The Plant Journal (2018) 94, 513–524 doi: 10.1111/tpj.

13872

CRISPR/Cas9-mediated mutagenesis of lncRNA1459 alters

tomato fruit ripening
Ran Li, Daqi Fu, Benzhong Zhu, Yunbo Luo and Hongliang Zhu*
The College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, China

Received 19 October 2017; revised 2 February 2018; accepted 12 February 2018; published online 15 February 2018.
*For correspondence (e-mail hlzhu@cau.edu.cn).

SUMMARY
With the development of high-throughput sequencing, many long non-coding RNAs (lncRNAs) have been
found to play important roles in diverse biological processes. However, the biological functions of most
plant lncRNAs are still unknown. We have previously discovered a tomato ripening-related lncRNA,
lncRNA1459. Here, we cloned the full-length lncRNA1459, giving two transcript isoforms. In addition,
lncRNA1459 exhibited a specific location in the nucleus. Furthermore, in order to fully identify the function
of lncRNA1459 in tomato ripening, loss-of-function mutants of lncRNA1459 were developed using clustered
regularly interspaced short palindromic repeats (CRISPR)/-associated protein 9 (Cas9)-induced genome edit-
ing technology. Compared with wild-type fruits, the tomato ripening process was significantly repressed in
lncRNA1459 mutants. Ethylene production and lycopene accumulation were largely repressed in
lncRNA1459 mutants. Additionally, genes related to ethylene and carotenoid biosynthesis were distinctly
downregulated in lncRNA1459 mutants compared with wild-type fruits. Moreover, expression of numerous
ripening-related genes was changed significantly when lncRNA1459 was knocked out. Expression of poten-
tial tomato ripening-related lncRNAs was also specifically changed after knocking out lncRNA1459. Taken
together, these results provide insight into the role of lncRNA1459 in tomato fruit ripening.

Keywords: tomato, fruit ripening, lncRNA, CRISPR/Cas9, full length, subcellular location.

INTRODUCTION

Tomato is an optimal model for development and ripening
of fleshy fruit due to its characteristics of simple diploid
genetics, ease of transformation, efficient propagation and
short life cycle. Fleshy fruit ripening is a developmental
process coordinated by a complex network of both
endogenous factors and exogenous elements, together
with a series of biochemical and physiological changes
such as color change, fruit softening, evolution of volatile
aromatics, accumulation of nutrients and so on (Giovan-
noni et al., 2017; Seymour et al., 2013). A large number of
transcription factors have been found to regulate tomato
fruit ripening in diverse biological processes, such as
MADS-RIN (Vrebalov et al., 2002), Nor (Giovannoni, 2007),
TAGL1 (Vrebalov et al., 2009) and the zinc finger transcrip-
tion factor SlAFP2 (Weng et al., 2015). Recently, long non-
coding RNAs (lncRNAs) have been proved to participate in
tomato fruit ripening (Sun and Xiao, 2015; Wang et al.,
2016a; Zhu et al., 2015).
Long non-coding RNAs are non-protein-coding tran-
scripts (longer than 200 nucleotides) often transcribed by
Pol ӀӀ (Guttman et al., 2013) and post-transcriptionally
modified by capping, polyadenylation and splicing (Beau-
lieu et al., 2012; Guttman et al., 2013; Ulitsky and Bartel,
2013). It has been demonstrated that lncRNAs interact with
DNA, RNA and protein to regulate gene expression by
DNA methylation, histone modification and chromatin
remodeling (Heo et al., 2013; Zhu and Wang, 2012). A large
number of lncRNAs have been definitely identified in
plants such as Arabidopsis (Wang et al., 2014; Zhu and
Wang, 2012), maize (Li et al., 2014), rice (Li et al., 2007),
wheat (Xin et al., 2011) and tomato (Zhu et al., 2015).
Moreover, the molecular functions of plant lncRNAs in
plants have been widely studied, especially in Arabidopsis.
Cold-induced long antisense intragenic non-coding RNA
(COOLAIR), transcribed from downstream of Flower Locus
C (FLC) in an antisense orientation is physically associated
with the FLC locus and promotes transcriptional shutdown
of FLC during vernalization, mediating the coordinated
switching of chromatin states at the FLC locus (Csorba
et al., 2014). Another lncRNA, ELF18-induced long-non-
coding RNA 1, directly interacted with Mediator subunit
19a (MED19a) and affected enrichment of MED19a on the

© 2018 The Authors 513

The Plant Journal © 2018 John Wiley & Sons Ltd

514 Ran Li et al.
PATHOGENESIS-RELATED GENE 1 (PR1) promoter (Seo
et al., 2017).
Unlike proteins and small non-coding RNA (ncRNAs),
the function of lncRNAs is complicated and obscure
because of their sequence or structure. Revealing the regu-
lation of lncRNAs in plants has proved difficult due to the
limitations of low expression levels and lack of sequence
conservation. Therefore, studies of plant lncRNAs were
stalled at the genome-wide identification stage rather than
discovery of the mechanism of biological function. Fortu-
nately, in recent years an explosion of technologies have
finally made it possible to directly address the where, what
and how of lncRNA function (Chu et al., 2015). Clustered
regularly interspaced short palindromic repeats (CRISPR)/-
associated protein 9 (Cas9)-induced genome editing tech-
nology has been an efficient and powerful technology for
the functional characterization of lncRNAs. The CRISPR/
Cas9 system comprises a Cas9 protein functioning as a
nuclease and sequence-specific single guide RNA (sgRNA)
which directs Cas9 to cut the target sequence, generating
double-strand breaks (DSBs) (Cong et al., 2013). The DSB
activates two independent endogenous DNA repair pro-
cesses: homologous recombination (HR) and non-homolo-
gous end joining (NHEJ) (Sonoda et al., 2006). In the
absence of a donor template, NHEJ is activated to repair
cleavage with insertion or deletion (indels) at the target
gene loci, resulting in loss of function (Cong et al., 2013;
Salomon and Puchta, 1998). The CRISPR/Cas9 technique
has several advantages in loss-of-function analysis: it
causes knockout of the target gene with relatively low off-
target activities, it efficiently disrupts the DNA sequence
and it causes heritable loss of function (Barrangou et al.,
2015). Hitherto, the multiplex CRISPR/Cas9 system has
been successfully applied to precisely edit tomato genomic
sequences and create multisite genome knockout muta-
tions (Li et al., 2017). However, there has been no report of
designing lncRNAs in tomato using CRISPR/Cas9
technology.
Our previous work annotated a ripening-related
lncRNA1459 in tomato (Zhu et al., 2015). Nevertheless, the
function of lncRNAs during fruit ripening is still unclear.
Here, we cloned the full length of lncRNA1459 and verified
the subcellular location of lncRNA1459. Furthermore, a
robust CRISPR/Cas9 system was chosen for its conve-
nience and high-efficiency multiplex genome editing ability
in plants (Ma et al., 2015) to obtain lncRNA1459 knock-out
mutants (CR-lncRNA1459). Two independent homozygous
CR-lncRNA1459 mutant lines showed significant delay of
ripening compared with wild-type (WT) fruits, with inhibi-
tion of ethylene production and lycopene accumulation. A
comprehensive set of differentially expressed genes
(DEGs) and differentially expressed lncRNAs (DELs) from
CR-lncRNA1459 mutants and WT fruits was discovered by
RNA sequencing (RNA-seq) analysis. These results proved
The Plant Journal © 2018 John Wiley & Sons Ltd, The Plant Journal, (2018), 94, 513–524
1365313x, 2018, 3, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/tpj.13872 by Indian Institute Of Technology, Kharagpur Central Library, Wiley Online Library on [07/11/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
that lncRNA1459 is a positive element during tomato fruit
ripening, providing a new insight into the function of
lncRNAs in fleshy fruit ripening.
RESULTS
Cloning the full length of lncRNA1459
Our previous study concerned a ripening-related lncRNA,
lncRNA1459, with a poly(A)+ tail and 50 7-methylguanylate
cap (Zhu et al., 2015). lncRNA1459 has been identified as
an sense intergenic lncRNA and a putative transcript with a
partial sequence. In this study, the 30 and 50 cDNA
sequences of lncRNA1459 were cloned by RNA ligase-
mediated rapid amplification of cDNA ends (RLM-RACE)
(Figure 1a). Due to its low expression in tomato,
lncRNA1459 was driven by the 35S promoter for transient
expression in Nicotiana benthamiana before 50 RACE. The
total RNA for 30 cDNA synthesis was extracted from WT
leaves. Two transcript variants, lncRNA1459 X1 (KT963310)
and lncRNA1459 X2 (KT963311), were obtained with only
one exon located on chromosome 3 (Figure 1b).
lncRNA1459 X2 was only 20 bp longer than lncRNA1459
X1 at the 30 region, but the rest of sequence was identical
between two transcript variants (see Figure S1 in the
online Supporting Information). Primers on the 30 and 50
regions were designed to amplify the full length of
lncRNA1459, obtaining a 834-bp fragment (Figure 1a). All
the lncRNA1459 sequences are shown in Figure S1. These
results indicate that lncRNA1459 is transcribed by Pol ӀӀ
and modified by capping and polyadenylation. Moreover,
it has recently been reported that several lncRNAs could
function as protein-coding genes (Anderson et al., 2015;
Nelson et al., 2016). lncRNA1459 does not seem to encode
a protein, although it has five short open reading frames
(ORFs) ranging from 11 to 30 amino acids (Figure S2).
Subcellular localization of lncRNA1459
To further examine the expression pattern of lncRNA1459,
quantitative PCR (qPCR) was used to detect the expression
pattern of lncRNA1459 between stem, leaf, flower and fruit.
The relative level of expression of lncRNA1459 in stem was
assigned as standard. Results showed that lncRNA1459
accumulated at the highest level in fruit, and no obvious
changes were detected between leaf and flower (with accu-
mulation approximately two-fold higher than in stem) (Fig-
ure 1c). As for proteins, the function of lncRNAs is
associated with their subcellular localization (Chen, 2016).
We therefore checked the subcellular localization of
lncRNA1459. Pure RNA was extracted from cytoplasmic
and nuclear fragments, respectively, and used for RT-
qPCR. U6 and Actin were used as internal controls for
nuclear and cytoplasmic extracts, respectively. U6 was
mainly detected in the nuclear fractions while Actin was
predominantly detected in the cytoplasmic fractions
© 2018 The Authors
 1365313x, 2018, 3, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/tpj.13872 by Indian Institute Of Technology, Kharagpur Central Library, Wiley Online Library on [07/11/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Tomato fruit ripening 515

Figure 1. Characteristics of lncRNA1459.

(a) The 50 and 30 end sequences of lncRNA1459 were obtained. 50 Rapid amplification of cDNA ends (50 RACE) was designed from Nicotiana benthamiana leaves
and 30 RACE was designed from tomato leaves. In addition, full-length of lncRNA1459 were obtained successfully. No RNA was used as control check (CK).
(b) Schematic diagram of the location of lncRNA1459 in a chromosome. Red boxes show the 50 RACE fragment sequences of lncRNA1459, black boxes show the
lncRNA1459 sequences from RNA-seq and blue boxes show the fragment sequences of lncRNA1459 by 30 RACE. The size of the diamond indicates the length of
lncRNA1459. Red letters indicate the overexpression portion for 50 RACE.
(c) Expression level of lncRNA1459 in different tomato tissues, determined by qPCR. The lncRNA1459 expression level was normalized by the Actin expression
level. The relative lncRNA1459 expression level in stem was assigned a value of 1. Error bars indicate SD of three biological replicates, with each measured in
triplicate. Samples marked with different letters show a significant difference at P < 0.05.
(d) Quantitative RT-PCR analysis from nuclear and cytoplasmic fractions of lncRNA1459; nuclear U6 and cytoplasmic Actin mRNA were used as controls. P1, P2
and P3 are three independent fragments for qRT-PCR analysis. Each sample was repeated with three biological replicates and each was measured in triplicate.

(Figure 1d). This indicated that there was minimal contami-
nation between the cytoplasm and nucleus. Three pair-
independent primers of lncRNA1459 were designed to
detect subcellular location (Figure 1b). The primers were
located in the common region between two transcripts
variants. All these qPCR results showed that lncRNA1459
was highly enriched in the nucleus (Figures 1d).
CRISPR/Cas9-engineered mutations in lncRNA1459 cause
great delay of ripening
To investigate the ripening-related function of lncRNA1459,
a CRISPR/Cas9 construct with one lncRNA1459 target was
designed and transformed into Solanum lycopersicum cv.
Ailsa Craig (AC) (Figure 2a). Three CRISPR/Cas9 (CR)-
lncRNA1459 mutant lines were detected from the first-gen-
eration transgenic plants (T0). A PCR amplification for the
presence of Cas9 and Hygromycin (Figure S3b) and T7
endonuclease Ӏ (T7EӀ) assay (Figure S3c), combined with
sequencing analysis, proved that all three transgenic lines
were heterozgyous mutants with small indels (Figure S3d).
Compared with the WT, CR-lncRNA1459-8 and CR-
lncRNA1459-21 obviously delayed fruit ripening until
35 days post-anthesis (DPA), while the maturation process
of CR-lncRNA1459-32 showed virtually no delay (Fig-
ure S3a). In addition, we selected four putative off-targets
to verify the specific knockout of lncRNA1459 in the modi-
fied T0 generation. No mutation was found in any putative
off-target site (Table S2).
CR-lncRNA1459-8 and CR-lncRNA1459-21 were chosen
for further research in the second generation (T1). Fifty-four
individual lines were detected in the T1 generation, with 31
lines of CR-lncRNA1459-8 and 23 lines of CR-lncRNA1459-
21 (Table S3). Two independent homozygous mutation
transgenic lines, CR-lncRNA1459-8-3 and CR-lncRNA1459-
21-1, showed similar and distinct ripening-delayed pheno-
types (Figure 2c, d). In addition, no mutation was found at
any putative off-target site in T1 generation (Table S4). Fur-
thermore, to confirm the specific repression of lncRNA1459

© 2018 The Authors

The Plant Journal © 2018 John Wiley & Sons Ltd, The Plant Journal, (2018), 94, 513–524
 1365313x, 2018, 3, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/tpj.13872 by Indian Institute Of Technology, Kharagpur Central Library, Wiley Online Library on [07/11/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
516 Ran Li et al.

Figure 2. Loss of function of lncRNA1459 specifically delayed tomato fruit ripening in T1 generation.
(a) One target site was designed on lncRNA1459 for clustered regularly interspaced short palindromic repeats (CRISPR)/-associated protein 9 (Cas9)-induced
genome editing technology. The target site was located on the blue box whose sequence were detected from 30 rapid amplification of cDNA ends (RACE) frag-
ment sequences of lncRNA1459. Red boxes show the 50 RACE fragment sequences of lncRNA1459 and black boxes show the RNA-seq data.
(b) Expression level of lncRNA1459 in wild-type (WT) and CR-lncRNA1459 mutants, determined by quantitative PCR. The lncRNA1459 expression level was nor-
malized by the Actin expression level. The relative lncRNA1459 expression level in WT was assigned a value of 1. Error bars indicate SD of three biological
replicates, with each measured in triplicate. Samples marked with different letters show a significant difference at P < 0.05.
(c) Phenotype of independent homozygous mutants CR-lncRNA1459-8-3 and CR-lncRNA1459-21-1 in the T1 generation. CR-lncRNA1459-8-3 and CR-lncRNA1459-
21-1 fruits were compared with WT fruits at 30, 35, 40, 45 and 50 DPA (days post-anthesis) ripening stages, showing a different color from 35 DPA.
(d) Genotype of CR-lncRNA1459-8-3 and CR-lncRNA1459-21-1 in the T1 generation. Both CR-lncRNA1459-8-3 and CR-lncRNA1459-21-1 were homozygous muta-
tions. Black dashed lines indicate base pair deletions. Red letters are target site sequences. Underlined letters are protospacer adjacent motif (PAM).

in the CR-lncRNA1459 mutants, fruits of the WT and trans- The CR-lncRNA1459 mutant exhibits impaired expression
genic mutants at the 35 DPA stage were used as samples of ripening-related genes and lncRNAs
to extract total RNA and submitted to qPCR analysis. The
The CR-lncRNA1459 mutants showed an obvious delayed-
transcript levels of lncRNA1459 were distinctly reduced in
ripening phenotype and inhibition of ethylene and
transgenic CR-lncRNA1459 mutants compared with the WT
lycopene production compared with the WT. It was
fruits (Figure 2b). These results proved that lncRNA1459 is
hypothesized that expression of some genes associated
necessary during tomato fruit ripening.

© 2018 The Authors

The Plant Journal © 2018 John Wiley & Sons Ltd, The Plant Journal, (2018), 94, 513–524

with ripening might be changed. Therefore, RNA-seq was
conducted to discover the DEGs and DELs from CR-
lncRNA1459-8-3, CR-lncRNA1459-21-1 and WT fruits at 35
DPA. The RNA-seq experiment was repeated with an inde-
pendent biological replicate. All clean reads were mapped
to the tomato reference genome (Table S5). Approximately
80% of clean reads were mapped to the tomato genome in
each sample file, ensuring that the RNA-seq results were
relatively credible. Thousands of genes, including coding
genes and lncRNAs, were specifically differently expressed
in CR-lncRNA1459-8-3 and CR-lncRNA1459-21-1 compared
with the WT (Figure 3a, b). There were 3829 upregulated
DEGs and 2435 downregulated DEGs in the CR-
lncRNA1459-8-3 mutant compared with the WT (Figure 3c)
and 3379 upregulated DEGs and 1584 downregulated DEGs
in the CR-lncRNA1459-21-1 mutant compared with the WT
(Figure 3d). Because of data redundancy between CR-
lncRNA1459-8-3/WT and CR-lncRNA1459-21-1/WT, overlap-
ping data were chosen for further analysis. Finally, 2579
upregulated DEGs and 1337 downregulated DEGs were
obtained by sequencing (Figure 3g, h). A number of signifi-
cant DEGs are listed in Table S6. Furthermore, qPCR vali-
dation of seven downregulated genes and one upregulated
gene was highly correlated with RNA-seq data (Figure S4).
Our previous research has revealed 3679 ripening-related
lncRNAs in tomato (Zhu et al., 2015). Here, a total of 629
and 521 lncRNAs were identified as significant DELs in
fruits of CR-lncRNA1459-8-3 and CR-lncRNA1459-21-1,
respectively, compared with the WT (Figure 3e, f). From
our data, 81 DELs were negatively regulated by
lncRNA1459 and 31 DELs were positively regulated by
lncRNA1459 (Figure 3i, j). A number of significant DELs are
listed in Table S7. These data suggest that lncRNA1459
modulates the transcription of genes and lncRNAs during
tomato fruit ripening.
Potential functional analysis of lncRNA1459
Gene Ontology (GO) analysis of DEGs revealed potential
functions of lncRNA1459 in three terms, including biologi-
cal process, molecular function and cellular component
(Figure 4). For cellular component, the major category was
cell part (GO:0044464). The most highly represented term
in biological process was metabolic process (GO:0008152),
including 201 downregulated DEGs and 365 upregulated
DEGs. The genes most significantly enriched among all
terms were in catalytic (GO:0003824), including 419 down-
regulated DEGs and 665 upregulated DEGs. In addition,
genes were also enriched in GO terms related to fruit
ripening, such as demethylase (GO:0032451), transcription
factors (GO:0003700), pigmentation (GO:0043473), pollina-
tion (GO:0009856), and reproductive process (GO:0022414),
ensuring that lncRNA1459 is ripening-related. More inter-
estingly, SlDML2 was significantly downregulated; this is
responsible for nearly all DNA demethylation during
© 2018 The Authors
The Plant Journal © 2018 John Wiley & Sons Ltd, The Plant Journal, (2018), 94, 513–524
1365313x, 2018, 3, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/tpj.13872 by Indian Institute Of Technology, Kharagpur Central Library, Wiley Online Library on [07/11/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Tomato fruit ripening 517
ripening and is critical for tomato fruit ripening (Fig-
ure S5a). In addition, transcription of PSY1, a phytoene
synthase, was seriously inhibited but not that of PSY,
PSY2 and PSY3 (Figure S5b). Furthermore, the transcrip-
tion factors NONRIPENING (NOR) and COLORLESSNONRI-
PENING (CNR) were both significantly downregulated after
loss of function of lncRNA1459 (Figure S4). Mapping
lncRNA1459 to the tomato reference genome showed there
to be four neighboring genes of lncRNA1459 (Figure S6a).
It was found that lncRNAs preferentially mediate expres-
sion of their neighboring genes. Therefore, expression of
these four genes was validated by qRT-PCR, indicating that
only one gene (Solyc03g095900.2) was downregulated by
loss of function of lncRNA1459 (Figure S6b). The other
three genes were not validated in either WT and CR-
lncRNA1459 mutants (Figure S6b).
Lycopene accumulation was inhibited after repression of
lncRNA1459
The fruit ripening process in tomato involves degradation
of chlorophyll and accumulation of lycopene, inducing a
color change from green to red in tomato. Different colors
were found in CR-lncRNA1459 mutants and WT fruits (Fig-
ure 2c and Figure S3a). We measured the lycopene content
at 45 and 50 DPA and found that the lycopene levels in CR-
lncRNA1459 fruits were distinctly lower than in WT fruits
(Figure 5a). Consistent with the lycopene content being
dramatically reduced due to loss of function of
lncRNA1459, the expression of genes related to carotenoid
biosynthesis was putatively changed. RNA-seq data were
used to identify expression profiles of genes involved in
the carotenoid biosynthesis pathway in the CR-lncRNA1459
mutants. Genes related to lycopene synthesis, namely PDS
(phytoene desaturase), PSY (phytoene synthase), ZDS
(f-carotene desaturase) were downregulated by knocking
out lncRNA1459. LCY-b (lycopene b-cyclase) and LCY-e (ly-
copene e-cyclase) catalyze the cyclization of lycopene to
produce b-carotene or a-carotene. Compared with WT
fruits, LCY-e was consistently invariant with knockout of
lncRNA1459 while LCY-b was upregulated (Figure 5b).
Quantitative PCR was also used to verify the expression of
these genes in loss of function lncRNA1459 mutants (Fig-
ure 5c). These results suggested that lncRNA1459 affected
the accumulation of lycopene during fruit ripening.
Ethylene production was inhibited in CR-lncRNA1459
mutants
Fruits undergo a series of chemical and physiological
changes during maturation, such as accumulation of nutri-
tional compounds and pigments, synthesis of volatile aro-
matics, cell wall degradation, and so on. In addition,
climacteric fruits require an ethylene burst during ripening.
Ethylene production in CR-lncRNA1459-8-3 and CR-
lncRNA1459-21-1 mutant and WT fruits was explored at 35,
 1365313x, 2018, 3, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/tpj.13872 by Indian Institute Of Technology, Kharagpur Central Library, Wiley Online Library on [07/11/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
518 Ran Li et al.

Figure 3. Expression of numerous differentially expressed genes (DEGs) and differentially expressed lncRNAs (DELs) was impaired due to loss of function of
lncRNA1459.
(a), (b) Volcano diagrams of DEGs and DELs in CR-lncRNA1459-8-3 and CR-lncRNA1459-21-1 fruits compared with wild-type (WT) fruits at 35 days post-anthesis
(DPA). A P-value < 0.05 indicates that differences in gene expression are significant. Blue spots show DEGs and DELs whose expression was less than half that
of the WT. Red spots display DEGs and DELs whose expression was more than double that of the WT. (c), (d) Number of up- and downregulated DEGs in CR-
lncRNA1459-8-1 and CR-lncRNA1459-21-1, respectively. (e), (f) Number of up- and downregulated DELs in CR-lncRNA1459-8-1 and CR-lncRNA1459-21-1, respec-
tively. (g), (h) Venn diagrams showing DEGs that are commonly expressed in CR-lncRNA1459-8-3 and CR-lncRNA1459-21-1 samples. The 2579 upregulated DEGs
and 1337 downregulated DEGs indicate credible DEGs for further research. (i), (j) Venn diagrams showing DELs that are commonly expressed in CR-
lncRNA1459-8-3 and CR-lncRNA1459-21-1 samples. Ripening-related DELs in rin were calculated between rin and WT, as long as |fold change| > 2 and P-value <
0.05. The 81 upregulated DELs and 31 downregulated DELs indicate credible DELs for further research.

© 2018 The Authors

The Plant Journal © 2018 John Wiley & Sons Ltd, The Plant Journal, (2018), 94, 513–524
 1365313x, 2018, 3, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/tpj.13872 by Indian Institute Of Technology, Kharagpur Central Library, Wiley Online Library on [07/11/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Tomato fruit ripening 519

Figure 4. Gene Ontology for the differentially expressed genes under molecular functions, cellular components and biological processes.

Figure 5. Lycopene accumulation in tomato fruits was impaired in CR-lncRNA1459 mutants.

(a) Lycopene accumulation in CR-lncRNA1459-8-3 and CR-lncRNA1459-21-1 fruits was significantly lower than in wild-type (WT) fruits at 45 and 50 DPA (days
post-anthesis) ripening stages. Error bars indicate SD of three biological replicates, with each measured in triplicate. Samples marked with different letters
show a significant difference at P < 0.05.
(b) Heat map of genes related to lycopene accumulation. Genes upstream of lycopene biosynthesis were downregulated in CR-lncRNA1459 mutants, while some
genes downstream of lycopene metabolism were upregulated, such as LCY-b and ZEP.
(c) Key genes in carotenoid biosynthesis were downregulated in CR-lncRNA1459, determined by qPCR, except for LCY-b. Relative expression levels were nor-
malized by the Actin expression level. The relative expression level in WT fruits was assigned a value of 1. Error bars indicate SD of three biological replicates,
with each measured in triplicate. Samples marked with different letters show a significant difference at P < 0.05.

40, 45 and 50 DPA. The results showed that CR-
lncRNA1459 fruits produced less ethylene than WT fruits in
all four ripening stages (Figure 6a). In addition, from RNA-
seq data, 10 genes associated with ethylene were chosen
for expression profiling (Figure 6b). Most of these genes
were downregulated, whereas the expression of a tran-
scription factor, EIN2, in the ethylene signal transduction
pathway was nearly same as in the WT (Figure 6b). Quanti-
tative PCR was also used to verify the expression of these
genes in loss of function of lncRNA1459 mutants

© 2018 The Authors

The Plant Journal © 2018 John Wiley & Sons Ltd, The Plant Journal, (2018), 94, 513–524
 1365313x, 2018, 3, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/tpj.13872 by Indian Institute Of Technology, Kharagpur Central Library, Wiley Online Library on [07/11/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
520 Ran Li et al.

Figure 6. Ethylene production in tomato fruits was inhibited in CR-lncRNA1459 mutants.

(a) Ethylene production in CR-lncRNA1459-8-3 and CR-lncRNA1459-21-1 fruits was much lower than in wild-type (WT) fruits at 35, 40, 45 and 50 DPA (days post-
anthesis) ripening stages. Error bars indicate SD of three biological replicates, with each measured in triplicate. Samples marked with different letters show a
significant difference at P < 0.05.
(b) Expression of genes and transcript factors associated with ethylene was reduced in CR-lncRNA1459 mutants compared with WT, whereas expression of EIN2
was upregulated in CR-lncRNA1459 mutants compared with WT.
(c) Key genes related to ethylene were downregulated in CR-lncRNA1459, determined by qPCR. Relative expression levels were normalized by the Actin expres-
sion level. The relative expression level in WT fruits was assigned a value of 1. Error bars indicate SD of three biological replicates, with each measured in trip-
licate. Samples marked with different letters show a significant difference at P < 0.05.

(Figure 6c). In turn, to investigate whether lncRNA1459
was ethylene-dependent, we treated green-matured WT
fruits with ethephon and 1-MCP, respectively. Expression
of ACS2 (positive control) increased about five times com-
pared with WT after ethephon treatment and decreased
obviously after 1-MCP treatment (Figure S7a). There was
actually no change in expression of lncRNA1459 (Fig-
ure S7b). These results suggested that lncRNA1459 regu-
lates ethylene production but cannot be induced by
ethylene. All these results showed that lncRNA1459 pro-
motes ethylene production by regulating the transcription
of ethylene-related genes.
DISCUSSION
Recently, the advent of deep sequencing as well as other
emerging technologies has greatly advanced our knowl-
edge of both the functional significance and mechanisms
of action of lncRNAs. With the limitation of low expression
of lncRNAs in plants it is difficult to obtain full-length plant
lncRNAs. In Solanum lycopersicum, the 50 -untranslated
region sequence of Dicer-like2b (DCL2b) has been cloned
by transient gene expression in N. benthamiana (Wang
et al., 2016b). In our research, we successfully obtained the
full-length sequence of lncRNA1459 (Figure 1a, b). A por-
tion of the 50 DNA sequence of lncRNA1459 combined with
about 1 kb of the DNA sequence upstream of lncRNA1459
was overexpressed before 50 RACE cloning (Figure 1b),
avoiding the influence of the low expression level of
lncRNA1459. Additionally, it has also been reported that
lncRNA has the potential to encode small peptides. The
non-protein-coding potential of lncRNA1459 was calculated
by three different online tools. Therefore, we speculated
that lncRNA1459 acts as a bona fide lncRNA. Moreover,
several works have found that the characteristic subcellular
localization of lncRNAs is relevant to their function (Chen,
2016). From our experiments, lncRNA1459 was found in
the nucleus, with a specific subcellular location (Figure 1d).
This information was helpful in the discovery of definite
functional mechanisms of lncRNA1459 in tomato fruit
ripening.
A complex regulatory system operates permanently dur-
ing fruit ripening. The general framework of the regulatory
system has been confirmed (Giovannoni, 2004). However,
existing research efforts have mainly focused on coding
genes relevant to fruit ripening and little is known about the
function of lncRNAs during fruit ripening. In the present
study we found that lncRNA1459 is a positive element dur-
ing fruit ripening in tomato. Fruit ripening was greatly
delayed due to repression of lncRNA1459 (Figure 2).
CRISPR/Cas9 was used to produce loss-of-function mutants

© 2018 The Authors

The Plant Journal © 2018 John Wiley & Sons Ltd, The Plant Journal, (2018), 94, 513–524

of lncRNA1459. Two independent homozygous transgenic
mutants (CR-lncRNA1459-8-3 and CR-lncRNA1459-21-1)
were detected in the T1 generation. Compared with RNA
interference (RNAi), CRISPRR/Cas9 is a genome editing sys-
tem that can modify a specific DNA sequence to induce loss
of function of genes. Therefore, the CRISPRR/Cas9 system
was an efficient method for knocking out interesting
lncRNAs for loss-of-function analysis, avoiding the limita-
tion of low expression. A previous study has reported that
lncRNAs interacted with proteins to modulate the level of
gene expression (Yoon et al., 2014). Protein has been
reported to be a vital partner for function of lncRNAs (Yang
et al., 2015) and structure is also an important element for
their function (Li et al., 2016). Therefore, the secondary
structure of lncRNA1459 was an important element in func-
tional analysis. The secondary structure of lncRNA1459 was
divided into two parts. Results showed that secondary
structure of CR-lncRNA1459-8 and CR-lncRNA1459-21 was
significantly changed compared with the WT in both parts
(Figure S8), especially in CR-lncRNA1459-8. However, there
was no difference between the secondary structure of
CR-lncRNA1459-32 and WT in the loop part (Figure S8).
Above all, it was predicted that lncRNA1459 indirectly regu-
lates gene transcription by interacting with a target protein.
Several key ripening-related genes were investigated by
RNA-seq analysis, and the expression of all of them was
dramatically decreased. Transcription of the lycopene
biosynthesis genes PSY1, PDS and ZDS has been shown
to be downregulated in loss-of-function lncRNA1459
mutants (Figure 5), while the lycopene metabolism gene
LCY-b was relatively upregulated in loss-of-function
lncRNA1459 mutants (Figure 5). Transcription of ethylene
biosynthesis genes and signal transduction genes has also
been shown to be specifically downregulated in loss-of-
function lncRNA1459 mutants (Figure 6). The process of
tomato fruit ripening involves loss of function of DNA
methylation almost genome-wide. SlDML2 was found nec-
essary for nearly all demethylation and covered about
30 000 genomic regions related to fruit ripening (Lang
et al., 2017). RNA-seq data showed expression of SlDML2
to be reduced instead of other DNA demethylases (Fig-
ure S5a). In addition, expression of lncRNAs was also sig-
nificantly altered after loss of function of lncRNA1459. A
total of 3679 lncRNAs were identified as being related to
ripening in tomato (Zhu et al., 2015). Further bioinformatic
analysis has discovered several lncRNAs that were pre-
dicted to be significantly related to tomato fruit ripening.
From our data, 81 upregulated DELs and 31 downregulated
DELs have been discovered with repression of
lncRNA1459. However, the function of these ripening-
related lncRNAs is still unclear. Increasing evidence has
shown that it is complicated to confirm the function of
lncRNAs on target genes. In general, comprehension of the
myriad roles of lncRNA1459 is still in its infancy. Currently,
© 2018 The Authors
The Plant Journal © 2018 John Wiley & Sons Ltd, The Plant Journal, (2018), 94, 513–524
1365313x, 2018, 3, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/tpj.13872 by Indian Institute Of Technology, Kharagpur Central Library, Wiley Online Library on [07/11/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Tomato fruit ripening 521
we are exploring the further functional mechanism of
ripening-related lncRNAs, especially lncRNA1459.
EXPERIMENTAL PROCEDURES
RNA extraction and real-time RT-PCR
RNA was extracted from tomato leaf or fruit using TRIZOL reagent.
Genomic DNA was removed by DNase treatment (Zhu et al.,
2015). RNA aliquots of 2 lg were used for cDNA synthesis with
the TranScript One-Step gDNA Removal and cDNA Synthesis
SuperMix kit (TransGen Biotech, http://www.transgenbiotech.com/
). Real-time PCR was performed using SYBR Green PCR Master
Mix (TransGen Biotech) on a CFX96 Touch Real-time PCR Detec-
tion System (Bio-Rad, http://www.bio-rad.com/). The specific
mRNA relative expression levels were measured by the 2ΔΔCt
analysis method, and samples were normalized using Actin. Three
biological repeats were performed for each experiment with three
technical replicates. The specific primers are listed in Table S1.
Cloning of the full-length lncRNA
Extracted total RNA was prepared for 30 and 50 cDNA synthesis by
oligo(dT) primer and specific primer, respectively. The 1351 bp of
lncRNA1459 combined with its upstream genomic sequence was
driven by the 35S promoter for transient expression in N. ben-
thamiana before 50 cDNA synthesis (Figure 1b). A FirstChoice
RLM-RACE kit was used for cloning the 50 7-methylguanylate cap
and 30 polyA tail sequences. Primers were designed to amplify 50
and 30 sequences based on the RNA-seq sequence of lncRNA1459.
The amplified fragment was finally determined by Sanger
sequencing. The primers are listed in Table S1.
Nuclear–cytoplasmic fractionation
Nuclear and cytoplasmic fragments was separated in Arabidopsis
(Wang et al., 2011) We used fruits at the 35-DPA stage for
nuclear–cytoplasmic separation with three biological replicates.
RNA elutions were used in qRT-PCR to measure the content of
lncRNA1459. U6 and Actin were used as internal controls for
nuclear and cytoplasmic extracts, respectively.
Selection of the target site and plasmid construction
Specific gRNA targeting lncRNA1459 was selected using the
online tool CRISPR-P (http://cbi.hzau.edu.cn/crispr/). To obtain
high editing efficiency, the GC% in specific target sequences
should be between 40% and 70%. In addition, four or more con-
secutive T nucleotides in the target sequence should be avoided
and there should be 5 bp between the target sequence and the
guide RNA (gRNA) sequence. The RNA Folding program was used
to fold the secondary structure (http://mfold.rna.albany.edu/?q=mf
old/RNA-Folding-Form2.3). The gRNA expression cassette was
generated by overlapping PCR. The target sequence was ligated
between the corresponding promoter and sgRNA by the first PCR.
The second PCR was used to induce a BsaӀ restriction site. The
amplified fragment was then assembled on the pYLCRISPR/Cas9-
Ubi-H binary plasmid through the Golden Gate ligation method
(Ma et al., 2015). The specific primers are listed in Table S1.
Growth conditions, plant transformation and plant
materials
Tomato and N. benthamiana were grown in a glasshouse with
16-h light at 20–25°C. The CRISPR/Cas9-lncRNA1459-expressing
plasmid was transformed into AC using the Agrobacterium

522 Ran Li et al.
tumefaciens-mediated transformation method (Joyce Van Eck,
2006). The transgenic tomato lines were selected by hygromycin
resistance. Leaves were collected from plants aged about 6 weeks,
and fruits were sampled at 30, 35, 40, 45 and 50 DPA.
Mutation analysis of transgenic lines
About 100 mg of fresh frozen tomato leaves were prepared for
genomic DNA extraction by a hi-DNA Secure Plant Kit (Tiangen,
http://www.tiangen.com/en/). The genomic DNA was then used as
a template to amplify the endogenous lncRNA1459 fragment by
PCR. The PCR fragments were digested by T7EӀ enzyme. More-
over, direct sequencing showed that PCR fragments contained
three mutation types, heterozygous, homozygous and biallelic.
The web-based tool DSDecode and the NTI vector were used to
decode the sequencing chromatograms of transgenic tomato
lines. The specific primers are listed in Table S1.
Measurement of ethylene production
To measure ethylene production by fruits, each fruit was placed at
room temperature (20°C) for about 4 h. Then each fruit was sealed
in a 330-ml container at room temperature for 2 h, and 1-ml gas
samples were taken for measurement by gas chromatography
(GC-14, Shimadzu, https://www.shimadzu.com/).
Treatment with ethephon and 1-MCP
Tomato fruits at the green mature stage were immersed into
100 ll l1 ethephon and 60 lM 1-MCP, respectively, followed by
with light culture for 24 h. Control fruits were placed in water for
24-h light culture in the same way.
Lycopene extraction and measurement
Fruit powder samples (approximately 0.5 g) were mixed with 1%
butylated hydroxytoluene (BHT). The powder was dissolved in 1 ml
of acetone and then pelleted at 12 000 g at 4°C for 5 min. The pellet
was resuspended in 1 mL of dichloromethane, with three or repeats
until the supernatant lost all of its color. All the organic phases were
collected and the same volume of diethyl ether and 20% volume of
NaCl (12%) was added to them. The mixture compound was
homogenized and centrifuged at 12 000 g at 4°C for 10 min. The
superorganic phase was gathered and dried under a stream of nitro-
gen. Finally, the extracted pigment was re-dissolved in acetone. Car-
otenoid pigments were measured using reverse-phase high-
performance liquid chromatography (HPLC) as described in a previ-
ous study, with some modifications (Neuman et al., 2014).
Library construction and RNA sequencing
Total RNA was extracted from tomato fruits of WT, CR-
lncRNA1459-8-3 and CR-lncRNA1459-21-1 at the 35-DPA ripening
stage. Total RNA was then submitted to Novogene in Beijing
(https://en.novogene.com/) for library construction and RNA
sequencing. Messenger RNA was enriched by oligo(dT)-attached
magnetic beads for cDNA synthesis. Size-selected and
adaptor-ligated cDNA fragments were then purified for library
construction. After library preparation, the libraries were
sequenced on an Illumina Hiseq 4000 platform and 150bp
paired-end reads were generated. Both generated raw data and
clean data from each library were no less than 6G in sequencing
depth. The clean data for this paper have been submitted to the
National Center for Biotechnology Information (NCBI) Sequence
Read Archive under accession number SRP108427 (http://www.
ncbi.nlm.nih.gov/sra/).
The Plant Journal © 2018 John Wiley & Sons Ltd, The Plant Journal, (2018), 94, 513–524
1365313x, 2018, 3, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/tpj.13872 by Indian Institute Of Technology, Kharagpur Central Library, Wiley Online Library on [07/11/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
RNA-seq analysis
The quality of clean reads obtained by RNA-seq was checked
by the FastQC program (v.0.11.3) to make sure that they were
credible for a series of analyses. Subsequently, all clean reads
were mapped to the tomato reference genomes (SGN release
v.SL2.50; ftp://ftp.sgn.cornell.edu/tomato_genome) combined
with tomato lncRNAs from our data using TopHat (v.1.4.6), and
fragments were assigned to genes by the feature count and
count program. Reads per kilobase per million mapped reads
(RPKM) was used to show the expression value. The fold
change was calculated by RPKMCR-lncRNA1459/RPKMWT). Tran-
scripts were identified as differential expression between CR-
mutants and WT, as long as |fold change| > 2 and P-value <
0.05.
Gene Ontology enrichment analysis
Firstly, GO-TermFinder (v.0.86) was used to analyze DEG enrich-
ment of GO terms. Secondly, the generated GO annotations were
plotted in WEGO (http://wego.genomics.org.cn/). Proteins were fil-
tered based on their grouping to cellular components, molecular
functions and biological function.
Statistical analysis
Statistical analysis of data was conducted using SPSS (v.20.0)
software. For three or more data sets, statistical significance was
calculated by Duncan’s test. Statistically significant differences
(P < 0.05) are indicated by different lowercase letters in figures.
ACKNOWLEDGEMENTS
We thank Dr. Yaoguang Liu for providing the binary vector pYL-
CRISPR/Cas9 system, Dr. Xiuying Liu for assistance of RNA-seq
data analysis, and Dr. Shijie Yan for providing assistance of Ethy-
lene production measurement. We thank Dr. Xiuren Zhang for
providing valuable comments. We also thank Yongfang Yang,
Tongtong Yu and Tian Wang for experimental assistance. In addi-
tion, this research was supported by a grant from the Chinese Uni-
versities Scientific Fund (31622050, 91540118, 31672208, and
31471921) to HZ.
CONFLICT OF INTEREST
The authors declare no conflict of interest.
SUPPORTING INFORMATION
Additional Supporting Information may be found in the online ver-
sion of this article.
Figure S1. Complementary DNA sequences of two lncRNA1459
transcript isoforms.
Figure S2. Protein-coding potential in lncRNA1459 transcript.
Figure S3. Loss of function of lncRNA1459 specifically delayed
tomato fruit ripening in T0 generation.
Figure S4. Quantitative PCR validation of RNA-seq results showing
the relative expression of seven down-regulated DEGs and one
up-regulated DEG.
Figure S5. Expression level of SlDML2 and PSY1 were down-regu-
lated in CR-lncRNA1459 mutants.
Figure S6. Expression of genes neighboring lncRNA1459 was
affected by loss of function of lncRNA1459.
Figure S7. Expression of lncRNA1459 was not affected by ethy-
lene.
© 2018 The Authors
 1365313x, 2018, 3, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/tpj.13872 by Indian Institute Of Technology, Kharagpur Central Library, Wiley Online Library on [07/11/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Tomato fruit ripening 523

Figure S8. Secondary structure of wild-type and mutant forms of Li, R., Zhu, H. and Luo, Y. (2016) Understanding the functions of long non-
lncRNA1459. coding RNAs through their higher-order structures. Int. J. Mol. Sci., 17,
702. https://doi.org/10.3390/ijms17050702
Table S1. Primers used in this study.
Li, R., Li, R., Li, X., Fu, D., Zhu, B., Tian, H., Luo, Y. and Zhu, H. (2017) Multi-
Table S2. Off-target detection in CR-lncRNA1459 mutants in the T0 plexed CRISPR/Cas9-mediated metabolic engineering of c-aminobutyric
generation. acid levels inSolanum lycopersicum. Plant Biotechnol. J., https://doi.org/
Table S3. Modification characteristic in the T1 generation. 10.1111/pbi.12781.
Ma, X., Zhang, Q., Zhu, Q. et al. (2015) A Robust CRISPR/Cas9 system for
Table S4. Off-target detection in CR-lncRNA1459 mutants in the T1 convenient, high-efficiency multiplex genome editing in Monocot and
generation. Dicot plants. Mol Plant., 8, 1274–1284. https://doi.org/10.1016/j.molp.
Table S5. Summary of clean read counts and percentage of 2015.04.007
unique mapped reads. Nelson, B.R., Makarewich, C.A., Anderson, D.M. et al. (2016) A peptide
Table S6. The differentially expressed genes between wild-type encoded by a transcript annotated as long noncoding RNA enhances
SERCA activity in muscle. Science, 351, 271–275. https://doi.org/10.
and CR-lncRNA1459 mutant fruits.
1126/science.aad4076
Table S7. The differentially expressed lncRNAs between wild-type Neuman, H., Galpaz, N., Cunningham, F.X., Zamir, D. and Hirschberg, J.
and CR-lncRNA1459 mutant fruits. (2014) The tomato mutationnxd1 reveals a gene necessary for neoxan-
thin biosynthesis and demonstrates that violaxanthin is a sufficient pre-
REFERENCES cursor for abscisic acid biosynthesis. Plant J., 78, 80–93. https://doi.org/
10.1111/tpj.12451
Anderson, D.M., Anderson, K.M., Chang, C. et al. (2015) A micropeptide
Salomon, S. and Puchta, H. (1998) Capture of genomic and T-DNA
encoded by a putative long noncoding RNA regulates muscle perfor-
sequences during double-strand break repair in somatic plant cells.
mance. Cell, 160, 595–606. https://doi.org/10.1016/j.cell.2015.01.009
EMBO J., 17, 6086–6095. https://doi.org/10.1093/emboj/17.20.6086
Barrangou, R., Birmingham, A., Wiemann, S., Beijersbergen, R.L., Hornung,
Seo, J.S., Sun, H., Park, B.S., Huang, C., Yeh, S., Jung, C. and Chua, N.
V. and Smith, A.V.B. (2015) Advances in CRISPR-Cas9 genome engineer-
(2017) ELF18-INDUCED LONG-NONCODING RNA Associates with
ing: lessons learned from RNA interference. Nucleic Acids Res., 43,
Mediator to Enhance Expression of Innate Immune Response Genes
3407–3419. https://doi.org/10.1093/nar/gkv226
in Arabidopsis. Plant Cell., 29, 1024–1038. https://doi.org/10.1105/tpc.
Beaulieu, Y.B., Kleinman, C.L., Landry-Voyer, A., Majewski, J. and Bachand,
16.00886
F. (2012) Polyadenylation-dependent control of long noncoding RNA
Seymour, G.B., Ostergaard, L., Chapman, N.H., Knapp, S. and Martin, C.
expression by the Poly(A)-Binding Protein Nuclear 1. PLoS Genet. 8,
(2013) Fruit Development and Ripening. Annu. Rev. Plant Biol., 64, 219–
e1003078. https://doi.org/10.1371/journal.pgen.1003078
241. https://doi.org/10.1146/annurev-arplant-050312-120057
Chen, L. (2016) Linking long noncoding RNA localization and function. Trends
Sonoda, E., Hochegger, H., Saberi, A., Taniguchi, Y. and Takeda, S. (2006)
Biochem. Sci., 41, 761–772. https://doi.org/10.1016/j.tibs.2016.07.003
Differential usage of non-homologous end-joining and homologous
Chu, C., Spitale, R.C. and Chang, H.Y. (2015) Technologies to probe func-
recombination in double strand break repair. DNA Repair, 5, 1021–1029.
tions and mechanisms of long noncoding RNAs. Na Struct Mol Biol., 22,
https://doi.org/10.1016/j.dnarep.2006.05.022
29–35. https://doi.org/10.1038/nsmb.2921
Sun, Y. and Xiao, H. (2015) Identification of alternative splicing events by
Cong, L., Ran, F.A., Cox, D. et al. (2013) Multiplex genome engineering
RNA sequencing in early growth tomato fruits. BMC Genom., 16, 16–
using CRISPR/Cas systems. Science, 339, 819–823. https://doi.org/10.
2015.
1126/science.1231143
Ulitsky, I. and Bartel, D.P. (2013) lincRNAs: genomics, evolution, and mech-
Csorba, T., Questa, J.I., Sun, Q. and Dean, C. (2014) Antisense COOLAIR
anisms. Cell, 154, 26–46. https://doi.org/10.1016/j.cell.2013.06.020
mediates the coordinated switching of chromatin states at FLC during
Vrebalov, J., Ruezinsky, D., Padmanabhan, V., White, R., Medrano, D.,
vernalization. P Natl Acad Sci USA, 111, 16160–16165. https://doi.org/10.
Drake, R., Schuch, W. and Giovannoni, J. (2002) A MADS-box gene nec-
1073/pnas.1419030111
essary for fruit ripening at the tomato ripening-inhibitor (rin) locus.
Giovannoni, J.J. (2004) Genetic regulation of fruit development and ripen-
Science, 296, 343–346. https://doi.org/10.1126/science.1068181
ing. Plant Cell., 16S, S170–S180. https://doi.org/10.1105/tpc.019158
Vrebalov, J., Pan, I.L., Arroyo, A.J.M. et al. (2009) Fleshy fruit expansion
Giovannoni, J.J. (2007) Fruit ripening mutants yield insights into ripening
and ripening are regulated by the tomato SHATTERPROOF Gene
control. Curr. Opin. Plant Biol., 10, 283–289. https://doi.org/10.1016/j.pbi.
TAGL1. Plant Cell., 21, 3041–3062. https://doi.org/10.1105/tpc.109.
2007.04.008
066936
Giovannoni, J., Nguyen, C., Ampofo, B., Zhong, S. and Fei, Z. (2017) The
Wang, W., Ye, R.Q., Xin, Y., Fang, X.F., Li, C.L., Shi, H.Q., Zhou, X.P. and Qi,
epigenome and transcriptional dynamics of fruit ripening. Annu. Rev.
Y.J. (2011) An Importin b protein negatively regulates MicroRNA activity
Plant Biol., 68, 61–84. https://doi.org/10.1146/annurev-arplant-042916-
in Arabidopsis. Plant Cell., 23, 3565–3576. https://doi.org/10.1105/tpc.111.
040906
091058
Guttman, M., Russell, P., Ingolia, N.T., Weissman, J.S. and Lander, E.S.
Wang, H., Chung, P.J., Liu, J., Jang, I., Kean, M.J., Xu, J. and Chua, N.
(2013) Ribosome profiling provides evidence that large non-coding RNAs
(2014) Genome-wide identification of long noncoding natural antisense
do not encode proteins. Cell, 154(1), 240–251. https://doi.org/10.1016/j.ce
transcripts and their responses to light in Arabidopsis. Cytogenet Gen-
ll.2013.06.009
ome Res., 24, 444–453. https://doi.org/10.1101/gr.165555.113
Heo, J.B., Lee, Y. and Sung, S. (2013) Epigenetic regulation by long noncod-
Wang, T., You, L., Li, R., Fu, D.Q., Zhu, B.Z., Luo, Y.B. and Zhu, H.L. (2016a)
ing RNAs in plants. Chromosome Res., 21, 685–693. https://doi.org/10.
Cloning, identification, and expression analysis of a Dicer-Like gene fam-
1007/s10577-013-9392-6
ily from Solanum lycopersicum. Biol Plantarum., 60, 410–418. https://doi.
Joyce Van Eck, D.D.K.A. (2006) Tomato (Lycopersicum esculentum).
org/10.1007/s10535-016-0620-8
Agrobacterium Protocols, 343, 474–495.
Wang, X., Ai, G., Zhang, C., Cui, L., Wang, J., Li, H., Zhang, J. and Ye, Z.
Lang, Z., Wang, Y., Tang, K., Tang, D., Datsenka, T., Cheng, J., Zhang, Y.,
(2016b) Expression and diversification analysis reveals transposable ele-
Handa, A.K. and Zhu, J. (2017) Critical roles of DNA demethylation in the
ments play important roles in the origin of Lycopersicon-specific
activation of ripening-induced genes and inhibition of ripening-repressed
lncRNAs in tomato. New Phytol., 209, 1442–1455. https://doi.org/10.1111/
genes in tomato fruit. P Natl A Sci INDIA A., 114, E4511–E4519. https://d
nph.13718
oi.org/10.1073/pnas.1705233114
Weng, L., Zhao, F., Li, R., Xu, C., Chen, K. and Xiao, H. (2015) The Zinc Fin-
Li, L., Wang, X., Sasidharan, R. et al. (2007) Global identification and charac-
ger Transcription FactorSlZFP2 negatively regulates abscisic acid biosyn-
terization of transcriptionally active regions in the rice genome. PLoS
thesis and fruit ripening in tomato. Plant Physiol., 167, 931–949. https://d
ONE, 2(3), e294. https://doi.org/10.1371/journal.pone.0000294
oi.org/10.1104/pp.114.255174
Li, L., Eichten, S.R., Shimizu, R. et al. (2014) Genome-wide discovery and
Xin, M., Wang, Y., Yao, Y., Song, N., Hu, Z., Qin, D., Xie, C., Peng, H., Ni, Z.
characterization of maize long non-coding RNAs. Genome Biol., 15, R40.
and Sun, Q. (2011) Identification and characterization of wheat long non-
https://doi.org/10.1186/gb-2014-15-2-r40

© 2018 The Authors

The Plant Journal © 2018 John Wiley & Sons Ltd, The Plant Journal, (2018), 94, 513–524

524 Ran Li et al.
protein coding RNAs responsive to powdery mildew infection and heat
stress by using microarray analysis and SBS sequencing. BMC Plant
Biol., 11, 61. https://doi.org/10.1186/1471-2229-11-61
Yang, Y.F., Wen, L. and Zhu, H. (2015) Unveiling the hidden function of long
non-coding RNA by identifying its major partner-protein. Cell Biosci., 5,
59. https://doi.org/10.1186/s13578-015-0050-x
Yoon, J.H., Abdelmohsen, K. and Gorospe, M. (2014) Functional interactions
among microRNAs and long noncoding RNAs. Semin. Cell Dev. Biol. 34,
9–14. https://doi.org/10.1016/j.semcdb.2014.05.015
The Plant Journal © 2018 John Wiley & Sons Ltd, The Plant Journal, (2018), 94, 513–524
1365313x, 2018, 3, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/tpj.13872 by Indian Institute Of Technology, Kharagpur Central Library, Wiley Online Library on [07/11/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Zhu, Q. and Wang, M. (2012) Molecular functions of long non-coding RNAs
in plants. Genes., 3, 176–190. https://doi.org/10.3390/genes3010176
Zhu, B., Yang, Y., Li, R., Fu, D., Wen, L., Luo, Y. and Zhu, H. (2015) RNA
sequencing and functional analysis implicate the regulatory role of long
non-coding RNAs in tomato fruit ripening. J. Exp. B., 66, 4483–4495.
https://doi.org/10.1093/jxb/erv203
© 2018 The Authors