour (Osorio, Carneiro et al. 2020), these hierarchical Ethylene production was measured as specified previously (Deng
transcriptional regulators can act as better breeding targets and et al., 2022) with a few minor adjustments. Before measurement,
offer a theoretical foundation for precision breeding. We also fruits gathered at different stages were placed in 120 mL open jars.
attest that SlERF.G3-Like can be an effective tool for phenylpro- After 0.5 h, the jars were sealed and incubated at room temperature
panoid metabolic engineering in processing tomatoes. for 2 h until measurement (Agilent 7890B gas chromatography).
The final ethylene production was obtained by comparing it with the
Materials and methods peak area of ethylene at known concentrations.
Plant materials and growth conditions Fruit firmness
A tomato (Solanum lycopersicum cv. MicroTom) was purchased More than twenty fruits from each line were harvested at the
from PanAmerican SeedTM. Tomato plants were grown in a Br + 15 stage, and the firmness was measured with a TA.XTC-18
greenhouse with standard conditions (16/8 h of light/dark cycle texture analyser (BOSIN, Shanghai) with a P/2 columnar probe
at 24 °C in 60% humidity and under 250 lmol/m2/s1 intense (2 mm diameter). The test parameters were set as follows: pre-
luminosity) and watered daily. Nicotiana Benthamiana was grown test speed 5 mm sec1; test speed 1 mm sec1; post-test speed
in appropriate greenhouses for the dual-luciferase reporter assay. 5 mm sec1; penetration depth 5 mm; trigger force 5 g. The
Plasmid construction and plant transformation value of firmness was calculated by BosinTech TAS.
The overexpression vector was assembled by the GoldenBraid2.0 Colour measurement

cloning strategy (Sarrion-Perdigones et al., 2013). The full length of The colour values are measured with a Konica Minolta chroma-
the coding sequence (CDS) of SlERF.G3-Like was introduced into the meter (CR-400) at various stages of ripening. The exact calculation
entry vector PUPD2, and one transcriptional unit was constructed method has been described in a previous study (Deng et al., 2018).
using the 35S promoter-transcription factor 35S terminator and the At least 20 fruits were measured for each line.
kanamycin resistance gene under the control of NOS promoter and
NOS terminator as an additional transcriptional unit. The CRISPR/ Analysis and identification of carotenoids and
Cas9 vector was kindly provided by Dr. Li Zhengguo (College of Life resveratrol
Sciences, Chongqing University). A target site was designed for the For LC–MS analysis of compounds, fruits were harvested at the
SlERF.G3-Like based on the online tool CRISPR-P 2.0 (http://cbi. Br + 10 stage and immediately placed in liquid nitrogen, then
hzau.edu.cn/crispr/). These constructed vectors were used for lyophilized and ground into a fine powder for extraction.
The carotenoids were extracted as described previously. All LUC/REN. For the Luciferase reporter assay, two different
the above operations must be carried out away from bright detection methods were used in this study. Another transactiva-
light. And the carotenoids were determined by LC–MS (the tion assay to test the regulation of SlERF.G3-Like on the SlACS2
Sciex TRIPLE QUADTM 5500 platform in Life Science Core and SlACO1 promoters, using D-Luciferin (Solarbio, D9390) as
Facilities, College of Life Sciences, Sichuan University) using a substrate, was performed using an IVIS Lumina Series III Pre-
YMC C30 chromatographic column (Thermo AccucoreTM C30 clinical In Vivo Imaging system (PerkinElmer), and the lumines-
chromatographic column, USA) and controlled by Analyst 1.6.3 cence intensity was detected by a luminometer.
software (AB Sciex). Mass spectrometry was performed using
Yeast One-Hybrid assays
an atmospheric pressure chemical ionization (APCI) source
operating in a negative ion mode. The carotenoids were The yeast onehybrid assay was performed as described previously
identified with standards and quantified by integrating the (Ying et al., 2020) with modifications. DNA fragments consisting
peak areas. Standards were purchased from Sigma-Aldrich (St of three copies of the proSlERF.G3-Like (1214 to 1184 bp)
Louis, MO; https://www.sigmaaldrich.com/). The detection was were chemically synthesized and cloned into the pHis2-Leu-GW
performed for lutein, zeaxanthin, a-carotene, b-carotene and through Gateway Cloning. And another upstream fragment
lycopene. Three independent replicates were used for the (688 to 589 bp) of the SlERF.G3-Like was cloned into the
analysis. pHis2-Leu-GW reporter vector. A full-length CDS of SlRIN was
Resveratrol was extracted as previously described (Zhang introduced into the pDEST22 vector. The pDEST22-EV (empty
et al., 2015a) with slight modifications. An amount of 0.01 g vector) was used as a negative control. Then, different
of dry powder was extracted with 1 mL 80% MeOH at 4 °C by combinations of effector and reporter were co-transformed into
ultrasonic extraction. The sample was then centrifuged at 3000 g the Y187 yeast strain. The transformants were grown on the SD/
at 4 °C for 10 min, and the supernatant was taken. The Leu/Trp selection medium, and the positive yeast cells were
supernatant was then centrifuged at 18 000 g at 4 °C for diluted with a 10 3 dilution series and dotted on SD/-Leu/-Trp/-
10 min. The supernatant was further diluted 20 times with 80% His medium with different concentrations of 3-AT as screening
MeOH. The samples were filtered through a 0.45 mm filter pressures.
before injection. For each line, three biological replicates were
Electrophoretic mobility shift assay (EMSA)
analysed. Moreover, resveratrol was determined by LC–MS using
a C18 chromatographic column (Thermo ScientificTM). Mass The full-length coding sequence of SlRIN was amplified by PCR
spectrometry was performed using an Electron Spray Ionization and cloned into pGEX-4 T-1–GST named pGEX-4 T-1–GST–SlRIN
(ESI) source operating in a negative ion mode. (Yuan et al., 2022). Oligonucleotide probes were synthesized and
labelled with 50 biotin (Sangon Biotech, China). EMSAs were
Cytological assessment of fruit pericarp
performed as previously described (Hellman and Fried, 2007;
The fresh fruits were trimmed from the target tissue with a scalpel Yuan et al., 2022). Briefly, biotin-labelled probes were incubated
and put into fixative (G1101, Wuhan Servicebio Technology Co., with GST-fusion proteins at room temperature for 20 min, and
Ltd, China) for more than 24 h. After dehydration with gradient free and bound DNAs were separated in an acrylamide gel.
alcohol, the tissue is embedded in paraffin wax at 65 °C. The slice Unlabelled wild-type probes were used as competitors. Electro-
transverse sections of 4 lm thickness were cut with the paraffin phoretic mobility shift assay binding reactions were detected
slicer and stained with Safranin O-Fast Green staining solution according to the manufacturer’s instructions (Chemiluminescent
(G1031, Servicebio). Oil-Red solution (G1015, Servicebio) stained Nucleic Acid Detection Module Kit, Thermo Scientific). The probes
slices were chosen for cuticle observation. Multiplex slice images used for EMSA are listed in Table S1.
were acquired through a whole slide imaging system (Olympus
VS200) at 209 objective. Cytological assessment, including
Acknowledgements
cuticle thickness, cell size and cell number, was performed using
ImageJ software. This study was funded by the Natural Science Foundation of
Sichuan Province, China (2023NSFSC1991), the National Natural
Luciferase reporter assay
Science Foundation of China (32100213), the Fundamental
The full-length CDS of SlERF.G3-Like and SlRIN under the control Research Funds for the Central Universities (SCU2023D0003)
of the 35S promoter were used as effectors and constructed into and the Institutional Research Fund of Sichuan University
the destination vector pEAQ-HT-DEST1 using Gateway Cloning. (2020SCUNL106). We acknowledge the Mass Spectrometry Core
The remaining promoters involved the promoters of SlACS2, Facility in the College of Life Sciences, Sichuan University, for
SlACO1, SlPG2a, SlPL and SlERF.G3-Like; each promoter fragment assistance in metabolic analysis.
was cloned upstream of the LUC (luciferase) CDS using the
GoldenBraid 2.0 cloning strategy. The internal control REN
Conflicts of interests
(Renilla luciferase), driven by the 35S promoter, was also
constructed to act as an internal reporter at the same level as All authors declare no competing interests.
the transcriptional unit. The pEAQ-HT-DEST1 empty vector was
used as a negative control (CK), and specific steps were
Author contributions
performed as described previously (Ying et al., 2020). The LUC
and REN activities were assayed using the Dual-Luciferase YZ and SJY designed the experiments. SJY, YW, WL, XL, QT, FKH,
Reporter Assay System (Promega, Madison, WI, USA; E1910) by YL and HW conducted the experiments. ML helped to analyse the
a SynergyTM H1 hybrid multimode microplate reader (BioTek). At data and contributed to supervising this study. SJY and YZ wrote
least 3 independent biological replicates were performed for the paper with the input of all the authors. All authors have
each experiment. Results were expressed as relative values of approved this manuscript.
RIPENING INHIBITOR reveals the regulation of fruit ripening. Plant Cell, 25,
Data availability statement 371–386.
Fujisawa, M., Shima, Y., Nakagawa, H., Kitagawa, M., Kimbara, J., Nakano, T.,
The data that supports the findings of this study are available in
Kasumi, T. et al. (2014) Transcriptional regulation of fruit ripening by tomato
the supplementary material of this article.
FRUITFULL homologs and associated MADS box proteins. Plant Cell, 26, 89–
101.
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Supporting Information section at the end of the article.
1649–1672. Figure S1 Identification and sequencing of CRISPR/Cas9-
Smith, C.J.S., Watson, C.F., Ray, J., Bird, C.R., Morris, P.C., Schuch, W. and SlERF.G3-Like lines.
Grierson, D. (1988) Antisense RNA inhibition of polygalacturonase gene
Figure S2 The expression levels of SlERF.G3-Like in p35S:
expression in transgenic tomatoes. Nature, 334, 724–726.
SlERF.G3-Like lines.
Sun, C., Deng, L., Du, M., Zhao, J., Chen, Q., Huang, T., Jiang, H. et al. (2020) A
transcriptional network promotes anthocyanin biosynthesis in tomato flesh.
Figure S3 Phenotypes of CR, OE and WT lines.
Mol. Plant, 13, 42–58. Figure S4 Analysis of transcriptome data for CR, OE and WT
Theologis, A., Thomas, Oeller, P.W., Liang, X. and Abel, S.J.P.p. (1992) lines.
Modification of Fruit Ripening by Suppressing. Gene Expr. 100, 549–551. Figure S5 SlERF.G3-Like directly regulates the expression of
Valletta, A., Iozia, L.M. and Leonelli, F. (2021) Impact of environmental factors SlACO1.
on stilbene biosynthesis. Plants (Basel), (10), 90. Figure S6 Response of SlERF.G3-Like to ethylene and ethylene
Vanholme, R., De Meester, B., Ralph, J. and Boerjan, W. (2019) Lignin triple-response.
biosynthesis and its integration into metabolism. Curr. Opin. Biotechnol. 56, Figure S7 Validation of RNA-seq results by RT-qPCR.
230–239.
Figure S8 SlRIN is a potential regulator for SlERF.G3-Like gene Figure S16 Trans-activation effects of SlERF.G3-Like on pro-
expression. moters of SlPDS and SlPSY1.
Figure S9 SlRIN directly binds to the promoter of SlERF.G3-Like. Table S1 Primers used in this study.
Figure S10. Screening of E8:VvStSy overexpression plants. Table S2 Carotenoid contents of tomato fruit in WT, OE and CR
Figure S11. PCR identifies hybrid tomato plants concurrently lines.
overexpressing SlERF.G3-Like and VvStSy. Table S3 The detailed contents of resveratrol and other
Figure S12 The different expression levels of SlERF.G3-Like and derivatives in the hybrid tomato fruit.
VvStSy were tested by RT-qPCR in different lines. Data S1 All sample_RNA-seq_TPM.
Figure S13 Identification of resveratrol and its derivatives. Data S2 Expression levels of genes upregulated in OE fruits but
Figure S14 Y2H assays for the interactions between SlERF.G3- downregulated in CR, or genes downregulated in OE fruits but
Like and SlFUL1. upregulated in CR lines.
Figure S15 Carotenoid contents in CR, OE and WT fruits.

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Plant Biotechnology Journal (2023), pp. 1–16 doi: 10.1111/pbi.

SlERF.G3-Like mediates a hierarchical transcriptional

Received 14 March 2023; Summary

factors in the regulation of ethylene biosyntheses, such as

WT (Zhong et al., 2013) (Figure S8c). RT-qPCR data showed the

The overexpression vector was assembled by the GoldenBraid2.0 Colour measurement

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