Abstract
Background
The non-climacteric ‘Yellow’ melon (Cucumis melo, inodorus group) is an
economically important crop and its quality is mainly determined by the
sugar content. Thus, knowledge of sugar metabolism and its related
pathways can contribute to the development of new field management and
post-harvest practices, making it possible to deliver better quality
fruits to consumers.
Results
The RNA-seq associated with RT-qPCR analyses of four maturation stages
were performed to identify important enzymes and pathways that are
involved in the ripening profile of non-climacteric ‘Yellow’ melon
fruit focusing on sugar metabolism. We identified 895 genes 10 days
after pollination (DAP)-biased and 909 genes 40 DAP-biased. The KEGG
pathway enrichment analysis of these differentially expressed (DE)
genes revealed that ‘hormone signal transduction’, ‘carbon metabolism’,
‘sucrose metabolism’, ‘protein processing in endoplasmic reticulum’ and
‘spliceosome’ were the most differentially regulated processes
occurring during melon development. In the sucrose metabolism, five DE
genes are up-regulated and 12 are down-regulated during fruit ripening.
Conclusions
The results demonstrated important enzymes in the sugar pathway that
are responsible for the sucrose content and maturation profile in
non-climacteric ‘Yellow’ melon. New DE genes were first detected for
melon in this study such as invertase inhibitor LIKE 3 (CmINH3),
trehalose phosphate phosphatase (CmTPP1) and trehalose phosphate
synthases (CmTPS5, CmTPS7, CmTPS9). Furthermore, the results of the
protein-protein network interaction demonstrated general
characteristics of the transcriptome of young and full-ripe melon and
provide new perspectives for the understanding of ripening.
Keywords: Cucumis melo, RNA-seq, Sucrose, Fruit ripening, Gene
expression
Background
Melon (Cucumis melo L., Cucurbitaceae) is an economically important
fruit crop worldwide that has an extensive polymorphism being
classified into 19 botanical groups [[43]1, [44]2]. This high
intra-specific genetic variation is reflected in fruit ripening
differences. In this regard, melon fruits present both climacteric and
non-climacteric phenotypes. Climacteric fruits are characterized by a
respiration peak followed by the autocatalytic synthesis of ethylene,
strong aroma, orange pulp, ripening abscission and short shelf life
with rapid loss of firmness and taste deterioration (e.g. cantalupensis
and reticulatus melon groups). On the other hand, non-climacteric melon
(e.g. inodorus melon group) has little ethylene synthesis, white pulp,
low aroma, no ripening abscission and a longer shelf life
[[45]3–[46]7].
During the ripening process, fruits undergo several biochemical and
physiological changes that are reflected in their organoleptic profile,
of which the alteration in sucrose accumulation is a determining
characteristic in melon quality and consumption [[47]6, [48]8, [49]9].
This characteristic is a developmentally regulated process that is
related to gene regulation, hormonal signalling and environmental
factors [[50]6, [51]9–[52]11]. Sucrose, glucose and fructose are the
major soluble sugars, and sucrose is the predominant sugar in melons at
maturity being stored in the vacuoles of the pericarp parenchyma cells
[[53]9, [54]12]. Both climacteric and non-climacteric melons accumulate
sugar during fruit ripening [[55]6]. However, the sugar content of C.
melo species differs according to the genetic variety and development
stage [[56]9, [57]13]. For example, the flexuosus melon group presents
non-sweet and non-aromatic fruits, and the cantalupensis melon group
has highly sweet and aromatic fruit [[58]14]. Additionally, in fruit
development, sugar is necessary for energy supply, it also generates
turgor for fruit cell enlargement and accumulates in late stages of
fruit (contributing to fruit taste) [[59]15].
Sucrose accumulation in melon fruit is determined by the metabolism of
carbohydrates in the fruit sink itself and can be provided from three
main sources: (1) photosynthetic product; (2) raffinose family
oligosaccharides (RFOs) catabolism; (3) sucrose resynthesis
(Fig. [60]1). In sucrose accumulation, melon plants export sucrose, as
well as raffinose family oligosaccharides (RFOs) such as raffinose and
stachyose from photosynthetic sources (leaves) to sink tissues
(developing melon fruit). RFOs are hydrolyzed by two different families
of α-galactosidase (neutral α-galactosidase/NAG or acid
α-galactosidase/AAG) producing sucrose and galactose. The synthesized
galactose is then phosphorylated by galactokinase (GK) and the
resulting galactose 1-phosphate (gal1P) can either participate in the
glycolysis pathway through the product glucose-6-phosphate or be used
for sucrose synthesis. In sucrose synthesis, galactose 1-phosphate is
transformed into glucose 1-phosphate (glc1P) by the actions of
UDP-gal/glc pyrophosphorylase (UGGP) and converted to other
hexose-phosphates, providing the substrates for the synthesis of
sucrose by sucrose-phosphate synthase (SPS) and sucrose-phosphate
phosphatase (SPP). Furthermore, sucrose resynthesis is an important
pathway and involves many enzymes of sugar metabolism. On the
afore-mentioned pathway, sucrose unloaded from the phloem can be
hydrolyzed in the apoplast by cell wall invertases (CINs), however, in
melon these enzymes may not have a crucial importance once cucurbits
have a symplastic phloem loading. Then, the hexose sugar (glucose and
fructose) products are imported into the cells by monosaccharide
transporters, phosphorylated by hexokinase (HXK) and fructokinase (FK)
and used for respiration or sucrose resynthesis. Within the cell,
sucrose can be resynthesized in the cytosol by sucrose synthase (SUS)
from fructose and UDP-glc. Sucrose can be hydrolyzed to fructose and
glucose for energy production also by neutral invertase (NIN), or
imported into the vacuole for storage or even hydrolyzed by vacuolar
acid invertase (AIN). The invertase activity can be
post-translationally regulated by invertase inhibitor proteins (INH)
[[61]3, [62]6, [63]9, [64]16–[65]18].
Fig. 1.
[66]Fig. 1
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Sugar pathway in Cucumis melo demonstrating different routes of sucrose
accumulation. UDPglc – Uracil diphosphate glucose; Fruc6P –
fructose-6-phosphate; Glc6P – glucose-6-phosphate; Glc1P –
glucose-1-phosphate; Gal1P – galactose-1-phosphate; UDP-gal – Uracil
diphosphate galactose . Adapted from Chayut et al. (2015) and Dai et
al. (2011) [[68]9, [69]16]. In (A) UDPglc substrate for synthesis of
trehalose (B) UDPglc substrate for synthesis of sucrose
The evidence that shelf life can be related to the sugar accumulation
metabolism as well as the relevance of sugar content as a ripeness
marker in non-climacteric melon, make sugar metabolism studies
important to develop new approaches that can improve its commercial
quality. Previous studies have elucidated the peculiarities of
carbohydrate metabolism, mainly in climacteric melons [[70]3, [71]9].
However, understanding of the biochemical aspects that govern the
different patterns of sucrose accumulation in the wide genetic variety
of melon during the ripening process as well as the identification of
new enzymes related to this pathway are limited. Comprehensive
molecular studies that could enlighten the complexity of this metabolic
pathway are essential. In the last decades, next-generation sequencing
(NGS) or high-throughput techniques and metabolomic technologies
allowed the generation of a vast amount of information that is
essential for the global understanding of metabolic networks. Thus, the
aim of our study was to comparatively analyze the transcriptomes of
different development and ripening stages of non-climacteric ‘Yellow’
melon (Inodorus group) fruits focusing on the sugar pathway. Our
analyses provide insights in gene expression ripening profiles of an
important Brazilian commercial melon ranked in second position for the
total amount of fruits exported by the country (197.60 million metric
tons in 2018) [[72]19].
Results
Variations in colour, pH and SS (soluble solids) during ripening of melon
(Inodorus group)
Colour, pH and SS are important characteristics to determine the fruit
development stage and changes in its chemical constituents. These
parameters were evaluated on non-climacteric melon fruit of the
‘Yellow’ commercial genotype (Inodorus group) at 10 days after
pollination (DAP), 20 DAP, 30 DAP and 40 DAP (Fig. [73]2a). Colour
measurement was expressed by the CIE (Commission Internationale de
l’Eclairage) and Hue angle. Colorimeters express colours in numerical
terms (see methods) along the L*, a* and b* axes (from white to black,
green to red and blue to yellow, respectively) [[74]20]. The results
showed that L* (brightness) of peel increases up to 20 DAP, declines up
to 30 DAP and remains stable until 40 DAP (Fig. [75]2b). For pulp
colour, there was a decline in brightness until 30 DAPS with later
stability (Fig. [76]2b). Coordinates on the a* axis increased during
ripening for peel and pulp, representing the change from green to red
(Fig. [77]2c). Coordinates on the b* axis increase during peel
maturation demonstrating a shift from blue to yellow colour, that it is
the opposite of the pulp profile (Fig. [78]2d). Hue angle (H°) is
variable as the true colour of the fruit and decreases with maturity,
corroborating the findings of Kasim and Kasim (2014) [[79]21] (Fig.
[80]2e). The pH fruit showed a subtle increase during melon maturation
(Table [81]1). Concerning soluble solids (SS) concentration, there is a
gradual increase during the ripening process (Table 1).
Fig. 2.
[82]Fig. 2
[83]Open in a new tab
Non-climacteric melon fruit of a ‘Yellow’ commercial genotype of four
development stages and its colour characteristics. From left to right
there are 10 DAP, 20 DAP, 30 DAP and 40 DAP (a). In (b) L* (brightness)
of peel fruit increases from 10 to 20 DAP and declines in 30 DAP and
remains constant until 40 DAP. In (c) the coordinates on the a* axis
increase during the maturation process in peel and pulp colour,
representing the trend change from green to red. In (d) coordinates on
the b * axis increase during maturation for peel colour, demonstrating
the shift from blue to yellow coloration, the opposite profile was
found for pulp colour. In (e) Hue angle (H°) decreased throughout
ripening, corroborating with Kasim and Kasim (2014)
Table 1.
pH and Soluble Solids (SS) (° Brix) mean for yellow melon (commercial
cultivar) with 10 DAP, 20 DAP, 30 DAP. and 40 DAP
10 D.A.P. 20 D.A.P. 30 D.A.P. 40 D.A.P.
pH 4,15 4,7 4,85 5,1
SS (°Brix) 5,0 8,5 10,9 13,3
[84]Open in a new tab
Transcriptome sequencing
RNA-seq (RNA sequencing) was carried out on the complementary DNA
libraries (cDNA) derived from 10 DAP (two biological replicates) and 40
DAP (three biological replicates) flesh mesocarp. The sequencing data
were evaluated for quality, and were subject to data filtering. The
results generated ~ 59 million clean single reads of ~ 100 bp in
length. A total of ~ 53 million filtered reads were mapped to the
Cucumis melo reference genome ([85]https://www.melonomics.net) [[86]22,
[87]23] using Bowtie2 [[88]24]. Most sample reads (79.65–97.88%) were
successfully aligned and for RNA-seq analysis, only the reads with
overlapping in a single gene were considered (Table [89]2 and
Additional file [90]1: Table S1).
Table 2.
Number of filtered reads from each sample sequenced and mapped to the
Cucumis melo ([91]https://www.melonomics.net) reference genome
Sample name Input reads (filtered) Mapped reads % of mapped reads
Detected genes
10DAP_V2 13,444,823 13,159,483 97.88% 16,865
10DAP_V3 11,805,793 10,577,826 89.60% 16,756
40DAP_M1 11,591,063 10,113,177 87.25% 16,161
40DAP_M2 12,635,568 11,246,225 89.00% 15,975
40DAP_M3 10,203,078 8,127,127 79.65% 15,090
[92]Open in a new tab
Ripening and development of fruit gene expression profile
RNA-seq is an efficient and powerful tool for studying gene expression.
The expression for each gene and differential expression (DE) analyses
were calculated by statistical test evaluating the negative binomial
distribution, being considered significant padj ≤0.05 (see Methods). In
this analysis, over 15,000 expressed genes were detected in each sample
(Table [93]2 and Additional file [94]1: Table S2) of the 29,980
annotated in the Cucumis melo genome [[95]22, [96]23]. However, a total
of 1804 genes showed significant DE between the evaluated stages of
fruit maturation (Additional file [97]1: Table S2). Of these, 895 were
10 DAP-biased and 909 were 40 DAP-biased as demonstrated in MA-plot
(Additional file [98]1: Table S2 and Additional file [99]2: Figure S1).
The RNA-seq data were validated by quantitative reverse transcription
PCR analysis (RT-qPCR) of 8 transcripts in the 10 DAP and 40 DAP melons
(genes related to the sugar pathway), using CmRPS15 and CmRPL as
reference genes (Fig. [100]3, Additional file [101]3: Table S3). From
pairwise comparison of RNA-seq and RT-qPCR analysis, Pearson’s
correlation coefficient was 0.98 (p = 0.0014) indicating positive
correlation between the two methods (Additional file [102]3: Figure
S2). The sample-to-sample distances that give an overview of
similarities and dissimilarities between samples demonstrated
clustering of young fruits (10 DAP) separately from the mature fruits
(40 DAP) (Additional file [103]4: Figure S3). Gene ontology (GO)
enrichment analysis was performed using FDR (false discovery rate)
adjusted p-value < 0.05 on DE genes to characterize the differences of
‘Yellow’ melon development and ripening. Figure [104]4 and
Additional File [105]5: Table S4 show the assigning of GO terms
according to the equivalent biological process (BP), molecular function
(MF) and cellular component (CC). We found that genes related to BP
such as metabolic, physiological, transport and signalling processes
were highly enriched in the 10 DAP stage DE genes. On the other hand,
DE genes of the 40 DAP fruit were more abundant in the cellular
process, cellular nitrogen compound and peptide metabolism BP
categories. Under the cellular component classification, the DE genes
of the young fruit were only significantly enriched within the
‘membrane’ category, while DE genes of the mature fruit were enriched
in several CC terms (e. g. ‘cytoplasm’, ‘chloroplast, ribosomes’). The
top 3 groups within the MF classification were ‘catalytic activity’,
‘ion binding’ and ‘hydrolase activity’ for the 10 DAP stage; and
‘binding, structural molecule activity’ and ‘structural constituent of
ribosome’ for the 40 DAP stage.
Fig. 3.
[106]Fig. 3
[107]Open in a new tab
The relative mRNA expression of 9 genes of the sucrose metabolism was
determined by 2^-ΔΔCt [[108]25]. Results are expressed as mean ± SEM
and significance of different developmental stages (10 DAP, 20 DAP, 30
DAP, 40 DAP) comparison is defined as p ≤ 0.05 by Tuckey test after
data normalization by Box-Cox method or by Kruskal-Wallis & Wilcoxon
(CmSUS1 and CmSUS2). Different letters indicate significant differences
Fig. 4.
[109]Fig. 4
[110]Open in a new tab
Gene ontology enrichment analysis of the DE genes in the young and
mature fruits within category: biological process (BP), cellular
component (CC) and molecular function (MF). The analysis was performed
using FDR (false discovery rate) adjusted p-value < 0.05 on DE genes
([111]http://cucurbitgenomics.org/goenrich)
Hierarchical clustering was performed on the 50 most significant DE
genes of the 10 DAP and 40 DAP fruits. The clustering of genes was
represented in a heatmap (Additional file [112]6: Table S5 and Figure
S4). The results showed 2 genes involved in ‘starch and sucrose
metabolism’ (cmo0500) that were more expressed in 10 DAP fruit
(beta-glucosidase and sucrose synthase 2); and 2 related to ‘hormone
signal transduction’ (cmo04075), being 2 genes more expressed in 10 DAP
fruit (xyloglucan endotransglucosylase/hydrolase) and 1 gene more
expressed in 40 DAP (pathogenesis-related protein 1-like).
KEGG enrichment analyses and network construction
The RNA-seq results were subjected to a KEGG pathway enrichment
analysis (DAVID software [[113]26]) to elucidate the main pathways
involved in fruit ripening and development. A total of 92% (1668/1804)
of the DE genes could be converted into UniProtID (available in the
DAVID software database). Table [114]3 shows the top 6 most
significantly enriched KEGG pathways for both development stages. The
young fruit was enriched with ‘plant hormone signal transduction’ and
energetics metabolisms including ‘starch and sucrose metabolism’. The
full-ripe melon presented more genes involved with ‘protein processing
in endoplasmic reticulum’ and ‘spliceosome’, in addition to energetic
metabolisms (Additional File [115]7: Table S6). Interestingly, the
ethylene receptor 1 (MELO3C003906.2) that is a gene of ethylene hormone
signal transduction was more expressed in 40 DAP than 10 DAP
(Table [116]4, Additional File [117]7: Table S6). In this study, we
focused on sucrose metabolism (related routes were also considered)
because this is an important pathway associated with fruit quality
traits. The other pathways will be analyzed in more detail in further
studies.
Table 3.
KEGG pathway analysis of fruit ripening and development candidates
genes
10 DAP fruit
KEGG pathway Gene count % Fisher Exact P-value*
1 Plant hormone signal transduction 21 2.5 4.3E-3
2 Carbon metabolism 16 1.9 6.7E-2
3 Starch and sucrose metabolism 11 1.3 5.7E-3
4 Photosynthesis 7 0.8 2.8E-3
5 Galactose metabolism 7 0.8 1.1E-2
6 Carbon fixation in photosynthetic organisms 6 0.7 7.5E-2
40 DAP fruit
KEGG pathway Gene count % Fisher Exact P-value*
1 Protein processing in endoplasmic reticulum 24 2.8 5.5E-7
2 Spliceosome 17 2 4.0E-4
3 Carbon metabolism 16 1.9 5.0E-2
4 Ribosome biogenesis in eukaryotes 11 1.3 4.8E-4
5 Carbon fixation in photosynthetic organisms 7 0.8 2.3E-2
6 Pyruvate metabolism 7 0.8 4.9E-2
[118]Open in a new tab
* Significant P-value ≤0.05
Table 4.
The differentially expressed genes (RNA-seq analysis) of the sugar
pathway (related routes were also considered) in two melon development
stages. Statistical test evaluating the negative binomial distribution
was applied using R package DeSeq2 (padj ≤0.05)
Melonomics ID (v4.0) Refseq ID Short Name Gene Name Pathway (KEEG)^a
Log2 FoldChange^b padj
MELO3C010698.2 [119]XP_008444380.1 CmAAG-LIKE1 Alpha-galactosidase
(Melibiase) Like 1 – 2.1853 3.723E-06
MELO3C004346.2 [120]XP_008448578.1 CmAGL2 Alpha-glucosidase 2 – −2.2809
3.160E-03
MELO3C005109.2 [121]XP_008465523.1 CmAMN Alpha-mannosidase – 2.4039
4.064E-06
MELO3C035167.2 [122]XP_008463923.1 CmAUXRF2 Auxin response factor 2 –
−2.1428 2.295E-02
MELO3C021281.2 [123]XP_008458374.2 CmBDXY Beta-D-xylosidase 1-like –
2.7858 1.523E-19
MELO3C020906.2 [124]XP_008438779.1 CmCSREM Chromatin
structure-remodeling complex protein SYD isoform X1 – −0.8568 3.099E-02
MELO3C034613.2 [125]XP_008459496.2 CmCLPP CLP protease regulatory
subunit CLPX3, mitochondrial isoform X2 – −1.5961 6.544E-05
MELO3C026854.2 [126]XP_008465290.2 CmRNApol1 DNA-directed RNA
polymerase subunit – −1.1522 2.372E-02
MELO3C016960.2 [127]XP_008452849.1 CmRNApol2 DNA-directed RNA
polymerase subunit beta – −0.9456 4.024E-02
MELO3C010495.2 [128]XP_008446732.1 CmDNAJ1 DnaJ protein homolog1 –
−3.5189 1.593E-06
MELO3C012052.2 [129]XP_008446732.1 CmDNAJ2 DnaJ protein homolog2 –
−1.6851 4.110E-13
MELO3C006726.2 [130]NP_001284475.1/[131]XP_008438969.1 CmGK
Galactokinase – 0.7983 9.738E-03
MELO3C002363.2 [132]XP_008437427.1 CmGLMT Glucuronoxylan
4-O-methyltransferase 1 – 0.9286 4.704E-02
MELO3C003459.2 [133]XP_008440310.1 CmGLYT Glycosyltransferases – 2.8172
2.076E-02
MELO3C021249.2 [134]XP_008454693.1 CmHEXT2 Hexosyltransferase 2 –
2.0767 1.605E-07
MELO3C015949.2 [135]XP_008447733.1 CmHEXT1 Hexosyltransferase 1 –
−2.2774 1.159E-03
MELO3C009735.2 [136]XP_008443230.1 CmNFKB NF-kappa-B-activating protein
– −1.0229 3.657E-03
MELO3C003497.2 [137]XP_008466126.1 CmPGLMT1 Phosphoglycerate
mutase-like protein 1 – 2.0468 2.103E-02
MELO3C022069.2 [138]XP_008459427.1 CmEBGLUC Probable
endo-1,3(4)-beta-glucanase – 1.1135 1.504E-02
MELO3C023253.2 [139]XP_008460901.1 CmPCE1 Probable
pectinesterase1/pectinesterase inhibitor 51 – 5.7293 3.178E-03
MELO3C023254.2 [140]XP_008460902.1 CmPCE2 Probable
pectinesterase2/pectinesterase inhibitor 51 – 4.8748 4.918E-03
MELO3C023627.2 [141]XP_008438007.1 CmPGLC1 Probable polygalacturonase1
– 6.3658 4.436E-02
MELO3C011986.2 [142]XP_008446196.1 CmPGLC2 Probable polygalacturonase2
– 2.1382 1.028E-14
MELO3C022542.2 [143]XP_016903497.1/XP_008466011.2 CmKAN2 Probable
transcription factor KAN2 – −4.4866 1.861E-02
MELO3C012479.2 [144]XP_008438929.1 CmPARG1 Protein argonaute 1 –
−3.0169 1.142E-24
MELO3C021378.2 [145]XP_008460254.1 CmRIK Protein RIK isoform X1 –
−1.3787 1.360E-03
MELO3C006266.2 – CmINH-LIKE3 Putative invertase inhibitor LIKE3 –
2.3543 1.750E-14
MELO3C008049.2 – CmINH2 Invertase inhibitor – −1.2754 5.661E-03
MELO3C014613.2 [146]XP_008449737.1 CmUP1 uncharacterized protein
LOC103491528 – 1.0875 3.548E-02
MELO3C004012.2 [147]XP_008451613.1 CmUP2 uncharacterized protein
LOC103492844 – −1.0783 6.416E-03
MELO3C027277.2 [148]XP_008462107.1 CmEXPGLC Exopolygalacturonase clone
cmo00040 2.4207 1.198E-03
MELO3C008202.2 [149]XP_008441351.1 CmRPE Ribulose-phosphate 3-epimerase
cmo00040 1.0046 1.448E-02
MELO3C004075.2 [150]XP_008452100.1 CmXISM Xylose isomerase cmo00040
0.8185 3.558E-02
MELO3C008467.2 [151]XP_008441609.2 CmUGGP UDP-sugar pyrophosphorylase
cmo00040/ cmo00052/
cmo00520
−1.0021 3.830E-03
MELO3C017213.2 [152]XP_008453254.1 CmUG6D UDP-glucose 6-dehydrogenase
cmo00040/ cmo00520 1.0501 4.919E-02
MELO3C023110.2 – CmNAG2 Neutral alpha galactosidase2 cmo00052 1.0713
2.967E-03
MELO3C011771.2 [153]XP_008445911.1 CmAAG2 Alpha-galactosidase
(Melibiase)2 cmo00052 1.5211 4.092E-02
MELO3C032910.2 [154]XP_008440953.1 CmATP-PPKN ATP-dependent
6-phosphofructokinase (Phosphofructokinase) cmo00052 1.1856 3.992E-02
MELO3C009979.2 [155]XP_008443553.1 Cm NAGLIKE2 Galactinol-sucrose
galactosyltransferase 5 cmo00052 2.4512 2.042E-04
MELO3C010314.2 [156]XP_008443958.1 CmNAG3 Galactinol-sucrose
galactosyltransferase 6 isoform X1 cmo00052 −0.9338 2.874E-02
MELO3C015912.2 [157]XP_008451468.1 CmSCS Stachyose synthase cmo00052
2.4118 7.679E-04
MELO3C005363.2 [158]NP_001284469.1 CmAIN2 Acid Invertase 2 (acid
beta-fructofuranosidase-like)
cmo00052/
cmo00500
2.3430 1.957E-08
MELO3C005293.2 [159]XP_008467118.1 CmPGIcyt Phosphoglucomutase,
cytoplasmic
cmo00052/
cmo00500
0.8393 1.918E-02
MELO3C017002.2 [160]XP_008452915.1 CmAAML Alpha-amylase
(1,4-alpha-D-glucan glucanohydrolase) cmo00500 0.9488 1.996E-02
MELO3C012010.2 [161]XP_008446229.1 CmTPS9 Alpha-trehalose-phosphate
synthase [UDP-forming] 9 cmo00500 1.1210 8.375E-03
MELO3C016121.2 [162]XP_008451866.1 CmBAML Beta-amylase cmo00500 −1.2463
6.429E-03
MELO3C034277.2 [163]XP_008453064.1 CmBGL18 Beta-glucosidase 18-like
cmo00500 1.7017 1.035E-04
MELO3C015214.2 [164]XP_008450452.1 CmBGL24 Beta-glucosidase 24 cmo00500
4.5276 6.270E-04
MELO3C021895.2 [165]XP_008459280.1 CmEGLC Endoglucanase-like cmo00500
7.8063 3.544E-05
MELO3C002024.2 [166]XP_008440956.1 CmGBGL1 Glucan
endo-1,3-beta-glucosidase 1 cmo00500 −1.8416 4.230E-02
MELO3C030768.2 [167]XP_016900389.1 CmIBAML Inactive Beta-amylase
cmo00500 1.7307 4.735E-02
MELO3C015552.2 [168]XP_008450968.1 CmSUS1 Sucrose synthase 1 cmo00500
−1.2647 5.108E-05
MELO3C025101.2 [169]XP_008463167.1 CmSUS2 Sucrose synthase 2 cmo00500
3.8280 1.430E-41
MELO3C009570.2 [170]XP_008442968.1 CmSPP1 Sucrose-phosphatase 1
cmo00500 0.8290 4.230E-02
MELO3C020357.2 [171]XP_008457154.1 CmSPS2 Sucrose-phosphate synthase 2
cmo00500 1.6220 4.051E-03
MELO3C006984.2 [172]XP_008439346.1 CmTPP1 Trehalose 6-phosphate
phosphatase 1 cmo00500 3.7901 4.521E-02
MELO3C018715.2 [173]XP_016901732.1 CmTPS7 Trehalose-6-phosphate
synthase 7 cmo00500 −0.6675 4.521E-02
MELO3C013838.2 [174]XP_008448661.1 CmTPS5 Trehalose-6-phosphate
synthase 5 cmo00500 −1.0657 6.854E-04
MELO3C005858.2 [175]XP_008437557.1 CmAEChit Acidic endochitinase
cmo00520 2.1904 4.663E-05
MELO3C009722.2 [176]XP_008443206.1 CmALAR Alpha-L-arabinofuranosidase
1-like isoform X2 cmo00520 2.5093 2.053E-17
MELO3C006704.2 [177]XP_008444611.1 CmEP3-Like Endochitinase EP3-like
cmo00520 2.1214 1.476E-02
MELO3C005859.2 [178]XP_016903343.1 CmHV-ALIKE Hevamine-A-like cmo00520
4.4816 5.239E-03
MELO3C019691.2 [179]XP_016902486.1 CmHEXT3 Hexosyltransferase 3
cmo00520 1.2974 1.711E-02
MELO3C005640.2 [180]XP_008451740.1 CmUGE3 UDP-glucose epimerase 3
cmo00520 1.5495 6.583E-07
MELO3C022932.2 [181]XP_008460595.1 CmAUXRF1 Auxin response factor1
cmo04075 −0.7397 5.303E-03
MELO3C003906.2 [182]XP_008450396.1 CmER1 Ethylene receptor 1 cmo04075
−1.2850 1.144e-06
MELO3C006371.2 [183]XP_008461049.1 CmAUXRS Auxin-resposive protein
cmo04075 −1.7529 1.147E-02
MELO3C011021.2 [184]XP_008444821.1 CmENDP Endoplasmin homolog cmo04141
−0.7411 4.808E-02
[185]Open in a new tab
^a cmo00040: pentose and glucuronate interconversions; cmo00052:
galactose metabolism; cmo00500: starch and sucrose metabolism;
cmo00520: amino sugar and nucleotide sugar metabolism; cmo04075: plant
hormone signal transduction; cmo04141: protein processing in
endoplasmic reticulum
^b The positive values are up-regulated genes and the negative values
are down-regulated genes when considerate the 10 DAP stage
For network construction, we used the STRING database
([186]https://string-db.org) that returned 417 nodes, 671 edges and the
p-value for protein-protein interaction (PPI) enrichment was < 1.0e-16
for 10 DAP fruit genes (Additional file [187]8: Figure S5, Table S7).
The 40 DAP fruit genes results showed 404 nodes, 1512 edges and the
p-value PPI enrichment was < 5.79e-08 (Additional file [188]8: Figure
S6, Table S8). The functional enrichment in the network demonstrated a
high number of proteins involved in metabolic pathways and protein
processing in the young and full-ripe fruit respectively (Additional
file [189]8: Figure S5, S6). Proteins related to the sugar pathway were
selected from total DE genes and the subnetwork generated was composed
of 38 nodes, 68 edges and PPI enrichment p-value < 1.0e-16 in young
fruit. The proteins with the highest interaction in this analysis were
alpha-N-arabinofuranosidase 1 ([190]XP_008443206.1), sucrose synthase
([191]XP_008463167.1) and acid invertase 2 ([192]NP_001284469.1)
(Fig. [193]5, Additional file [194]9: Tables S9, S10). Regarding the
mature fruit, the subnetwork generated was characterized by 22 nodes,
27 edges and PPI enrichment p-value < 1.0e-16. The protein argonaute 1
([195]XP_008438929.1) and probable galacturonosyltransferase 10
([196]XP_008447733.1) presented the highest interactions number (Fig.
[197]5, Additional file [198]9: Tables S11, S12).
Fig. 5.
[199]Fig. 5
[200]Open in a new tab
Protein–protein interaction network of sugar pathway and associated
routes in the 10 DAP (a) and 40 DAP (b) melon fruits obtained by STRING
analyses. Nodes represent related proteins and edges represent
protein–protein associations. In A) red is “Starch and sucrose
metabolism”, blue is “Amino and nucleotide sugar metabolism”, light
green is “Galactose metabolism”, yellow is “Pentose and glucoronate
interconversions”, pink is “Cyanoamino acid metabolism” and dark green
is “Pentose phosphate pathway”. In B) red is “Starch and sucrose
metabolism”, blue is “Auxin signalling pathway”, green is
“Transcription”, yellow is “Nucleotidyltransferase” and pink is
“DNA-directed RNA polymerase”. The white nodes are genes not classified
within a pathway or protein group
Sugar pathway and associated proteins
Seventeen DE genes are associated with the sucrose metabolism by KEGG
analyses (Fig. [201]6, Table [202]4, Additional file [203]10: Figure
S7, Additional file [204]11: Figure S10). The genes that present higher
interaction with these enzymes (STRING database) by PPI analyses and
those that are important in sucrose metabolism described in previous
studies (not available in the KEGG database) were also considered
[[205]9, [206]16] (Fig. [207]6, Table [208]4, Additional file [209]10:
Figures S8, S9, Additional file [210]11: Figure S10). Some enzymes
associated with this pathway are encoded by multiple genes and their
amino acid sequences were aligned using the MUSCLE algorithm [[211]27]
as well as submitted to percentage similarity analysis
([212]http://imed.med.ucm.es/Tools/sias.html software). The results
showed a wide difference between the isoenzymes; and the alpha
galactosidases, invertase inhibitor and hexosyltransferase sequences
were the most dissimilar (Additional file [213]12: Figure S11).
Fig. 6.
[214]Fig. 6
[215]Open in a new tab
Hierarchical clustering analyses of DE genes of sugar and associated
pathways of young (10 DPA) and mature (40 DAP) fruit samples. The log2
fold change values were converted by rlog (regularized logarithm)
function in Deseq2. Each line represents one gene and the rows are the
samples. The colour bar represents the rlog values and ranges from blue
(low expression) to red (high expression)
Considering RNA-seq analysis, in the ‘starch and sucrose metabolism’
(KEGG: cmo00500) 12 genes are more expressed in young fruit (acid
invertase 2/ CmAIN2, phosphoglucomutase/CmPGIcyt, alpha-amylase/CmAAML,
alpha-trehalose-phosphate synthase9/CmTPS9, beta-glucosidase
18-like/CmBGL18, beta-glucosidase 24/CmBGL24,
endoglucanase-like/CmEGLC, inactive beta-amylase/CmIBAML, sucrose
synthase 2/CmSUS2, sucrose-phosphatase1/CmSPP1, sucrose-phosphate
synthase 2/CmSPS2, trehalose 6-phosphate phosphatase 1/CmTPP1); and 5
genes are more expressed in full-ripe fruit (beta-amylase/CmBAML,
glucan endo-1,3-beta-glucosidase 1/CmGBGL1, sucrose synthase 1/CmSUS1,
trehalose-6-phosphate synthase 7/CmTPS7, trehalose-6-phosphate synthase
5/CmTPS5) (Table [216]4, Additional file [217]11: Figure S10). The
highest log2 fold change values were to CmEGLC (7.8063) and CmBGL24
(4.5276) in young melon. For mature melon they were to CmGBGL1 (1.8416)
and CmSUS1 (1.2647) (Table [218]4). The RT-qPCR (quantitative reverse
transcription PCR analysis) was conducted for some of these genes in
the 10 DAP, 20 DAP, 30 DAP and 40 DAP stages (Fig. [219]3). In this
analysis, the CmAIN2 gene has a markedly increased expression from 10
to 20 DAP fruit, declining rapidly in subsequent stages (Fig. [220]3).
The two sucrose synthase isoenzymes showed different expression
patterns in fruit maturation as also observed in RNA-seq. CmSUS1
relative expression has a continuous increase from 10 DAP to 40 DAP
fruit. In contrast, the CmSUS2 gene has a higher expression level in
younger fruit and gradually decreased in the following ripening stages
(Fig. [221]3). The expression level of CmSPS2 was more remarkable in 30
DAP fruits when compared to other maturation stages (Fig. [222]3).
CmSPP1 expression increased from 10 DAP to 20 DAP and then decreased in
the following developmental stages (Fig. [223]3). The CmINH-LIKE3 is
not presented in the KEGG pathway; however, it has been included in
RT-qPCR analyses because the literature reports its function in
invertase inhibition. The expression profile of this gene demonstrated
a marked expression only in younger fruit when compared to other
development stages (Fig. [224]3). However, the CmINH2 isoform presented
higher expression in 40 DAP fruit when compared to 10 DAP fruit
(RNA-seq analysis).
In the ‘amino sugar and nucleotide sugar metabolism’ (cmo00520), 7
genes are more expressed in 10 DAP fruit (UDP-glucose
6-dehydrogenase/CmUG6D, Acidic endochitinase/CmAEChit,
Alpha-L-arabinofuranosidase 1-like isoform/CmALAR, Endochitinase
EP3-like/CmEP3-Like, Hevamine-A-like/CmHV-ALIKE, Hexosyltransferase
3/CmHEXT3, UDP-glucose epimerase 3/CmUGE3) and 1 gene is more expressed
in 40 DAP (UDP-sugar pyrophosphorylase/CmUGGP) (Table [225]4,
Additional file [226]11: Figure S10). The most representative
expression level was to CmHV-ALIKE (4.4816). In the RT-qPCR analysis,
the gene expression of CmUGE3 was relatively low in young fruit,
increased rapidly in the 20 DAP stage and decreased in the following
developmental stages (Fig. [227]3).
The ‘galactose metabolism’ (cmo00052) has 9 DE genes, 6 of them more
expressed in young fruit (Alkaline alpha-galactosidase/CmNAG2,
Alpha-galactosidase 2/CmAAG2, Galactinol-sucrose galactosyltransferase
5/CmNAGLIKE2, Stachyose synthase/CmSCS, Acid Invertase 2/CmAIN2,
Phosphoglucomutase/CmPGIcyt) and 3 more expressed in mature fruit
(UDP-sugar pyrophosphorylase/CmUGGP, ATP-dependent
6-phosphofructokinase/CmATP-PPKN, Galactinol-sucrose
galactosyltransferase 6 isoform X1/CmNAG3) (Table [228]4,
Additional file [229]11: Figure S10). The relative expression of the
CmNAG2 gene showed a rapid increase from 10 DAP to 20 DAP decreasing in
30 DAP and keeping constant in 40 DAP (Fig. [230]3). The RT-qPCR of
CmAIN2 has been previously discussed.
Also, another 32 DEGs were identified in network analyses that are
potentially associated with the sugar pathway (Table [231]4,
Additional file [232]11: Figure S10). The more expressed genes were:
probable polygalacturonase1 (6.3658), probable pectinesterase1
(5.7293), probable pectinesterase 2 (4.8748) for young fruits and
probable transcription factor KAN2 (4.48), DnaJ protein homolog1
(3.51), Protein argonaute 1 (3.016) for full-ripe fruits.
Discussion
Global characteristics of the ‘yellow’ non-climacteric melon ripening
Fruit ripening and development is a genetically programmed and
irreversible process that involves physiological, biochemical and
organoleptic changes influencing the fruit quality such as flavour,
texture, colour and aroma [[233]28]. However, the study of the
metabolic networks is complex and the central signal of genic cascade
is not completely understood. In our study, we used an important
commercial non-climacteric ‘Yellow’ melon fruit (Cucumis melo, inodorus
group) as experimental material to comprehend the main metabolic
processes that involve maturation in this phenotype, focusing on the
sugar pathway study that is a main quality attribute in melon fruits.
RNA-seq technology was used to analyze the transcriptomic differences
between young (10 DAP) and mature (40 DAP) non-climacteric melon fruit.
A total of 895 DE genes are down-regulated and 909 are up-regulated
during melon ripening. GO enrichment analysis showed that the DE genes
in young fruit were more related to molecular transport and metabolic
processes including the ‘carbohydrate metabolism’; while in ripe fruit
the most DE genes are required for peptide metabolism and protein
biosynthesis. In addition, the integrative KEGG analysis conducted for
metabolic pathways demonstrated that ‘carbon fixation in photosynthetic
organisms’ and ‘carbon metabolism’ pathways were enriched in both fruit
development stages; however different genes or isoforms are DE. At the
beginning of fruit development there is high anabolism and catabolism
of sugar that is the metabolic process required for carbon skeleton
construction and energy supply in plants. In strawberry fruit, an
important role of oxidative phosphorylation in ripening was
demonstrated [[234]29]. The DE genes enriched in 40 DAP melon fruits
are related to the sucrose accumulation function [[235]6]. The protein
processes in the endoplasmic reticulum, spliceosome mechanism and
ribosome biogenesis were also significantly enriched in KEGG analysis
in the late development of melon indicating high transcription and
translation rate. Moreover, the high splicing process is reflected in
the production of different proteins that can act and control a
specific metabolic route. This characteristic associated with the
activation of different protein isoforms can also explain the KEGG
enrichment of the same pathways in both maturation stages as previously
mentioned. Studies have reported the presence of paralogous copies
acting in diverse metabolic pathways in plants, including in sugar
metabolism, that in melon have definite functionalization concerning
both development stages and tissue specificity [[236]9]. The high
activity of photosynthesis in young melon when compared to full-ripe
fruit has also been described for grape and other melon varieties
[[237]6, [238]30].
The ‘plant hormone signal transduction’ is an important process in
fruit ripening [[239]31, [240]32], and this pathway was significantly
enriched in the early melon fruit development. Ethylene (ETH), abscisic
acid (ABA) and brassinosteroids (BRs) have been suggested to promote
ripening through complex interactions; while auxin (IAA), cytokinins
(CYT), gibberellin (GA) and jasmonic acid (JA) are putative inhibitors
of ripening [[241]29, [242]33]. In our study, the DE genes present in
IAA, JA, GA and CYT signal transduction decreased during maturation
which also occurs in other non-climacteric fruits [[243]29, [244]34,
[245]35]. In the ABA pathway, only the ‘protein phosphatase 2C 77’ gene
(repressor of the abscisic acid signalling pathway [[246]36]) was DE in
the 10 DAP fruit. Studies have been suggested that ABA plays an
important role in the regulation of non-climacteric fruits [[247]37,
[248]38] and the key gene for its biosynthesis is 9-cis-epoxycarotenoid
dioxygenase (CmNCED) that was significantly more expressed in full-ripe
fruit (Additional file [249]7: Table S6). This can indicate that ABA
might be involved in the regulation of melon maturation and senescence.
Interestingly, there are intimate connections between sugar and ABA
signalling [[250]39]. The BR burst production generally occurs in the
colour change stage in late fruit development [[251]29, [252]33,
[253]40]. In our study, the genes related to BR signal transduction are
more expressed in young ‘Yellow’ melon fruit; however the colour change
occurs from 20 DAP to 30 DAP fruits. Thus further studies should be
conducted to understand the transcriptome profile of these stages. The
expression of some genes present in the ethylene and salicylic acid
metabolism were highest in mature fruit (Additional file [254]7: Table
S6). One of these genes is the ethylene receptor 1 that has been shown
to negatively regulate ethylene signal transduction and suppress
ethylene responses [[255]41]. Thus, it can be a candidate gene in
non-climacteric and climacteric melon comparative study.
In the subnetwork protein-protein interaction (PPI), the results of the
10 DAP fruits showed the interaction of 6 metabolic pathways: ‘Starch
and sucrose metabolism’; ‘Amino and nucleotide sugar metabolism’;
‘Galactose metabolism’; ‘Pentose and glucuronate interconversions’;
‘Cynoamino acid metabolism’; and ‘Pentose phosphate pathway’.
Furthermore, enzymes related to cell wall degradation were identified
such as pectinesterase and polygalacturonase that are mainly
responsible for the pectin changes. The up-regulation of these genes
and those associated with sucrose synthesis in the early stage of
development are involved with progressive fruit softening and sucrose
accumulation. In flesh watermelon, some isoforms of pectinesterase and
polygalacturonase also show an increase in the first development
stages, decreasing in the full-ripe fruit [[256]42]. Another cell wall
enzyme was alpha-L-arabinofuranosidase that catalyzes the breaks in the
arabinoxylan (major component of cell wall plant hemicellulose)
[[257]43]. Saladié et al. (2015) demonstrated that several genes
related to cell wall degradation were more strongly up-regulated in
climacteric melon (cv. Védrantais) than non-climacteric (cv. Piel de
Sapo) [[258]6]. The sugar metabolism is an important process in fruit
ripening and development and sucrose accumulation is the major
determinant of melon sweetness [[259]6, [260]44]. One enzyme of this
pathway is the acid invertase (CmAIN2) that had the second highest
number of interactions in the young melon fruit subnetwork (Fig.
[261]5; Additional file [262]9: Table S9). This reinforces the idea of
its key function in the catabolism of sucrose [[263]6]. Two
beta-glucosidases (CmGL18, CmGL24) have an interaction with CmAIN2,
these enzymes have the function of hydrolyzing the terminal,
non-reducing beta-D-glucosyl residues (final reaction in cellulose
hydrolysis) with the release of beta-D-glucose (primary energy source
in plants) [[264]45] that suggest a high sugar conversion to energy in
the early fruit development stage. Another important enzyme in the
subnetwork is sucrose synthase 2 (CmSUS2) that has a strong interaction
with alpha-trehalose phosphate synthase 9 (CmTPS9) followed by
trehalose phosphate phosphatase (CmTPP1). These enzymes and others of
sugar metabolism will be discussed in more detail below in the next
topic.
Although the majority of DE genes of auxin and sugar metabolism are
up-regulated in 10 DAP melons, some isoforms or different genes from
these pathways are more expressed in 40 DAP. The subnetwork generated
for mature fruit is represented by a different sucrose synthase
(CmSUS1) which also has high interaction with two alpha-trehalose
phosphate synthase isoenzymes (CmTPS7, CmTPS5). The trehalose phosphate
synthases (TPS) convert glucose-6-phosphate and uridine diphosphate
(UDP) glucose into trehalose-6phosphate (T6P) and the subsequent
dephosphorization of T6P is catalyzed by trehalose-phosphate
phosphatases. A recent study reported that threalose-6-phosphate
inhibited sucrose synthase and consequently the sucrose cleavage in
castor bean [[265]46]. The T6P may be undergoing a higher conversion
into trehalose in young melon due to the greater trehalose-phosphate
phosphatase gene expression. Thus T6P accumulation is expected in
full-ripe fruit, once that TPP is down-regulated, contributing to the
increase of sucrose content [[266]47]. In addition, genes involved with
the auxin pathway such as auxin response factor (CmAUXRF1, CmAUXRF2)
and responsive auxin protein (CmAUXRS) are present in this subnetwork
and have interaction through hexosyltransferase and argonaute proteins
with the CmSUS1, CmTPS7 and CmTPS5 (Fig. [267]5). In that respect,
previous studies reported that auxin reduces the sugar content in
fruits [[268]48]. However, the precise association of the genes
CmAUXRF1, CmAUXRF2 and CmAUXRS with the sugar pathway requires further
studies. Regarding the argonaute proteins, they bind to micro RNAs
(miRNA) and act in transcript cleavage [[269]49]. Plant miRNAs
typically target transcription factors including the auxin-response
factor [[270]49]. A weak interaction was detected between
hexosyltransferases, unknown proteins and argonaute proteins and
further studies should be conducted to better understand this
association. It is also noteworthy that the chromatin
structure-remodelling complex protein SYD (CmCSREM) gene present in
this subnetwork is related to a promotor regulation of several genes
downstream of the jasmonate and ethylene signalling pathways [[271]50].
Sucrose metabolism
Sugar metabolism is an important pathway related to the sweetness of
fruits and it is the most attractive characteristic for consumers
[[272]51]. Furthermore, studies have reported that sugars may serve as
important signals that modulate a wide range of processes in plant
physiology including fruit maturation [[273]39, [274]52, [275]53]. In
our study, a total of 17 genes were DEs in the sucrose; amino and
nucleotide sugar; and galactosidase pathways and 8 were evaluated in
two additional development stages (20 and 30 DAP). Sucrose is the main
sugar component that gives the sweet taste in melon and its high
content at the mature stage could be used as a marker [[276]6]. Only
sucrose synthases and invertases are known enzymes responsible for
sucrose cleavage [[277]10]. The sucrose synthases convert sucrose to
fructose and UDP glucose that is a reversible reaction [[278]10]. In
our study, two isoforms were DEs by RNA-seq analysis, the CmSUS1 that
was up-regulated in full-ripe fruit while CmSUS2 had a burst of gene
expression in 10 DAP fruit. In fruit maturation, there is a gradual
expression increase of CmSUS1 and decrease of CmSUS2 in the ‘Yellow’
melon. In non-climacteric melons, ‘Hami’ [[279]51] and ‘Piel del Sapo’
[[280]6], the same expression profile was observed. In ‘Dulce’
climacteric melon, the CmSUS1 was more expressed in young fruit,
followed by near-silencing in mature fruit. CmSUS2 showed low levels of
expression throughout fruit development and the third sucrose synthase
(CmSUS3) was DE being weakly expressed in the young fruit and increased
in the maturing fruit [[281]9]. Thus, it may be suggested that in
non-climacteric melons, CmSUS1 is mainly responsible for the synthesis
of sucrose for storage in the vacuole, contributing to ripe fruit
taste, while CmSUS2 acts in an opposite way providing the substrate for
energy production by sucrose catabolism during early development
(Fig. [282]7). Also, the TPS and TPP have an important function in the
sucrose synthase activities contributing to sucrose content in the
fruit as previously described (Fig. [283]7).
Fig. 7.
[284]Fig. 7
[285]Open in a new tab
Differential expression of sugar metabolism related genes in the melon
ripening process. The green arrows represent 10 DAP or 20 DAP fruits
and the yellow arrows 30 DAP or 40 DAP fruits. The genes with burst of
expression in the intermediate stages (20 DAP and 30 DAP) have the
phase indicated in parentheses
Invertases produce glucose instead of UDP-glucose and fructose in a
non-reversible reaction. Acid invertases have been attributed to
vacuole localization while neutral invertases have generally been
located in the cytosol, consistent with the optimal neutral pH activity
and absence of glycosylation [[286]9]. In the RNA-seq analysis, only
the acid invertase (CmAIN2) was DE in non-climacteric ‘Yellow’ melon.
Previous studies with ‘Piel del Sapo’ [[287]6] and ‘Hami’
non-climacteric melon fruit [[288]51] also showed only transcriptional
activity of acid invertase 2 (CmAIN2). In ‘Dulce’ climacteric melons,
four neutral invertase (CmNIN1, CmNIN2, CmNIN3 and CmNIN4) were DE, as
well as the acid invertase 2 (CmAIN2) [[289]9]. The peak of CmAIN2
expression occurs in the 20 DAP ‘Yellow’ melon fruits and consistently
decreased in the following developmental stages (Fig. [290]7). Studies
have demonstrated that acid and neutral invertase genes are highly
expressed in young developing fruit, and subsequently declined
substantially at the sucrose accumulation stage [[291]6, [292]9,
[293]16, [294]51]. This reduction of soluble acid invertase activity
signals the metabolic transition from fruit growth to sucrose
accumulation [[295]3, [296]18]. The higher expression of neutral
invertases in climacteric melon fruit suggests that cytoplasmatic sugar
catabolism might be an additional source of energy, supporting the
hypothesis that climacteric melon fruit spends more energy during fruit
development, due to respiration, than non-climacteric ones. In the
non-climacteric and climacteric melon comparison, studies demonstrated
that the acid invertase gene (CmAIN2) was almost 10-fold higher in
‘Védrantais’ (climacteric) than in ‘Piel del Sapo’ (non-climacteric).
The high activity of soluble acid invertase (CmAIN2) might limit the
accumulation of sucrose during climacteric ripening and increase
organic acids, such as malate, that impart a stale flavour to the fruit
[[297]3, [298]6, [299]9, [300]18]. Thus, the inhibition of CmAIN2 can
be a key process in the differences of sugar content and melon quality.
Invertase inhibitors are responsible for decreasing the activity of
soluble acid invertases through post-translational regulation, reducing
sugar consumed in respiration and regulating the accumulation of
sucrose during melon development and ripening [[301]6, [302]18]. Two
invertase inhibitors (CmINH2; CmINH-LIKE3) were DE by RNA-seq analysis
and are characterized by the presence of plant invertase/pectin
methyltransferase inhibitor domain (Additional file [303]12: Figure
S11). The invertase inhibitor 2 presented higher transcription level in
full-ripe (40 DAP) than in youngest melon (10 DAP) (Fig. [304]7). On
the other hand the putative invertase inhibitor 3 (CmINH-LIKE3) had
high activity in the beginning of development and low expression in the
following stages (Fig. [305]7). Hence, in the 20 DAP stage the
inactivation of CmAIN2 by CmINH-LIKE3 protein interaction may be
occurring. Subsequently, the CmAIN2 expression rapidly decreases and
the CmINH-LIKE3 transcription is not necessary anymore. The CmINH2 can
have affinity with other invertases that have not been evaluated on the
intermediate stages (20 DAP and 30 DAP). The putative CmINH-LIKE3
expression was not reported in previous studies. The DE of CmINH2 was
also detected in climacteric ‘Dulce’ melon; however, it is more
strongly expressed in the first stages of development decreasing during
ripening [[306]9]. One other DE isoform detected for this same variety
was CmINH1 that was expressed at high levels in the 30 DAP stage
[[307]9]. The results demonstrated different expression profiles for
non-climacteric ‘Yellow’ melon and climacteric ‘Dulce’ melon,
indicating recruitment of distinct INH isoforms in the regulation of
the invertases or its activation in different stages of ripening.
Moreover, two invertase inhibitors (cCL2226Contig1 and
c15d_02-B02-M13R_c) are about 30 times higher in non-climacteric ‘Piel
del Sapo’ melon than in climacteric ‘Védrantais’ melon [[308]6]. These
characteristics are in accordance with invertase expression
differences.
The raffinose oligosaccharides (RFOs) synthesized by photosynthesis can
be converted into sucrose and galactose by α-galactosidases in fruit
tissues. In melon, α-galactosidase includes acid and neutral isoenzymes
and in young fruit, both can be used to provide energy for the growth
metabolism. In mature fruits, sucrose can be stored in the vacuole
while galactose can be metabolized to sucrose [[309]3, [310]6, [311]9].
In our study, three neutral α-galactosidases (CmNAG2, CmNAGLIKE2,
CmNAG3) were DEs (Fig. [312]7). In the early development stages of
‘Yellow’ melon, there is an increase of the CmNAG2 expression, that has
the highest level in the 20 DAP stage and decreases in 30 and 40 DAP.
Though the RNA-seq analyses demonstrated that in the 40 DAP stage the
expression of this enzyme was higher than in 10 DAP, the log2 fold
change was low (1.07). These differences might be due to individual
variations. The CmNAGLIKE2 also has high activity in the beginning of
fruit development, while CmNAG3 peaks in the 40 DAP stage. Previous
studies showed no DE of neutral α-galactosidases in non-climacteric
melon. In climacteric ‘Dulce’ melon the high activity transcriptional
of CmNAG2 was also detected in early stages and low activity of acid
α-galactosidases in the maturation process [[313]9]. We suggest that
the sucrose produced in the catabolic reaction of CmNAG2 and CmNAGLIKE2
in young fruit is recruited in respiration by its conversion to
hexoses, evidenced by the high activity of CmAIN2 and CmSUS2 in the
same stages. The CmNAG3 has a function in the sucrose accumulation in
full-ripe melon.
Another important route of sucrose synthesis is by conversion of
galactose-1P into glucose-1P by UDP-glucose epimerase (UGE) [[314]3,
[315]6, [316]9, [317]17]. In the present study, we observed the
increase of CmUGE3 expression in the initial development, followed by
subsequent decrease until the full-ripe stage, implying a concomitant
gene expression with CmNAG2 (Fig. [318]7). The activity of both enzymes
denotes high sucrose production in young fruits. In climacteric ‘Dulce’
melon, three UGEs were DE throughout fruit development (CmUGE1, CmUGE2
and CmUGE3), but CmUGE3 expression increased significantly during fruit
maturation [[319]9]. The CmUGE3 gene expression or its enzyme activity
were not reported in other non-climacteric melon in previous studies
[[320]6, [321]51]. Antagonistic profile of CmUGE3 is observed in the
two melon phenotypes that can be associated with differences in sugar
accumulation.
Sucrose-P synthase (SPS) is considered the key gene for sucrose
accumulation in fruit ripening [[322]9, [323]16, [324]18]. This enzyme
catalyzes the reversible transfer of a hexosyl group from UDP-glucose
to D-fructose 6-phosphate to form UDP and D-sucrose-6-phosphate
[[325]54]. Only CmSPS2 was DE in our study, which has an increase
during fruit maturation, reaching the highest levels in colour change
melons (30 DAP) (Fig. [326]7). This is in agreement with gene
expression observed in ‘Hami’ non-climacteric melon [[327]51]. In
climacteric ‘Dulce’ melon, CmSPS2 is weakly expressed during fruit
development, but CmSPS1 rapidly increased from 20 DAP, peaking at late
developmental stages [[328]9]. The gene expression of SPS was similar
when comparing climacteric and non-climacteric melon, however different
isoenzymes are responsible for syntheses D-sucrose-6-phosphate. The
sucrose phosphate phosphatase (SPP) has complementary activity with SPS
by conversion of sucrose-6-phosphate to sucrose [[329]3, [330]9,
[331]16, [332]18, [333]51]. In our study only CmSPP1 was DE, having a
moderate expression without pronounced differences in the first two
stages but with evident decrease in the full-ripe fruit. In
non-climacteric ‘Hami’ melon, the DE of sucrose-P phosphatase was not
observed [[334]51]. In climacteric ‘Dulce’ melon, the CmSPP1 gene was
weakly expressed with a slight increase in the 40 DAP stage [[335]9].
Conclusion
Considering the limited knowledge about molecular mechanisms that act
in the ripening process in non-climacteric melon, studies that involve
high-throughput analyses like RNA-seq are paramount to open new
perspectives on this matter. Sucrose-cleaving enzymes perform essential
mechanisms for the distribution and use of sucrose in fruits. Only
sucrose synthase and invertase enzymes can cleave sucrose. CmSUS2 and
CmAIN2 are up-regulated in the early development stages of the ‘Yellow’
melon (Fig. [336]7), indicating high hexose production, which in turn
increases the respiration metabolism and the generation of hexose-based
signals. Studies demonstrated that these signals are involved in
development processes such as cell division [[337]10]. In addition, the
UDP glucose product of sucrose synthase has been implicated in the
formation of diverse cell wall polysaccharides [[338]55]. The sucrose
substrate for these enzymes is provided by CmNAG2, CmNAGLIKE2 and
CmUGE3 transcriptional activity that is high in the same stages. The
new putative invertase inhibitor CmINH-LIKE3 (exclusively expressed in
‘Yellow’ melon) decreases invertase activity in young non-climacteric
fruit (Fig. [339]7), suggesting its importance during non-climacteric
melon fruit development, sucrose accumulation and organic acid content.
SPP1 has the highest expression in 20 DAP fruit and CmSPS2 in 30 DAP,
both enzymes have a complementary role in sucrose biosynthesis in the
intermediate stages (Fig. [340]7). Finally, CmNAG3 and CmSUS1 have a
crucial function in sucrose accumulation in the late stages of fruit
development (Fig. [341]7). Also, the higher expression of CmTPP in the
early stages increases the trehalose-6-phosphate conversion to
trehalose preventing sucrose synthase inhibition (CmSUS2) (Fig.
[342]7). In contrast, trehalose-6-phosphate accumulation by TPS
activity inhibits sucrose cleavage in full-ripe melons (Fig. [343]7).
Many genes within hormone pathways showed differential expression
detected by RNA-seq analysis. The hormones also play an essential
function in fruit ripening, but the mechanisms are complex and poorly
understood. In our study, for IAA, JA, GA, CYT, ABA and BRs signal
transduction, most genes are more expressed in young than full-ripe
fruit. This characteristic is observed in previous studies [[344]29,
[345]34, [346]35, [347]56]. In mature fruit, the auxin response factor,
responsive auxin, 9-cis-epoxycarotenoid dioxygenase and receptor of
ethylene 1 with the highest transcriptional activity were detected and
are interesting genes for further melon ripening studies. Furthermore,
several studies have demonstrated the integration of sucrose and
hormonal pathways including auxin and abscisic acid, as well as
epigenetic control (microRNAs and chromosomal modification) acting on
fruit development.
This is the first study conducted for non-climacteric ‘Yellow’
Brazilian commercial melon and the results on sugar metabolism and
related pathways during development and ripening contribute to new
perspectives in management practices and molecular tools to improve
fruit quality.
Methods
Plant material
Non-climacteric melon fruit of a ‘Yellow’ commercial genotype (Cucumis
melo, Inodorus group) was cultivated and provided in the different
ripening stages by Itaueira Agropecuária SA company in São Paulo
(Brazil). Fruit was manually pollinated, and three biological
replicates were harvested at different development stages: 10 days
after pollination (DAP), 20 DAP, 30 DAP and 40 DAP. For each sampling
time, flesh mesocarp was collected, immediately frozen in liquid
nitrogen and stored at − 80 °C until analysis.
Colour, SS (soluble solids) and pH measurement
Peel and pulp colour at different ripening stages were measured using a
Minolta CR400 colorimeter. The CIE (Commission Internationale de
l’Eclairage) L* (lightness), a* (green/red coordinate), b* (blue/yellow
coordinate) colour scale was adopted. The angle Hue was calculated by
the equation
[MATH: h°=tan−1b∗a∗<
/mo> :MATH]
if a* > 0 and b* > 0 or by equation
[MATH: h°=180+tan−1b∗a∗<
/mo> :MATH]
if a* < 0 or b* > 0 [[348]20]. The soluble solids content (SS° Brix)
and the pH were measured using digital refractometer and automatic pH
matter respectively. Three measurements were made for each fruit and a
mean was obtained.
RNA extraction
Total RNA was extracted in biological triplicate (different fruit) of
four development stages using the sodium perchlorate method as
described for melon by Campos et al. (2017) [[349]57]. The RNA quality
and quantity were determined using Nanovue™ spectrophotometer and 1%
agarose gel electrophoresis. Only RNAs that presented A260/A280 ratio
~ 2.0, A260/A230 ratio ~ 1.80 and no discernible degradation were used
for RNA-seq and qPCR analyses.
Preparation of cDNA libraries and RNA-seq
The cDNA library preparation for RNA-seq analyses was performed to 10
DAP and 40 DAP fruits. Sample RNA quality and concentration for RNA-seq
were assessed with the Agilent 2100 Bioanalyzer (Thermo Scientific).
Messenger RNA (mRNA) was isolated using the Dynabeads mRNA Direct Micro
kit (Life Technologies). Single-end libraries were prepared with the
Ion Total RNA-Seq Kit v2, barcoded with the PI™ Chip Kit v3 at the
Federal University of Paraná (Curitiba, BR). After pooling into
two-sample groups, the libraries were sequenced (five technical
replicas) on an Ion Torrent™ (Life Technologies™) using the PI
Template 200 bp v3 and Ion PI Sequencing 200 Kit v3. A total of ~ 27
million reads were obtained for each sample. The raw sequencing data
has been deposited in the NCBI sequence read archive (SRA) under the
accession number SRP230494
([350]https://trace.ncbi.nlm.nih.gov/Traces/sra/?study=SRP230494) .
Gene expression analysis of RNA-Seq data
RNA-Seq reads from each biological replicate (2 per 10 DAP and 3 per 40
DAP) were filtered and submitted to adaptor trimming using
fastx-toolkits ([351]hannonlab.cshl.edu/fastx_toolkit/) and cutadpt
[[352]58] respectively. Reads showing ≥80% of sequenced bases with
Phred scores over 20 and more than 50 bp in length were selected. The
melon genome (Cucumis melo version v3.6.1) and annotation (gff3 file)
provided at [353]https://www.melonomics.net were used as a reference in
differential expression analysis. Transcriptome mapping was achieved by
using the software Bowtie2 aligner [[354]24]. Gene counts were
calculated using featureCounts and only reads with overlapping in a
single gene were considered for RNA-seq analysis [[355]59].
Differential expression analyses were carried out applying a
statistical test evaluating the negative binomial distribution provided
in the R package DeSeq2 [[356]60]. For each gene, the padj ≤0.05 was
considered as the significant threshold. The hierarchical clustering
was performed to sample clustering analysis and to evaluate the profile
of the top 50 DE genes using the gplots package and heatmap.2 function
available in R.
Gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG)
Gene ontology term enrichment analysis of DE genes was performed using
[357]http://cucurbitgenomics.org/goenrich software (dataset melon DHL92
v3.61), with FDR (false discovery rate) adjusted p-value < 0.05. KEGG
pathway enrichment analysis was carried out using DAVID according to
the default actions [[358]26]. The pathways of differentially expressed
genes were visualized using the ‘Pathview’ software based on the
KO-gene-assignment file and fold change value for each gene under
pairwise comparisons [[359]61]. The degree of log2 fold changes was
highlighted in different colours.
Protein-protein interaction (PPI) network construction and modules mining
Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) is
a database of protein-protein interaction [[360]25]. This database
contains direct and physically related interactions between known and
predicted proteins and genes. The sources are mainly from (a)
experimentally determined (b) text mining in scientific articles and
other databases, (c) gene-neighbourhood, (d) gene fusions, (e)
co-expression, (f) gene co-occurrence, and (g) protein homology. The
system uses a scoring mechanism to give a certain weight to the results
and finally gives a comprehensive high throughput analyses [[361]62].
For this analysis we setting the minimum required interaction score
0.400, none max number of interactors and all interaction sources were
selected. The Cytoscape software [[362]63] was used to analyze
protein-protein interaction (PPI) generated by STRING. In this study,
we set as input the genes 10 DAP-biased fruit separately from genes of
40 DAP-biased fruit. We selected the proteins interactions more
relationship with sucrose metabolism from the general network.
Quantitative reverse transcription PCR analysis (RT-qPCR)
To validate the accuracy of transcriptome profiling, the gene
expression of eight transcripts related to the sugar pathway was
evaluated by quantitative reverse transcription PCR. Gene specific
primers were designed using the ‘PrimerBlast’ database
([363]https://www.ncbi.nlm.nih.gov/tools/primer-blast/) (Additional
file [364]3: Table S3). The RPS15 and RPL genes were used with internal
control according to Kong et al. (2014) [[365]64]. Also, in these
analyses all fruit development stages were considered (10 DAP, 20 DAP,
30 DAP and 40 DAP). Total RNA was treated with the TURBO™ DNase kit
(Invitrogen) to remove genomic DNA residues from the extraction and it
was submitted to cDNA conversion by Maxima H minus First Strand cDNA
Synthesis kit (Thermo Scientific) following the manufacturer’s
instructions.
RT-qPCR was performed on a LightCycler® Nano platform (Roche
Diagnostics GmbH, Mannheim, Germany) using 100 ng of cDNA in a reaction
containing 1 μL of forward and reverse primer (10 μM), 10 μL FastStart
Essential DNA Green Master 2X (Roche), in a final volume of 20 μL. The
amplification conditions were performed at 94 °C for 10 min and then
cycled at 95 °C for 15 s, 55–60 °C for 20s, 72 °C for 20s for
45 cycles. A melting curve analysis (60 °C to 99 °C) was performed
after the thermal profile to ensure specificity in the amplification.
Each assay was performed in triplicates. Relative gene expression
analysis was performed using the 2^-∆∆Ct method according to Livak and
Schmittgen (2001) [[366]65]. Data were converted to a log2 fold change
scale to make the data comparable with the RNA-seq results. Pearson’s
correlation distance was calculated across 10 DAP and 40 DAP
developmental stages.
RT-qPCR statistical analysis
The R was used for the statistical analyses. Normality was
statistically assessed by the Shapiro–Wilk test [[367]66]. Values that
were not normally distributed were transformed by the Box–Cox method
[[368]67]. Significant differences among means were determined with the
ANOVA (P ≤ 0.05) and Tukey’s test (P ≤ 0.05). It was not possible to
normalize the sucrose synthase 1 (CmSUS1) and sucrose synthase 2
(CmSUS2) genes in RT-qPCR, and the Kruskal-Wallis & Wilcoxon tests
(P ≤ 0.05) were applied in this case.
Supplementary information
[369]12864_2020_6667_MOESM1_ESM.xlsx^ (5.1MB, xlsx)
Additional file 1: Table S1. Count of number of reads per gene obtained
by featureCounts software. Only reads with overlapping in a sigle gene
were considered for RNA-seq analysis; Table S2. RNA-seq data analysis
(diferential expression and statistical test).
[370]12864_2020_6667_MOESM2_ESM.tiff^ (7.9MB, tiff)
Additional file 2: Figure S1. PlotMA (Deseq2 R package) shows the log2
fold changes of young fruits (positive values) and full-ripe fruit
(negative values) over the mean of normalized counts for all the
samples. Points in red are genes that have significant differential
expression (adjusted p-value ≤0.05). Points that fall out of the window
are plotted as open triangles pointing either up or down.
[371]12864_2020_6667_MOESM3_ESM.pdf^ (288.9KB, pdf)
Additional File 3: Table S3. Target genes and reference genes used in
RT-qPCR analysis; Figure S2. Pearson’s correlation between the 10 DAP
and 40 DAP development stage. The expression ratio for RNA-seq and
RT-qPCR analysis are represented by log2 fold change.
[372]12864_2020_6667_MOESM4_ESM.tiff^ (10MB, tiff)
Additional file 4: Figure S3. Heatmap of the sample-to-sample distances
that gives an overview over similarities and dissimilarities between
samples (V is 10 DAP fruit and M is 40 DAP fruit). Dark blue shade
indicates higher levels of similarity and light blue indicates higher
levels of dissimilarities.
[373]12864_2020_6667_MOESM5_ESM.xlsx^ (42.1KB, xlsx)
Additional file 5: Table S4. Gene ontology enrichment analysis of young
and mature melon DE genes. This analysis was performed using FDR (false
discovery rate) adjusted p-value < 0.05 on DE genes
([374]http://cucurbitgenomics.org/goenrich).
[375]12864_2020_6667_MOESM6_ESM.pdf^ (478.9KB, pdf)
Additional file 6: Table S5. The top 50 DE genes between young (10 DPA)
and mature (40 DAP) fruit samples; Figure S4. Hierarchical clustering
analyses of DE top 50 genes between young (10 DPA) and mature (40 DAP)
fruit samples. The log2 fold change values were converted by rlog
(regularized logarithm) function in Deseq2. Each line represents one
gene and the rows are the samples. The colour bar represents the rlog
values and ranges from blue (low expression) to red (high expression).
[376]12864_2020_6667_MOESM7_ESM.pdf^ (225.4KB, pdf)
Additional file 7: Table S6. Differential expressed genes present on
KEGG enrichment pathways (Fisher exact test ≤0.05).
[377]12864_2020_6667_MOESM8_ESM.pdf^ (2.9MB, pdf)
Additional file 8: Figures S5, S6 and Tables S7, S8. Figures represent
protein–protein interaction network of young (Figure S[378]4) and
mature melon (Figure S5) fruit generated by STRING and Cytoscape
analyses. The tables represent the characteristics of the network
interaction.
[379]12864_2020_6667_MOESM9_ESM.pdf^ (265.5KB, pdf)
Additional File 9: Tables S9, S10, S11, S12. Characteristics of PPI
network interaction of sugar and associated pathways.
[380]12864_2020_6667_MOESM10_ESM.pdf^ (419.1KB, pdf)
Additional File 10: Figures S7, S8, S9. KEGG (Kyoto Encyclopedia of
Genes and Genomes) analyses using Pathview software
([381]https://pathview.uncc.edu/) of “starch and sucrose metabolism”,
“galactose metabolism” and “amino sugar and nucleotide sugar
metabolism”. The colour bar represents de log2 fold change of the
maturation process and ranges from green (up-regulated genes in 40 DAP
fruit) to red (up-regulated genes in 10 DAP fruit). The blue letters
are enzyme short names described in the KEGG pathway and the purple are
enzyme short names that were described in the literature associated
with the sugar pathway [[382]6, [383]9]. There are protein isoforms
that act in the same metabolic route and the information of all log2
fold change were included.
[384]12864_2020_6667_MOESM11_ESM.pdf^ (775.8KB, pdf)
Additional File 11: Figure S10. Graphics of the normalized gene counts
obtained by RNA-seq results (plotCounts function of DESeq2 analysis –
differential gene expression analysis based on negative binomial
distribution).
[385]12864_2020_6667_MOESM12_ESM.pdf^ (936.6KB, pdf)
Additional File 12: Figure S11. Alignment of protein isoforms related
to sugar metabolism or associated pathways. The domains were detected
by Pfam database ([386]https://pfam.xfam.org/) and the amino acid
sequences were aligned by the MUSCLE algorithm.
Acknowledgements