Abstract
To fully utilize the characteristic climatic conditions in the southern
region of China, a two-crop-a-year cultivation system technique for
‘Kyoho’ grape was developed during the past decade. After summer
harvest in June, appropriate pruning and chemical treatments promote
flowering and fruiting, which enables a second harvest in late
December. Due to climatic differences between the two crop growing
seasons, grape phenol and carotenoid metabolism differ greatly. The
reported study analyzed the transcriptome of the carotenoid and
phenylpropanoid/flavonoid pathways in grapes at four different stages
during the two growing seasons. Compared with those in summer grapes,
expression levels of the majority of genes involved in the carotenoid
metabolic pathway in winter grapes were generally upregulated. This
result was associated with lower rainfall and much more abundant
sunlight during the second growing season. On the other hand, summer
cropping strongly triggered the expression of upstream genes in the
phenylpropanoid/flavonoid pathway at E-L 33 and E-L 35. Transcript
levels of flavonoid 3’,5’-hydroxylase (F3’5’H), flavonoid
3’-hydroxylase (F3’H), flavonoid 3-hydroxylase (F3H) and glutathione
S-transferase (GST) were upregulated in winter grapes at the mature
stage. Together, these results might indicate that more flavonoids
would be synthesized in ripe winter grapes during the mature stage of
the second crop under much drier conditions, longer sunlight hours and
lower temperature. These data provide a theoretical foundation for the
secondary metabolism of berries grown under two-crop-a-year cultivation
systems.
Introduction
The regions of southern China are considered to be suboptimal for grape
cultivation because of extremely high temperature and concentrated
rainfall [[42]1]. Fortunately, because of the promotion of rain shelter
cultivation technology and two-crop-a-year cultivation in recent years,
southern China has become a booming grape production region [[43]2].
Statistical data show that the total cultivated area of Guangxi (a
province in southern China) has increased by three times, and the
annual production value has increased from 246 million to 2.6 billion
yuan (RMB) since two-crop-a-year techniques have applied. In most parts
of Guangxi, the active accumulated temperature of the first half and
second half of a year exceeds 3000°C accumulative temperature, which
can meet the needs of outdoor grape growing for two seasons. In
addition, the rainfall and extremely high temperature are lower during
the second half of the year than during the first half of the year.
Moreover, greater temperature differences between day and night during
the second half of the year than during the first half of the year, and
more sunlight hours occur in the former [[44]3]. Therefore, the quality
of winter grapes is much better than that of summer grapes for the same
cultivar, which is mainly manifested in the greater content of
anthocyanins and soluble solids concentration [[45]4,[46]5].
Carotenoids are a family of C40 isoprenoid pigments that have critical
functions in plants, including harvesting light during photosynthesis
and providing cleavage products such as the well-known phytohormone
abscisic acid (ABA) [[47]6]. The carotenoid cleavage dioxygenase (CCD)
enzyme catalyzes the formation of carotene into norisoprenoids. In
grape berries, most norisoprenoids are released to a free state by
glycosidase or acid hydrolysis from flavorless glycosidically bound
forms; these free-state norisoprenoids have extremely low perception
thresholds and pleasant floral characteristics [[48]7]. ABA generally
plays important roles in regulating the onset of veraison and maturity
in grapes [[49]8]. The enzyme 9-cis-epoxycarotenoid dioxygenase (NCED)
is thought to be the key enzyme involved in ABA biosynthesis.
Polyphenolic compounds play an important role in grape quality.
Phenolic compounds containing flavonoids and nonflavonoids,
anthocyanins, flavonols, and flavan-3-ols belong to flavonoids, whereas
nonflavonoids include stilbenes, hydroxybenzoic and hydroxycinnamic
acids. All of these compounds are derived from the
phenylpropanoid/flavonoid pathway. The concentration of phenolic
compounds in grapes depends on the variety and is also influenced by
viticulture techniques and climatic conditions [[50]9–[51]11]. The
influence of two growing seasons on the phenolic compound composition
and concentrations in grapes has been studied, and the results showed
that the concentrations of phenolic compounds in winter grapes were
significantly greater than those in summer grapes [[52]12].
The climatic conditions in southern China constitute the foundation of
two-crop-a-year cultivation development. This technique exploits the
rich heat and sunlight during the second half of the year. Thus, the
effects of climatic conditions on grape quality are emphasized in
two-crop-a-year research. Many studies have shown that both phenolic
compounds and carotenoid metabolism in grape berries are greatly
influenced by climatic conditions [[53]13–[54]14]. High temperature
often increases the degradation of anthocyanins, resulting in poor
coloration [[55]15–[56]16]. Additionally, the carotenoid metabolic flux
in grapes is influenced by distinct climatic conditions among wine
regions [[57]17].
The two-crop-a-year cultivation system has solved the problems of
budbreak in the spring, flower bud differentiation during the second
season and fruit maturation, but poor coloration and insufficient
flavor of summer grapes as well as differences in grape quality between
the two crops have not been explained thoroughly. Previous studies have
shown that flavonoid composition, content and metabolism are often
distinct between the two crops [[58]12,[59]17]. However, a
transcriptomic analysis of grape phenolic compounds and carotenoids in
two crops grown within the same year has not been reported from a
climatic difference perspective.
Thus, the present research was designed to analyze the transcriptome of
phenolic compounds and carotenoids in summer and winter grapes of Vitis
labrusca × Vitis vinifera L. cv. ‘Kyoho’ by RNA-seq. This paper
provides new insights into the understanding of the mechanisms of
secondary metabolism influenced by the growing season.
Materials and methods
Experimental vineyard and two-crop-a-year viticulture practices
This experiment was conducted during two growing seasons in 2016 on
4-year-old self-rooted ‘Kyoho’ grapevines in the vineyards of the Grape
and Wine Research Institute, Guangxi Academy of Agricultural Sciences,
located in Nanning, Guangxi Province (22°, 36’39”N, 108°, 13’51”E). The
vines in this vineyard were managed on a canopy frame with a single
trunk and were planted in north-south-oriented rows spaced 2.0 m
(between vines) × 6.0 m (between rows). Nutrition, pest, water and
fertilizer management was carried out in accordance with uniform
standards for two-crop-a-year as previously described [[60]18].
The key techniques of two-crop-a-year cultivation systems include
dormancy breaking, rain shelter cultivation, soil management,
irrigation, fertilizer, pruning, flower and fruit management, and
disease and pest control. Two-crop-a-year cultivation systems involve
two modes referred to as nonoverlap and overlap cultivation systems
([61]Fig 1).
Fig 1.
[62]Fig 1
[63]Open in a new tab
Nonoverlap (A) and overlap (B) cultivation systems.
In two-crop-a-year grape cultivation systems, the growing season for
the first crop usually occurs from February to June, and that of the
second crop occurs from August to December. The nonoverlap cultivation
system is suitable for regions where the mean annual temperature > 20°C
([64]Fig 1A), and the overlap cultivation system is suitable for
regions where the mean annual temperature < 20°C ([65]Fig 1B). In this
research, the experimental vines were grown in a nonoverlap cultivation
system.
Berry sampling and physical chemical index analysis
Grape berries in three biological replicates were collected at four E-L
stages [[66]19]: berries still hard and green (E-L 33), the onset of
veraison (E-L 35), berries not quite ripe (E-L 37), and the harvest
stage (E-L 38). For each biological replicate, 150 berries were
randomly separated from at least 100 clusters within 9 vines. The
sampling time was fixed at 9:00 to 10:00 a.m., and three biological
replicates were collected via the same method at each sampling date.
After being transported to the laboratory, a subsample of 100 berries
from each biological replicate was subjected to physiological
measurements, including berry fresh weight, total soluble solids (TSS)
content and titratable acidity (TA). The remaining berries were
immediately frozen in liquid nitrogen and transported to the laboratory
on dry ice for transcriptional analysis. The TSS concentrations in the
juices were measured with a digital pocket handheld refractometer
(Digital Hand-held Pocket Refractometer PAL-1, Atago, Tokyo, Japan),
and the TA was determined by titration with NaOH to the end point of pH
8.2 and was expressed as tartaric acid equivalents [[67]16].
Transcriptome sequencing and data analysis
A subsample of 50 berries was randomly selected from each biological
replicate for RNA extraction. Transcriptome sequencing and data
analysis were performed as described previously [[68]20]. Heatmap
visualizations were performed using MetaboAnalyst 3.0. From the
24-sample transcriptome analysis, we identified 175.81 Gb of clean
data, and each sample produced at least 6.02 Gb. The base percentage
was greater than 86.47%. The contrast efficiency of all the sample
clean reads and specified reference genomes was between 58.69% and
67.16%. Based on the experimental results, alternative splicing
determination, genetic structural optimization analysis, and novel gene
discovery were performed. According to the expression levels in the
different samples, different genes were identified, and gene functional
annotation and enrichment analysis were performed. Gene expression
levels were estimated via the fragments per kilobase of transcript per
million fragments mapped (FPKM). The formula is as follows:
[MATH: FPKM=cDNAFragmentsMappedFragments(Millions)×TranscriptLength(kb) :MATH]
Validation of RNA-seq based quantitative real-time PCR
To verify the reliability of the RNA-Seq-based transcript
quantification, quantitative real-time PCR (qRT-PCR) was carried out
using 14 genes related to the carotenoid and phenol pathways. The
remnant RNAs from the RNA-seq experiment were used to synthesize cDNA,
after which qRT-PCR was performed as previously described in
[[69]20–[70]21]. VvUbiquitin1 and VvActin were selected as two
reference genes for qRT-PCR. The sequences of the specific primers used
are provided in the supplementary data ([71]S1 Table). All reactions
were run in triplicate, and the normalized relative expression levels
of the target genes were calculated by 2^−ΔCt, where ΔCt = Ct (target
gene) - Ct (geometric mean of two reference genes) and Ct is the mean
cycle threshold [[72]22]. Pearson correlation coefficients (p-value ≤
0.01) were calculated to assess the correlations between the different
expression patterns obtained by qRT-PCR and RNA-seq. The correlation
coefficients of 0.763 for summer grapes and 0.716 for winter grapes
indicated the reliability of the results of the RNA-Seq-based gene
expression ([73]Fig 2).
Fig 2.
[74]Fig 2
[75]Open in a new tab
Validation of the gene expression results of qRT-PCR and RNA-seq in the
summer crop (A) and winter crop (B). The data were obtained from
fourteen unigenes at four developmental stages. The expression values
of both qRT-PCR and RNA-seq were log2 transformed. Coefficients of
linear regression were also calculated.
Statistical analysis
Significant differences were determined when p < 0.05 according to
independent t-tests. Partial least squares-discriminant analysis
(PLS-DA) was performed with MetaboAnalyst 3.0, and statistical analysis
was performed with SPSS (SPSS Inc., Chicago, IL, USA) for Windows,
version 20.0. Line graphs were constructed with Origin 8.0 software
(OriginLab Corporation, Northampton, MA, United States).
Results
Meteorological characteristics
It is well known that the synthesis and accumulation of grape
carotenoids and phenolic compounds are strongly affected by
environmental conditions such as temperature, sunlight and rainfall
[[76]16, [77]23]. [78]Table 1 displays large differences in climatic
conditions between the two crop growing seasons. An active accumulated
temperature of 2800°C has been suggested as the minimum for grape
production [[79]1]. In the present study, the active accumulated
temperatures for both growing seasons were greater than 3200°C
([80]Table 1), meaning that the temperatures are sufficient to
guarantee normal grape maturity. In addition, the average maximum
temperature from veraison to harvest during the winter season was
approximately 8°C less than that during the summer season. During the
growing seasons, the sunlight duration of summer grape was lower than
that of winter grape. Moreover, the rainfall also differed between the
two growing seasons. Specifically, there was much more rainfall during
the summer growing season than during the winter growing season. For
the first crop, the temperature increased gradually during the
developmental stages ([81]Fig 3). During the second growing season, the
temperature decreased from E-L 33 to E-L 38. According to the berry
developmental phases of the two crops, the daily maximum temperature
exceeded 35°C at E-L 37 and E-L 38 for the summer crop. However, the
result was the opposite for the winter crop, for which the daily
maximum temperature exceeded 35°C mainly in E-L 33 and E-L 35.
Table 1. Climatic factors during the growing seasons of summer and winter
grapes in Nanning in 2016[82]^a.
Year Active-T[83]^b (°C) Sunlight-D[84]^c (h) Rain[85]^d (mm)
Summer Winter Summer Winter Summer Winter
2016 3274.6 3551.1 574.4 779.6 249.2 34.4
Average-30[86]^e 3242.4 3486.1 478.4 785.6 207.1 24.5
[87]Open in a new tab
^a Data from the Nanning Meteorological Administration.
^b Active accumulated temperature during the grape growing seasons,
calculated as T = ∑ti (ti ≥ 10°C); ti is the average daily temperature
from February to June for the summer crop and from August to December
for the winter crop.
^c Sunshine duration during the grape growing seasons calculated by
adding the sunshine hours from February to June for the summer crop and
from August to December for the winter crop.
^d Average monthly rainfall. Values in June for the summer crop and
December for the winter crop.
^e Average of 30 years (from 1971 to 2000).
Fig 3. Meteorological data for both crops in 2016.
[88]Fig 3
[89]Open in a new tab
Tm is the average daily temperature from February to June for the
summer crop and from August to December for the winter crop. Tmax is
the average daily maximum temperature from February to June for the
summer crop and from August to December for the winter crop, and Tmin
is the average daily minimum temperature from February to June for the
summer crop and from August to December for the winter crop.
Grape berry development
Samples that corresponded to the four stages of the E-L system [[90]19]
were measured for berry fresh weight, skin-to-berry ratio, TSS and TA
([91]Fig 4). Summer and winter grapes presented different variation
trends in physicochemical parameters. Compared with the winter grapes,
the summer grapes exhibited significantly greater berry fresh weight at
all developmental stages ([92]Fig 4A). In addition, compared with the
winter grapes, the summer grapes showed a significantly greater
skin-to-berry ratio at E-L 33, 35 and 37 but showed the opposite result
at E-L 38 ([93]Fig 4B). As grapes ripen, their TSS content and TA
gradually increases and decreases, respectively [[94]24]. The TSS
content in the winter grapes was significantly higher than that in the
summer grapes at E-L 38, although no significant difference was
observed at E-L 35, 36 or 37 ([95]Fig 4C). The TA content in the winter
grapes was always higher than that in the summer grapes; these contents
reached approximately 11 g/L and 6 g/L titratable acids at harvest,
respectively ([96]Fig 4D).
Fig 4.
[97]Fig 4
[98]Open in a new tab
Evolution of berry fresh weight (A), the skin-to-berry ratio (B), TSS
(C) and TA (D) in grapes grown under a two-crop-a-year cultivation
system in 2016. The four points refer to stages E-L 33, 35, 37 and 38,
respectively. The asterisk indicates significant differences between
the samples from the same stage.
Global analysis of differential gene expression
Based on the FPKM values of the unigenes in each sample, the
differentially expressed genes (DEGs) between two samples were screened
([99]Fig 5A). Compared to the summer grapes, the winter grapes
presented 6783 upregulated transcripts and 947 downregulated
transcripts in the four stages. Generally, fewer transcripts were
upregulated in grapes at the late stage from the same growing season
than were downregulated. As shown in the Venn diagrams illustrating the
relationships among DEGs at the different developmental stages
([100]Fig 5B), 1191, 42, 155 and 1013 genes were specifically expressed
in E-L 33, 35, 37 and 38, respectively, whereas only five DEGs were
shared by all four stages. The DEGs from the eight samples were
subsequently processed by hierarchical clustering. The results showed
that samples at the same developmental stage were clustered together,
indicating their similarities in gene expression ([101]Fig 5C).
Fig 5. Differential gene expression in grapes under a two-crop-a-year
cultivation system.
[102]Fig 5
[103]Open in a new tab
(A) Numbers of DEGs in pairwise comparisons of eight samples. (B) Venn
diagram showing DEG distributions. (C) Expression profile clustering.
PLS-DA
PLS-DA was performed on the whole normalized gene expression data of
the carotenoid and phenolic pathway ([104]Fig 6). The data were
normalized by the autoscaling method in MetaboAnalyst 3.0. As shown in
[105]Fig 6A, grape samples from the two seasons were completely
separated from each other. Variables whose variable importance in
projection (VIP) values > 1.0 were considered important contributors
([106]Fig 6B). Compared with the summer grapes, the winter grapes
displayed a distinct and significant upregulation of the unigenes (CCD,
VIT_02s0087g00910; VIT_02s0087g00930), beta-carotene 3-hydroxylase
(BCH, VIT_02s0025g00240), glutathione S-transferase (GST,
VIT_15s0024g01540; VIT_12s0028g00920; VIT_12s0028g00930), flavonoid
3’5’-hydroxylase (F3’5’H, VIT_06s0009g03050; VIT_06s0009g02810),
flavonol synthase (FLS, VIT_18s0001g03430), flavonoid 3-hydroxylase
(F3H, VIT_18s0001g14310) and lycopene-beta-cyclase (LBCY,
VIT_18s0001g14310). In contrast, the summer grapes presented
upregulated levels of stilbene synthase (STS, VIT_16s0100g01100;
VIT_16s0100g01200; VIT_16s0100g01150; VIT_16s0100g00750), caffeic acid
3-O-methyltransferase (COMT, VIT_03s0063g00140), phenylalanine
ammonialyase (PAL, VIT_08s0040g01710), NCED (VIT_19s0093g00550) and
dihydroflavonol reductase (DFR, VIT_16s0039g02350). The GO annotations
of the transcripts were supplemented in the supplementary data ([107]S2
Table).
Fig 6. Results of PLS-DA.
[108]Fig 6
[109]Open in a new tab
(A) Score plot. (B) Selected genes based on VIP scores. The four points
refer to stages E-L 33, 35, 37 and 38, respectively.
Transcriptomic changes in the carotenoid biosynthesis pathway under a
two-crop-a-year cultivation system
To explore the differences in gene expression patterns in the two
seasons, a Kyoto Encyclopedia of Genes and Genomes (KEGG,
[110]http://www.genome.jp/kegg/) pathway enrichment analysis revealed
that 18 unigenes encoding 12 enzymes were involved in carotenoid and
ABA metabolism ([111]Fig 7).
Fig 7. Transcriptomic profile of the structural genes involved in the
carotenoid and ABA biosynthetic pathways in summer and winter grape berries.
[112]Fig 7
[113]Open in a new tab
PSY, phytoene synthase; Z-ISO, zeta-carotene isomerase; PISO,
prolycopene isomerase; LBCY, lycopene-beta-cyclase; LECY, lycopene
epsilon-cyclase; CCD, carotenoid cleavage dioxygenase; ZEP, zeaxanthin
epoxidase; NSY, neoxanthin synthase; NCED, 9-cis-epoxycarotenoid
dioxygenase; BCH, beta-carotene 3-hydroxylase; XO, xanthosin
dehydrogenase; ABA8ox, ABA 8’-hydroxylase; ABAO, abscisic-aldehyde
oxidase; ABA-GE, ABA glucosyl-ester; ABA-GT, ABA glucosyltransferase;
PA, phaseic acid. Each square in the heatmap located beside its gene
names corresponds to the average FPKM value of the gene in each sample,
as illustrated in the legend. SK, summer ‘Kyoho’; WK, winter ‘Kyoho’.
In this study, the expression of most of the unigenes participating in
carotenoid biosynthesis peaked at E-L 31 or E-L 35, which are proposed
to encompass the period of large-scale carotenoid production. However,
the unigene encoding zeta-carotene isomerase (Z-ISO, EC:5.2.1.12)
presented its maximum FPKM level at E-L 38. PSY (EC:2.5.1.32), Z-ISO,
prolycopene isomerase (PISO, EC:5.2.1.13), LBCY (EC:5.5.1.19) and
lycopene epsilon-cyclase (LECY, EC:5.5.1.18) were found to be involved
in the upstream flux of carotenoid synthesis, and compared with that in
the summer grapes, the expression of all the unigenes in the winter
grapes was upregulated at the E-L 35, 37 and 38 stages. These results
might suggest that the carotenoids contents in the winter grapes higher
than summer grapes.
The downstream flux of carotenoid metabolism in grape berries includes
the synthesis of norisoprenoids, a group of potent flavor and aromatic
compounds. In plants, CCD (EC:1.13.11.51) catalyzes the breakdown of
carotenoids into volatile norisoprenoids [[114]7, [115]25]. Two CCD
unigenes were identified and exhibited similar expression patterns
between the two crop growing seasons, and compared with that in the
summer grapes, the expression of all CCD members in the winter grapes
was upregulated throughout the whole period.
In our study, one unigene annotated as ZEP and three annotated as NCED
were identified. The ZEP unigenes showed similar expression profiles
between the two crop growing seasons; compared with that at other
stages, the expression of these unigenes at E-L 33 was upregulated.
Young et al. [[116]25] also reported a similar decreased expression
pattern for two ZEPs from the green to harvest stages. In the present
study, the three NCED transcripts exhibited different expression
profiles. The NCED6 expression level increased gradually in both the
summer and winter grapes throughout their development, and compared
with that in the summer grapes, NCED6 expression in winter grapes was
upregulated at E-L 38. Expression of the NCED1 and NCED2 transcripts
peaked at E-L 33, and their expression levels in the summer grapes were
higher than those in the winter grapes at E-L 33.
Compared with those in summer grapes, the expression levels of
xanthosin dehydrogenase (XO, EC:1.1.1.288) and ABA glucosyltransferase
(ABA-GT, EC:2.4.1.263) in winter grapes were upregulated throughout the
whole period. Most transcripts of ABA 8’-hydroxylase (ABA8ox,
EC:1.14.13.93) and BCH (EC:1.14.13.129) were expressed the most at E-L
33 for summer.
Transcriptomic changes in the phenolic biosynthetic pathway under a
two-crop-a-year cultivation system
To investigate the differences in phenylpropanoid/flavonoid
biosynthesis pathway-related structural genes between the summer and
winter grapes at four distinct developmental stages (E-L 33, 35, 37,
and 38) during the 2016 growing season, we used RNA-seq to characterize
the changes in gene expression at the transcript level ([117]Fig 8).
The results showed that the expression of the general structural genes
of the phenylpropanoid metabolic pathways, which included PAL
(EC:4.3.1.24), trans-cinnamate 4-monooxygenase (C4H, EC:1.14.13.11),
chalcone synthase (CHS, EC:2.3.1.74), F3H (EC:1.14.11.9), flavonoid
3’-hydroxylase (F3’H, EC: 2.3.1.133), DFR (EC:1.1.1.219),
leucoanthocyanidin reductase 1 (LAR1, EC:1.17.1.3) and anthocyanidin
reductase (ANR, EC:1.3.1.77), was significantly or moderately
upregulated in the summer grapes at the E-L 33 stage.
Fig 8. Transcriptomic profile of the structural genes involved in phenolic
biosynthesis in summer and winter grape berries.
[118]Fig 8
[119]Open in a new tab
PAL, phenylalanine ammonia-lyase; C4H, trans-cinnamate 4-monooxygenase;
COMT, caffeic acid 3-O-methyltransferase; F5H, ferulate-5-hydroxylase;
4CL, 4-coumarate: CoA ligase; STS, stilbene synthase; CHS, chalcone
synthase; CHI, chalcone isomerase; F3H, flavonoid 3-hydroxylase; F3’H,
flavonoid 3’-hydroxylase; F3’5’H, flavonoid 3’,5’-hydroxylase; FLS,
flavonol synthase; DFR, dihydroflavonol reductase; LAR,
leucoanthocyanidin reductase; LDOX, leucoanthocyanidin dioxygenase;
ANR, anthocyanidin reductase; UFGT, UDP-glucose: flavonoid
3-O-glucosyltransferase; GST, glutathione S-transferase. Each square in
the heatmap located beside its gene names corresponds to the average
FPKM value of the gene in each sample, as shown in the legend. SK,
summer ‘Kyoho’; WK, winter ‘Kyoho’.
STS is a pivotal enzyme that catalyzes the biosynthesis of resveratrol
and is known to be induced by ultraviolet (UV) irradiation and Botrytis
cinerea infection [[120]26]. Compared with that in the winter grapes,
the expression of almost all STS (EC 2.3.1.74) members in the summer
grapes was significantly upregulated at the E-L 35 stage (the beginning
of veraison).
The hydroxylation pattern of flavonoids is known to be mediated by the
enzymatic activity of F3’H (EC 1.14.13.21), F3H (EC:1.14.11.9) and
flavonoid 3’,5’-hydroxylase (F3’5’H, EC:1.14.13.88), which catalyze the
hydroxylation of naringenin and dihydrokaempferol at the 3’, 3’ and
3’,5’ positions of the B-ring, respectively [[121]27–[122]28]. Our
results showed that, compared with that in the summer grapes, the
transcriptional abundance of several F3’5’H, F3’H and F3H family
members in the winter grapes was significantly upregulated at the E-L
37 and E-L 38 stages. This result suggested that many more flavonoids
containing flavonols, flavan-3-ols and anthocyanins would be
synthesized in winter grapes than in summer grapes at the mature stage.
Compared with those during the summer ripening of berries, the higher
sunshine duration and lower rainfall during the winter ripening of
berries were presumed to result in greater expression of some members
of the F3’5’H, F3’H and F3H gene families in berries at E-L 37 and 38
stages and subsequently might contribute to the greater concentration
of flavonoids.
Furthermore, FLS is responsible for catalyzing the formation of
dihydroflavonols into flavonols. Two FLS unigenes exhibited different
variation trends between developmental stages of the two crops, and
higher expression levels were found in winter grapes than in summer
grapes at each stage. LAR and ANR are key regulators of flavan-3-ol and
proanthocyanidin biosynthesis [[123]20], and the expression levels of
both were highest at E-L 33 for both crops, after which the levels
decreased sharply from veraison (E-L 35) to harvest (E-L 38). In
addition, the expression of both LAR and ANR in the winter grapes was
upregulated at E-L 33.
UDP-glucose: flavonoid 3-O-glucosyltransferase (UFGT) can catalyze the
formation of anthocyanin-3-O-glucosides, and 5GT is critical for the
synthesis of anthocyanin-3,5-O-diglucosides [[124]29]. In our study,
the expression of UFGT was higher at the later stages of grape
maturity. Members of the GST family are believed to participate in
vacuolar trafficking and the sequestration of anthocyanins from the
endoplasmic reticulum to the vacuole [[125]30]. In the present
research, the expression of GST was significantly upregulated in winter
grapes at the E-L 33, 35 and 38 stages.
Discussion
The metabolism of carotenoids and phenols in grapes is influenced by
variety, environmental factors, developmental stage, and plant hormone
regulation. In recent years, transcriptomic studies involving secondary
metabolites that respond to internal and external factors have been
prevalent [[126]20,[127]31–[128]34]. A two-crop-a-year cultivation
system based on innovations in terms of pruning and pregermination was
developed in southern China. Some works discussed the two-crop-a-year
cultivation system in wine grape cultivars, such as 'NW196' (V.
quinquangularis Rehd. × V. vinifera L.), ‘Cabernet Sauvignon’ (V.
vinifera L.), ‘Riesling’ (V. vinifera L.) [[129]12, [130]35]. ‘Kyoho’
was the first variety subjected to two-crop-a-year techniques in
southern China. Here, the transcriptional expression patterns of
phenols and carotenoids in ‘Kyoho’ grapes under a two-crop-a-year
cultivation system were analyzed and compared for the first time. To
our knowledge, this is the first study of the transcriptomic sequencing
of two-crop-a-year ‘Kyoho’ grapes, providing a theoretical basis for
future research on the quality of grapes grown during different growing
seasons.
The climatic conditions of summer and winter grapes differed greatly.
During the first growing season, the temperature gradually increased
from germination until harvest. However, the second crop showed the
opposite results. During berry development, the number of sunshine
hours was clearly greater for the winter grapes than for the summer
grapes. Additionally, the rainfall during the summer growing season was
much greater than that during the winter growing season. Therefore, the
differences in climatic factors strongly affect the variation in
quality-related indicators between the two crops. During the maturation
of grapes, increased sugar contents are favored by sufficient sunlight
[[131]36]. On the other hand, excessive humidity is unfavorable to
sugar accumulation [[132]37]. Thus, compared with the summer grapes,
the winter grapes had a higher TSS content in the present study because
of lower amounts of rainfall and richer sunlight conditions.
Carotenoids are precursors of C[13]-norisoprenoids and ABA, and when
they occur mainly as glycoconjugated forms, C[13]-norisoprenoids
contribute to the characteristic aromas of many varieties
[[133]38–[134]39]. The carotenoids were mostly synthesized from E-L 33
until E-L 35, after which they degraded. The genes encoding nearly all
the enzymes of carotenoid biosynthesis in plants have been identified
[[135]17]. Phytoene synthase (PSY), CCD, and NCED are widely believed
to be the most important regulatory nodes in the separate biosyntheses
of carotenoids, norisoprenoids, and ABA; these nodes are tightly
controlled by environmental factors and development stage [[136]23,
[137]40]; compared with those in the summer grapes, these unigenes in
the winter grapes were upregulated at grape-ripening stage. The
expression of PSY is induced by light, water deficit, and hormones
[[138]40]. In the present study, compared with that in the summer
grapes, the expression of PSY in the winter grapes was upregulated at
the E-L 35, 37 and 38 stages as a result of drier climatic conditions
and more sunlight during the winter season. In the present study, the
transcriptional analysis of CCD genes in the two-crop-a-year grape
berries revealed that, compared with that in the summer grapes, the
expression of two unigenes in the winter grapes was upregulated. This
observation was in agreement with the upregulated expression of CCD
under much drier climatic conditions [[139]17]. ABA is a plant hormone
involved in environmental stress responses, and the production of ABA
represents another flux of carotenoids within plants. The epoxidation
of zeaxanthin to violaxanthin catalyzed by zeaxanthin epoxidase (ZEP)
and the oxidative cleavage by NCED are two crucial steps in ABA
biosynthesis, whereas ABA8ox and ABA-GT are the major enzymes for ABA
glucosyl-ester (ABA-GE) and phaseic acid (PA) [[140]17]. The expression
patterns of NCED were similar between the summer and winter grapes, but
the three transcripts showed different expression profiles during the
different developmental stages. ABA8ox was predominantly expressed in
the summer grapes at E-L 33, and ABA-GT expression peaked in the winter
grapes at the same stage, indicating that ABA mainly broke down to PA
in the summer grapes and was stored as ABA-GE in the winter grapes.
Longer sunshine hours and stronger light intensity result in relatively
greater amounts of carotenoids synthesized at the green stage and lower
amounts synthesized at the harvest stage [[141]17]. Furthermore, many
studies have shown that water deficit can increase carotenoid and
norisoprenoid contents in grape berries [[142]41]. In the present
study, compared with those during first growing season, the green berry
and veraison stages during the second growing season received lower
amounts of rainfall but had a longer sunlight duration. These phenomena
might explain the more active carotenoid and norisoprenoid synthesis
pathway in the winter grapes during E-L 33 and E-L 35. Notably, these
environmental factors are hard to separate. Thus, carotenoid metabolism
is influenced by many integrated factors rather than by a single factor
[[143]17].
Phenolic compounds, mainly phenolic acids, stilbenes and flavonoids,
are among the most abundant secondary metabolites in grapes. Phenolic
acids accumulate in grape berry skin and flesh, while stilbenes have
been detected in the skin, fruit flesh and seeds [[144]20, [145]42].
Phenolic compounds are synthesized via the phenylpropanoid/flavonoid
pathway. STS, or resveratrol synthase (EC 2.3.1.95), catalyzes the
formation of resveratrol from p-coumaroyl-CoA; resveratrol is related
to biotic and abiotic stresses in plants [[146]43]. In the present
study, compared with that in winter grapes, the expression of six STS
unigenes in summer grapes was upregulated. Whether this difference was
caused by sharp temperature increases at the veraison stage for summer
grapes, needs to be further researched.
Flavan-3-ols, which share common upstream steps with both flavonols and
anthocyanins, accumulated from E-L 27 (fruit set) until E-L 35
(veraison), after which they decreased [[147]22]. In the present study,
the expression of both LAR and DFR was highest at E-L 33 for both
crops, and both genes were upregulated in the summer grapes. However,
anthocyanins in winter ‘Kyoho’ grapes did not accumulate at the expense
of flavan-3-ols in previous study [[148]12], even though both compounds
share common precursors. Thus, this finding may be associated with
higher gene expression levels upstream of the phenolic biosynthesis
pathway in winter grapes than in summer grapes.
F3’5’H, F3’H and F3H catalyze naringenin flavanones to form flavonoids
containing flavonols, flavan-3-ols and anthocyanins [[149]20]. The
expression levels of F3’5’H, F3’H and F3H were upregulated in winter
grapes at the E-L 37 and E-L 38 stages. These results also indicate
that many more flavonoids accumulate in winter grapes than in summer
grapes.
As a hybrid of V. labrusca and V. vinifera, ‘Kyoho’ grapes contain
3-O-glucosides and 3,5-O-diglucosides in their skin [[150]11]. Although
the transcripts of UFGT showed higher expression levels at some stages
in the summer grapes than in the winter grapes, transcripts of GST were
more abundant in the winter grapes than in the summer grapes at the E-L
33, 35 and 38 stages. Previous research has shown that, compared with
summer grapes, winter grapes contain greater amounts of anthocyanins
[[151]12]. High temperature (35°C days) from veraison to harvest
reduced the total anthocyanin content to less than half
[[152]44–[153]45]. This conclusion can be explained by the higher
number of extreme-temperature (> 35°C) days during the summer
grape-ripening stage.
Conclusions
The two-crop-a-year cultivation system is based on unique weather
conditions in southern China. This work investigated the transcriptomic
dynamics of ‘Kyoho’, which was the first variety cultivated via this
technique. These results mainly revealed differences in the
transcriptional expression patterns of phenols and carotenoids in
‘Kyoho’ grapes during the four stages of grape berry development in a
two-crop-a-year cultivation system. The results showed that the second
crop produced winter grapes that were smaller than the summer grapes
and had greater contents of TSS and TA. Compared with the summer
grapes, the winter grapes overall showed higher expression levels of
genes required for the synthesis of carotenoids, which could be
attributed to lower rainfall and much more abundant sunlight during the
second growing season. In addition, the expression of some genes
involved in the upstream portion of the phenylpropanoid/flavonoid
pathway was upregulated in the summer grapes at E-L 33 and E-L 35.
Although the expression of some genes directly related to the synthesis
of flavan-3-ols and anthocyanidins was upregulated in the summer grapes
at E-L 37 and E-L 38, the expression of three hydroxylase genes and GST
was higher in the winter grapes than in the summer grapes. All of these
factors could result in the production of less flavonoids and could be
explained by extremely hot weather during the mature stage of the first
crop. We anticipate that the present study will enrich theoretical
research on two-crop-a-year cultivation systems and will facilitate
additional investigations into the differences in grape berry quality
between two crops grown during the same year.
Supporting information
S1 Table. List of the primers used for quantitative real-time RT-PCR
validation experiments.
(PDF)
[154]Click here for additional data file.^ (188.7KB, pdf)
S2 Table. The GO annotations of the transcripts were supplemented for
[155]Fig 6B.
(PDF)
[156]Click here for additional data file.^ (91KB, pdf)
Data Availability
The RNA-seq rawdata has been uploaded at NCBI SRA, SRA: SRP161853,
BioProject: PRJNA491334.
Funding Statement
This work was supported by the Basal Research Fund of Guangxi Academy
of Agricultural Sciences (Guinongke Grant Nos. 2015YT82 and 2015YT85),
and the Natural Science Foundation of Guangxi (Grant No.
2017GXNSFBA198156). Guinongke Grant Nos. 2015YT82 and 2015YT85 have
roles in data collection and analysis; Grant No. 2017GXNSFBA198156 has
a role in the study design and preparation of the manuscript.
References