Download Developmental, transcriptome, and genetic alterations associated

Journal of Experimental Botany, Vol. 67, No. 1 pp. 259–273, 2016
doi:10.1093/jxb/erv452 Advance Access publication 9 October 2015
RESEARCH PAPER
Developmental, transcriptome, and genetic alterations
associated with parthenocarpy in the grapevine seedless
somatic variant Corinto bianco
Carolina Royo1,*, Pablo Carbonell-Bejerano1,*,† Rafael Torres-Pérez1, Anna Nebish2, Óscar Martínez3,
Manuel Rey3, Rouben Aroutiounian2, Javier Ibáñez1 and José M. Martínez-Zapater1
1 Instituto de Ciencias de la Vid y del Vino (Consejo Superior de Investigaciones Científicas-Universidad de La Rioja-Gobierno de La
Rioja), Finca La Grajera, Carretera LO-20 - salida 13, Autovía del Camino de Santiago, 26007, Spain
2 Department of Genetics and Cytology, Yerevan State University, 1 Alex Manoogian str., 0025 Yerevan, Armenia
3 Departamento de Biología Vegetal y Ciencia del Suelo. Facultad de Biología. Universidad de Vigo, 36310 Vigo, Spain
* These authors contributed equally to this work.
To whom correspondence should be addressed. E-mail: [email protected]
†
Received 7 July 2015; Revised 15 September 2015; Accepted 25 September 2015
Editor: Ariel Vicente
Abstract
Seedlessness is a relevant trait in grapevine cultivars intended for fresh consumption or raisin production.
Previous DNA marker analysis indicated that Corinto bianco (CB) is a parthenocarpic somatic variant of the
seeded cultivar Pedro Ximenes (PX). This study compared both variant lines to determine the basis of this parthenocarpic phenotype. At maturity, CB seedless berries were 6-fold smaller than PX berries. The macrogametophyte was absent from CB ovules, and CB was also pollen sterile. Occasionally, one seed developed in 1.6%
of CB berries. Microsatellite genotyping and flow cytometry analyses of seedlings generated from these seeds
showed that most CB viable seeds were formed by fertilization of unreduced gametes generated by meiotic
diplospory, a process that has not been described previously in grapevine. Microarray and RNA-sequencing
analyses identified 1958 genes that were differentially expressed between CB and PX developing flowers.
Genes downregulated in CB were enriched in gametophyte-preferentially expressed transcripts, indicating the
absence of regular post-meiotic germline development in CB. RNA-sequencing was also used for genetic variant calling and 14 single-nucleotide polymorphisms distinguishing the CB and PX variant lines were detected.
Among these, CB-specific polymorphisms were considered as candidate parthenocarpy-responsible mutations, including a putative deleterious substitution in a HAL2-like protein. Collectively, these results revealed
that the absence of a mature macrogametophyte, probably due to meiosis arrest, coupled with a process of
fertilization-independent fruit growth, caused parthenocarpy in CB. This study provides a number of grapevine parthenocarpy-responsible candidate genes and shows how genomic approaches can shed light on the
genetic origin of woody crop somatic variants.
Key words: Diplospory, embryo sac, gametogenesis, grapevine, meiosis, microarray, parthenocarpy, pollen sterility, polyploidy,
RNA-seq, seedlessness, SNP, somatic variation, transcriptomics, unreduced gamete, Vitis vinifera.
Abbreviations: C, cytotype; CB, Corinto bianco; CB-SDD, Corinto bianco seeded; CB-SDL, Corinto bianco seedless; dba, days before anthesis; DEG, differentially expressed gene; ENC, Encín; FPKM, fragments per kb of exon per million fragments mapped; GRAJ, Grajera; PCA, principal component analysis; PX, Pedro
Ximenes; RMA, robust multiarray average; RNA-seq, RNA sequencing; SNP, single-nucleotide polymorphism; TMM, trimmed mean of M-values; VALD, Valdegón.
© The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved.
For permissions, please email: [email protected]
260 | Royo et al.
Introduction
Fruits appeared in angiosperms as innovative structures
directed to protect and disseminate their sexual propagules,
the seeds. Accordingly, fruit set is induced soon after fertilization, when developing seeds release growth substances
to sporophytic tissues of the mother plant, generally carpellary tissues, triggering their development into a fruit (Dorcey
et al., 2009; Sotelo-Silveira et al., 2014). Exceptionally, in
parthenocarpic mutants, fruit development proceeds without
fertilization, which results in seedless fruits. In contrast to
facultative parthenocarpy, requiring an external intervention
to avoid pollination/fertilization, obligatory parthenocarpic
mutants are male and/or female sterile (Varoquaux et al.,
2000). Although these mutants are not usually spread in
nature due to their inability for sexual reproduction, seedlessness has been selected in many vegetatively propagated fruit
crops as a highly desirable agronomic trait that allows easier
fruit preparation and consumption by humans.
Seedless variants are preferred as grapevine (Vitis vinifera L.) cultivars used for table grapes and raisin production. Given the high heterozygosity of the grapevine genome
(Laucou et al., 2011), varietal characteristics are maintained
through asexual propagation and thus a grapevine cultivar
consists of an array of lines or clones descended by vegetative propagation from the original seedling (Pelsy, 2010).
Asexual propagation of a cultivar over long periods favours
the accumulation of somatic mutations, which sometimes
give rise to plants showing new phenotypes (somatic variants) (Torregrosa et al., 2011). In this way, asexual propagation has enabled the appearance and maintenance of seedless
grape variants. Two main mechanisms resulting in seedlessness have been identified among grapevine germplasm: (i)
stenospermocarpy, in which there is fertilization and embryo
formation but seed development aborts giving rise to seed
traces; (ii) parthenocarpy, in which small berries without seed
traces develop in the absence of fertilization (Stout, 1936;
Varoquaux et al., 2000). The first variation is widely used in
the production of seedless table grape cultivars because berry
size is less compromised, while the second one is exploited in
cultivars used for raisin production.
Regardless of their genetic or geographical origin, parthenocarpic grapes used for dry fruit production are classically
denominated Corinth (=Corinto=currant) grapes. Historical
reports indicate that some current Corinth grape cultivars were
cultivated by ancient Greeks (Weaver, 1960). Recent molecular analyses have elucidated that different Corinth cultivars
including Black Corinth, Cape Currant, Corinto bianco, and
Corinthe Blanc are not genetically related (Adam-Blondon
et al., 2001; Vargas et al., 2007). Thus, independent somatic
mutations affecting sexual reproduction are expected in different Corinth cultivars.
In plants, successive mitosis of haploid cells produced
by meiosis results in multicellular gametophytes, which is
the basis for the double fertilization process (Chasan and
Walbot, 1993). Female (macrogametophyte or embryo sac)
and male (microgametophyte or pollen grain) gametophytes
are produced in specialized sexual organs (ovule and anther,
respectively). In grapevine, the female reproductive unit is
a bicarpellary pistil with two anatropous ovules per locule,
containing a Polygonum type embryo sac each (Pratt, 1971).
The grapevine mature pollen is tricolpate and bicellular, consisting of a large vegetative cell and a small generative cell,
which divides into two haploid sperm cells only after pollen
germination (Cresti and Ciampolini, 1999). At fertilization,
egg (haploid) and central (diploid) embryo sac cells each fuse
with sperm cells giving rise to embryo and endosperm development, respectively, whereas seed coats develop from ovule
sporophytic tissues. Failure of fertilization due to defects in
any of these processes coupled with a capability for fertilization-independent fruit development is expected to occur in
parthenocarpic grapes such as Corinth cultivars (Varoquaux
et al., 2000). In fact, some extent of pollen sterility and occasional development of seeds, usually lacking embryos, was
documented previously in certain Corinth cultivars (Pratt,
1971). Mutations responsible for parthenocarpy have been
identified in fruit crops such as apple or tomato (Yao et al.,
2001; Carrera et al., 2012). However, almost no information is
available on the genetic basis of parthenocarpy in grapevine.
A recent DNA microsatellite analysis identified Corinto
bianco (CB) as a parthenocarpic seedless grape somatic variant of the white wine Spanish cultivar Pedro Ximenes (PX)
(Vargas et al., 2007). Although the existence of macrogametogenesis alterations in CB was proposed previously (Pratt,
1971), the actual genetic and reproductive defects responsible
for parthenocarpy of CB remain unknown. To investigate the
developmental and genetic causes of seedlessness in CB, this
study compared flower and fruit development between CB
and its original seeded cultivar PX. Transcriptomic comparisons were directed to characterize the variant syndrome and
to screen for mutations in candidate genes, involving either
misregulation of gene expression or nucleotide sequence
polymorphisms. Additionally, a genetic study of offspring
obtained from infrequent CB seeds was useful to clarify their
origin. Although the male and female sterility found in CB
restricts the opportunities for genetic analysis, the overall
results permitted us to postulate likely cytological and molecular explanations for the parthenocarpy of CB.
Materials and methods
Plant material
All CB and PX plants used were grafted on Richter-110 rootstocks,
trellized in double cordon Royat system, and cultivated in a similar
way in three different grapevine plots (5–10 plants per cultivar and
plot). Two plots belonged to the Grapevine Germplasm Collection
of the Instituto de Ciencias de la Vid y del Vino (ICVV) (ESP217) and are maintained under the same agronomical conditions
in two separate experimental locations: ‘Finca Valdegón’ (VALD)
in Agoncillo (La Rioja, Spain) and ‘Finca La Grajera’ (GRAJ) in
Logroño (La Rioja, Spain). Plants at GRAJ were grafted in 2010
from scions taken from VALD (20–30 years old). The third plot is
the ‘Vitis Germplasm Bank’ (ESP-080) of the Instituto Madrileño
de Investigación y Desarrollo Rural, Agrario y Alimentario
(IMIDRA) located in ‘Finca El Encín’ (ENC) in Alcalá de Henares
(Madrid, Spain) where PX and CB were planted in 2003 and 2004,
respectively.
Alterations associated with parthenocarpy in grapevine | 261
Morphometric analyses
Fruit and seed features were characterized in both cultivars. Berry
length and maximum diameter were estimated using an Absolute
Digital Caliper (Mitutoyo, Japan). Berry and seed fresh weight were
measured with a precision balance. The number of seeds per berry
and the percentage of floating seeds were calculated in both cultivars, and the percentage of seeded berries only in CB (CB-SDD).
Phenotyping analyses were carried out in three different seasons,
2011 at VALD, 2012 at VALD and ENC, and 2013 at GRAJ.
1962) supplemented with 20 g l–1 of sucrose, 1 µM gibberellic acid,
10 µM indoleacetic acid, 2.5 g l–1 of active charcoal and 8 g l–1 of
agar. Plates were maintained at 23 °C under continuous illumination provided by cool white fluorescent tubes in a growth phytotron
(Grow-RH, Ing. Climas, Barcelona, Spain). When true leaves developed, the seedlings were transplanted to a tube with growth medium
without charcoal or hormones. After 1–2 months, the plants were
transplanted to soil in pots, acclimated, and grown under field conditions at VALD.
Macrogametogenesis analysis
Ovule and macrogametophyte cytohistological observations were
carried out on PX and CB flower buds (closed flowers) collected at
VALD on 24/05/2011, at approximately 1 d before anthesis (dba),
when few flowers of the same inflorescence were starting to open.
Closed flowers were fixed in 37% formaldehyde, glacial acetic acid,
95% ethanol and water (10:5:50:35) at 4 °C. The material was resin
embedded and sectioned in serial longitudinal and cross-sections,
as described previously (Chevalier et al., 2014). Digital images were
acquired with a Photometrics CoolSNAPcf camera connected to an
Axioskop 2 plus microscopy (Zeiss, Germany). For each cultivar, the
total number of ovules and number of ovules with a mature embryo
sac per pistil were counted in sections of 70 and 30 flowers, respectively, collected weekly in 2013 at GRAJ between inflorescence swelling and anthesis stages, fixed with formalin/acetic acid/alcohol, and
treated by common cytohistological techniques for sectioning after
paraffin embedding (Ruzin, 1999).
Microsatellite genotyping
Young leaves were used for DNA extraction using a DNeasy Plant
Mini kit (Qiagen, Hilden, Germany). They were genotyped for nine
unlinked microsatellite loci, as described previously (Ibáñez et al.,
2009).
Microgametogenesis analysis
Pollen viability was analysed in PX and CB over 3 consecutive years
using Alexander’s modified staining (Peterson et al., 2010). Pollen
was collected from inflorescences at 50% bloom and more than 1000
pollen grains per three biological replicates were analysed each year.
Pollen germination was evaluated in vitro in 2014. Fresh pollen
was collected from slightly opened flowers at anthesis and was sown
immediately. Pollen grains were spread in germination medium (10%
sucrose and 100 ppm boric acid dissolved in deionized water and
solidified with 2% agar) on 40 × 12 mm uncovered Petri dishes, which
were placed floating in humid chambers and incubated for 16 h at
25 °C. Pollen grains were considered germinated when the length
of the pollen tube was more than twice the diameter of the pollen
grain (>500 grains per three biological replicates per genotype were
analysed).
Images of pollen viability and germination assays were taken using
an AxioCam camera (Zeiss) connected to a SteREO Discovery.V20
stereo-microscope (Zeiss) and were analysed using ImageJ software
(Schneider et al., 2012).
Statistical analysis of phenotypic data
One-way ANOVA was conducted to test for significant differences
between PX, CB-SDD and CB seedless (CB-SDL) berry measures.
Games–Howell was used as a post hoc test because variances were
unequal according to Levene’s tests. A t-test was performed for twoclass comparisons. A significance level of P<0.01 was set in all cases.
Every statistical test was performed using SPSS software (v.19.0 for
Windows; IBM Corp., Somers, NY, USA).
In vitro embryo rescue
Every CB-SDD berry identified from VALD in 2011 was collected,
as well as from all three plots in 2012 and 2013. Berries were sterilized before seed extraction by consecutively washing with Tween
20 diluted in water, 70% ethanol, and 1.5% sodium hypochlorite
for 13 min and rinsed three times with sterilized distilled water.
Seeds were cut at the distal extreme and sown on sterile Petri plates
containing half-strength MS medium (Murashige and Skoog,
Ploidy analysis
Fresh young leaves were treated as described previously (Acanda
et al., 2013), with minor modifications. Briefly, 50 mg of leaves was
chopped with a sharp razor blade in 0.5 ml of 0.5× WPB buffer
(Loureiro et al., 2007) and 1 ml of the same buffer was added. The
homogenate was filtered through a 50 μM nylon mesh and incubated
with 50 μg ml–1 of RNase (Sigma-Aldrich) and 100 μg ml–1 of propidium iodide for 10 min. The resulting samples were analysed using
an FC500 cytometer (Beckman Coulter, Miami, USA) in 2013 and
a BD Accuri C6 cytometer (BD Biosciences, San Jose, USA) in 2014
to estimate the cytotype (C).
Transcriptomic analysis
RNA extraction Total RNA was extracted from frozen flower buds
using a Spectrum™ Plant Total RNA kit (Sigma-Aldrich). An
in-column DNase digestion step with the RNase-Free DNase Set
(Qiagen) was added.
Microarrays Three biological replicates of CB and PX flowers at
1 dba were hybridized to NimbleGen microarrays. Inflorescences
collected from different VALD plants on 24/05/2011 were used for
each replicate. Synthesis, labelling, and hybridization of cDNA
to NimbleGen microarray 090818 Vitis exp HX12 (NimbleGenRoche) and robust multiarray average (RMA) normalization were
performed as indicated elsewhere (Carbonell-Bejerano et al., 2014).
Linear models for microarray data (limma) were run in Babelomics
(Medina et al., 2010) to search for differentially expressed genes
(DEGs) between CB and PX. DEGs were identified considering a
Benjamini–Hochberg corrected P≤0.05 and ≥2-fold change as significance thresholds.
RNA-sequencing (RNA-seq) analysis Six libraries were constructed from three replicates of CB and PX flowers at 10 dba.
Inflorescences collected from different VALD plants on 23/05/2012
were used for each replicate. Libraries were prepared using the
Illumina TruSeq Stranded mRNA Sample Prep kit starting from
1 µg of total RNA. The adapter-ligated library was amplified
with 15 PCR cycles, followed by purification with Ampure beads.
Finally, the libraries were run on a Bioanalyser DNA1000 chip
and quantified using a real-time PCR kit (Kapa Biosciences).
Sequencing was performed on an Illumina HiSeq 2000 using v.3
chemistry (flowcells and sequencing reagents). Paired-end strandspecific reads of 101 nt were produced. Gapped alignment of
reads to the PN40024 12× grapevine reference genome assembly (http://www.genoscope.cns.fr/externe/GenomeBrowser/Vitis/)
was carried out using TopHat2 v.2.0.13 (Kim et al., 2013). Only
uniquely mapped single copy, ≤1 polymorphism per 25 bp reads
with quality ≥20, paired in the expected orientation and aligned
with a maximum insert size of 10 078 [mean fragment size in
the libraries (277 bp)+95 percentile of gene size in the PN40024
12× V1 genome assembly (9801 bp)] were kept for further analysis. The htseq-count tool (v.0.5.4p5) from HTSeq (Anders et al.,
262 | Royo et al.
2015) was used to estimate unambiguous read count per 12× V1
annotated transcript. Normalization following the trimmed mean
of M-values (TMM) method (Robinson and Oshlack, 2010),
as well as a CB versus PX DEGs search (adjusted Benjamini–
Hochberg P≤0.05 and ≥2-fold change) were performed in edgeR
v.2.2.6 (Robinson et al., 2010). Finally, fragments per kb of exon
per million fragments mapped (FPKM) was calculated using
Cuffdiff v.2.2.1 (Trapnell et al., 2013) and low-expressed transcripts were filtered out when FPKM was <1 in both samples.
Reads aligned to DEGs were inspected visually for allele-specific
expression with the Integrative Genomics Viewer (IGV) software
(Thorvaldsdóttir et al., 2012).
Principal component analysis (PCA) PCA was directed using
Qlucore Omics Explorer v.3.0 (Lund, Sweden). The RMAnormalized signal and BAM format alignments from NimbleGen
microarray and RNA-seq experiments, respectively, were used as
input data.
Functional analysis DEGs at 10 and 1 dba were compared using
Venny (http://bioinfogp.cnb.csic.es/tools/venny/). The obtained
gene lists were further analysed for functional enrichment in
comparison with the whole set of transcripts predicted in the
12× V1 annotation of the grapevine reference genome following a grapevine-specific functional classification (Grimplet et al.,
2012). The analysis was carried out in FatiGO, as described elsewhere (Carbonell-Bejerano et al., 2014). Additionally, enrichment meta-analysis of DEG lists in organ-specific genes and floral
organ-detected genes, considered accordingly to the expression
atlas published in the cultivar Corvina (Fasoli et al., 2012), were
performed in FatiGO following the same procedures described
above.
Database deposit The microarray and RNA-seq data have been
deposited in NCBI under Gene Expression Omnibus (GEO)
GSE69282 and Sequence Read Archive SRP058799 (BioProject
PRJNA285072) accession numbers, respectively.
Search for sequence polymorphisms To detect polymorphisms
[single-nucleotide polymorphisms (SNPs) and insertions/deletions] between CB or PX and the PN40024 12× reference genome,
RNA-seq alignments used for differential expression analysis
were also analysed with the variant caller utility implemented
in the SAMtools package v.0.1.18 (Li et al., 2009). Alignments
in all three replicates of the same cultivar were analysed simultaneously. Then, to identify differential polymorphisms between
CB and PX, the sequence of polymorphic sites identified in one
cultivar was compared with the sequence at the same position in
the other cultivar following ad hoc Bash shell and Perl scripts
(Supplementary File S1, available at JXB online). Briefly, filters
for the average depth per replicate (≥10 counts) and frequency
for cultivar-distinctive alleles (≥35%), and a frequency of spurious alleles of <2.5% in both samples, were set. In addition, only
polymorphisms confirmed in all replicates after checking in IGV
software were considered. The effect of detected polymorphisms
considering grapevine 12× V1 gene prediction annotations was
estimated using SnpEff v.2.0.3 (Cingolani et al., 2012), while the
effects of amino acid substitutions on the protein function were
predicted in PROVEAN (Choi et al., 2012).
Selected polymorphisms were checked by PCR from CB and PX
genomic DNA obtained as described above. PN40024 DNA was
also analysed as a homozygous positive control. The PCR primers used (Supplementary Table S1, available at JXB online) were
designed according to the PN40024 12× reference genome sequence.
Primer specificity was checked by BLAST against this genome version in the Genoscope website (http://www.genoscope.cns.fr/externe/
GenomeBrowser/Vitis/). PCRs were carried out with approximately 50 ng of genomic DNA using MyTaq™ DNA polymerase
(Bioline, Meridian Life Science, Memphis, USA). Amplification
products were purified with ExoSAP-IT (USB Products Affymetrix,
Cleveland, OH, USA) following the manufacturer’s instructions and
then sequenced by capillary electrophoresis sequencing using the
same primers as in PCR.
Results
Development of parthenocarpic and occasional
seeded fruits in CB
CB presents a seedless syndrome resulting in fruit and cluster phenotypes clearly different from those observed on its
ancestor seeded cultivar PX (Fig. 1A). CB parthenocarpic
berries were about six times lighter and smaller than PX berries (Fig. 1B–D). Occasionally, CB developed seeded berries
(CB-SDD), which were identified in almost every CB cluster,
in 1.6 ± 1.2% of CB berries as an average of all seasons and
locations analysed. Generally, CB-SDD berries displayed a
comparable size to that of PX, although in 2013 they were
significantly smaller (P<0.01) because the PX berries were
larger than in other years (Fig. 1B–D). Concerning the number of seeds per berry, almost every CB-SDD berry bore
one seed, while PX berries carried 1.7 ± 0.8 seeds in both
analysed years (P<0.01, Table 1). The fresh weight of individual seeds was not significantly different between CB-SDD
and PX berries, although the mean value was higher in PX
(Fig. 1E). Grapevine frequently produces normal-sized hard
seeds that are ‘empty’ and unable to germinate (Stout, 1936).
Empty seed rate was estimated by floatability as an indication of non-viable seed rate production. This rate was 10-fold
higher in CB-SDD (49.5 ± 23.1%) than in PX (4.5 ± 6.6%)
seeds. Despite this high rate, a few CB seeds with developed
embryos were identified (Supplementary Fig. S1, available at
JXB online). These CB seeds did not germinate when sown
directly in soil (n=99), whereas ~50% of PX seeds did germinate under these conditions (n=300). Using an in vitro
germination protocol, germination of approximately 15%
of non-empty CB seeds was obtained allowing their genetic
characterization.
Pollination of CB flowers with PX pollen did not significantly change either the rate of CB-SDD berries or the number of seeds in these berries. Only six out of 796 berries were
seeded in the three CB clusters that were pollinated with PX
pollen in 2012 at VALD, while the six seeded berries bore
one seed. This result indicated that female defects impeded
seed formation in CB and thereby determined its parthenocarpic phenotype. Pollination with exogenous pollen was
not required for CB parthenocarpy since seedless fruit setting took place in CB inflorescences that were covered with
a paper bag during pollination. Moreover, this experiment
carried out at GRAJ in 2013 showed that the proportion of
seeded berries was not significantly different between selfpollinated (1.7 ± 1.3%) and free-pollinated (2.0 ± 1.0%) CB
clusters.
Embryo sac development is absent in CB
Ovule development and macrogametogenesis was analysed
to search for defects preventing seed formation in CB. As in
PX, four normal inverted anatropous ovules were developed
in each CB flower, including regular development of nucellus
and both outer and inner integuments (Fig. 2A–C). However,
while a fully developed embryo sac was identified in most PX
ovules (Fig. 2D, G), disorders in CB ovules could be observed
Alterations associated with parthenocarpy in grapevine | 263
Fig. 1. Fruit and seed features of PX and CB. (A) Representative clusters in the field at véraison. Arrowheads indicated CB-SDD berries. (B–D)
Phenotypic traits measured in ripe grapes: berry fresh weight (B), berry length (C), berry diameter (D), and seed fresh weight (E) in 2011 and 2013. PX,
yellow; CB, green. CB berries were classified according to the presence (CB-SDD) or absence (CB-SDL) of seeds. Bars represent means±SD. Different
letters (a, b, c) indicate mean values statistically different among the groups at P≤0.01.
Table 1. Number of seeds per berry in PX and CB-SDD berries
Year
2011
2013
a
PX
CB-SDD
Mean±SD
na
Mean±SD
na
1.66 ± 0.75
1.72 ± 0.84
109
150
1.00 ± 0
1.04 ± 0.23
104
133
Number of berries analysed.
before anthesis or after full bloom. In a sample of 30 CB flowers analysed, all but one of the ovules displayed an arrested
embryo sac (Fig. 2F–I) and four dying cells were located at
the position of the embryo sac in most CB ovules just prior to
anthesis (Fig. 2F). According to their linear polar distribution
and their short distance, these four degenerating cells could
correspond to the macrospores resulting from the reduction
division of the megaspore mother cell. Cells in anaphase
were also identified at the same sampling time in CB, which
further indicates that degeneration takes place after meiosis
onset (Fig. 2H). Concurrently, distinctive inner integument
cells wrinkled and separated from the outer integument, and
degeneration of the nucellus appeared in CB ovules (Fig. 2I).
CB is pollen sterile
To define whether reproductive defects in CB extended to the
male gametophyte, pollen viability was analysed. No viable
CB pollen grain was observed in 2012 and 2013, and only
1.4 ± 0.5% viable pollen grains were detected in 2014. In contrast, the PX seeded cultivar showed a pollen viability rate of
between 78 and 95% for the three seasons analysed (Fig. 3A,
B). Furthermore, pollen germination was negligible in CB,
while germination was observed in 43% of PX pollen grains
(Fig. 3C, D). Thus, functional pollen grains are hardly ever
produced in CB.
Fertilization of unreduced gametes triggers seed set
and polyploid embryo development in CB
To further characterize the phenotype of CB and to elucidate the origin of CB-SDD berries, a set of plants derived
from these seeds was obtained. A total of 58 plants were
genotyped for nine microsatellite loci to study the segregation of CB alleles. CB and PX shared the same genotype for
all nine loci, confirming the previous result by Vargas et al.
(2007) (allelic combinations are defined in Supplementary
Table S2, available at JXB online). Genotyped CB offspring
fell into four different genotype groups: 21 individuals
(36%) displayed the same genotype as CB/PX plants (type
1 in Supplementary Table S2); 26 (44.8%) showed the same
CB/PX genotype plus additional non-CB/PX alleles in several loci, suggesting that they could be triploid (type 2 in
Supplementary Table S2); nine plants (15.5%) showed loss
of CB/PX heterozygosity in one or two analysed microsatellite loci as well as additional non-CB/PX alleles in several loci (type 3 in Supplementary Table S2); and finally,
two plants (3.4%) showed loss of heterozygosity at three
and five loci, respectively, but did not display any non-CB/
PX allele, as could be expected for offspring plants derived
from autofertilization of regular haploid gametes (type 4 in
Supplementary Table S2).
264 | Royo et al.
Fig. 2. Ovule and macrogametophyte development. (A, B) Comparison of ovary transversal sections at 1 dba showing four ovules per pistil in PX (A) and
CB (B). (C) Average number of ovules per flower (n=70 per cultivar). (D, E, G, H) Longitudinal sections of ovules in 1 dba ovaries. (D) A mature embryo
sac with egg cell (ec), central cell (cc), and synergids (s) developed in PX. (E) A schematic diagram of a normal mature embryo sac within an ovule. (F) CB
ovule showing degeneration in the four megaspores after meiosis (md). (G) Number of ovules with a mature embryo sac per flower (n=30). The dot in CB
represents an outlier. (H) Detail of a CB ovule with one dividing germline cell at an apparent anaphase (a). (I) Longitudinal section of a CB ovary showing one
ovule with nucellus degeneration (nd) and other ovule with megaspores degeneration (md). Bars, 100 µm. (This figure is available in colour at JXB online.)
Given the possible existence of ploidy changes, the ploidy
level was analysed in the CB offspring by flow cytometry. Out
of 39 plants analysed, 28 were 3C (probable triploid), nine
were 4C (probable tetraploid), and only two were 2C (probable diploid) (Supplementary Fig. S2, available at JXB online,
and Supplementary Table S2). The selection of individuals
analysed for ploidy level was consciously biased towards individuals that carried non-CB alleles in the microsatellite genotyping, which explains the higher proportion of triploids in
comparison with that suggested by the microsatellite genotyping (Supplementary Table S2). Comparison between ploidy
level and microsatellite genotypes in 38 individuals analysed
with both techniques showed that all type 1 offspring plants
with identical microsatellite genotypes as CB/PX were 4C
(Table 2), suggesting that they were generated by fertilization
of an unreduced CB female gamete by an unreduced CB male
gamete. Type 2 and type 3 plants bearing additional non-CB/
PX alleles were confirmed as 3C, indicating the fertilization
of an unreduced egg cell by a haploid non-CB sperm cell.
Remarkably, the two type 4 plants showing segregation of CB
alleles at three and five loci were both 2C like CB, PX, PX
selfed-cross offspring plants, and other cultivars analysed as
ploidy level controls. Their presence suggested that these two
plants could result from a reversion of male and female gamete formation defects.
To discard the possibility that polyploid plants might result
from the in vitro germination procedure, 68 F1 plants from a
self-cross of the Chasselas cultivar germinated in vitro were
genotyped for the same nine microsatellites. All 68 plants
showed a genotype compatible with the expected segregation
for meiotic haploid gametes (data not shown), indicating that
polyploidy in CB descendant seedlings is not an artefact of
the in vitro germination procedure.
Transcriptome alterations during CB flower
development
In order to characterize the developmental processes altered
in CB and to identify candidate mutations responsible for
CB parthenocarpy, transcriptomic comparisons were carried
out between CB and PX developing flower buds. In 2011,
NimbleGen grapevine whole-genome microarrays were used to
compare flower buds just prior to anthesis (1 dba), a stage with
aborted pollen grains and degenerating macrogametophytes
Alterations associated with parthenocarpy in grapevine | 265
Fig. 3. Pollen viability and germination. (A) Alexander staining. Dark pollen is viable, while empty (pale) pollen is sterile. (B) Percentage of pollen viability in
three consecutive years. (C) In vitro pollen germination images. (D) Percentage of germination after 24 h of incubation at 25 °C. In (B) and (D), the data are
means of three replicates. *, Significant differences between cultivars in the given year (P<0.01; t-test). Bars, 100 µm. (This figure is available in colour at
JXB online.)
Table 2. Ploidy level within CB offspring genotype groups
Only individuals analysed by both methods were considered in this
table. Genotype and ploidy level from all analysed individuals can be
consulted in the Supplementary Table S2. n, Number of individuals
analysed.
Genotype group
n
Cytotype (C)
frequency
2C
Type 1. CB-like
Type 2. CB-like + exogenous alleles
Type 3. CB segregant + exogenous alleles
Type 4. CB selfed segregant
8
22
6
2
3C
4C
100%
100%
100%
100%
in CB and mature gametophytes in PX (Figs 2 and 3). In
2012, RNA-seq was used to analyse 10 dba flower buds as the
expected stage when microgametogenesis is being completed
and macrogametogenesis is initiating (Pratt, 1971; Lebon
et al., 2008). Despite the larger library size in one RNAseq PX replicate (Supplementary Table S3, available at JXB
online), PCA showed that the first component of variation
in both experiments separated the samples by the variant line
(component 1 explaining 46 and 69% of variation in RNA-seq
and microarray experiments, respectively; Fig. 4A, B). A total
of 1958 DEGs (≥2-fold change and Benjamini–Hochberg
adjusted P≤0.05) between CB and PX were identified (Fig. 4C
and Supplementary Table S4, available at JXB online). In both
analysed stages, a higher number of CB-downregulated (625
and 969 at 10 and 1 dba, respectively) than CB-upregulated
(179 and 430 at 10 and 1 dba, respectively) genes was identified. In spite of developmental, seasonal, and technical divergences, 35% of CB-downregulated genes at 10 dba were also
downregulated at 1 dba. In contrast, only 11 genes were CB
upregulated in both stages, while 17 DEGs showed inverted
expression profiles between both stages.
Functional enrichment analysis was performed to understand the biological meaning underlying detected DEGs.
Genes that were upregulated in CB flowers at 10 or 1 dba
were enriched in dissimilar functional categories (Fig. 4D
and Supplementary Table S5, available at JXB online). While
CB-upregulated genes at 10 dba were enriched in ‘Sugar
transport’, ‘MYB family transcription factor’, and ‘Protein
disulfide bond rearrangement’ categories, CB-upregulated
genes at 1 dba were mostly enriched in categories related
to biotic stress (‘Protein kinase’, ‘Biotic stress response’,
‘Salicylic acid signalling’, and ‘NBS-LRR superfamily’) and
oxidative stress (‘Ascorbate and aldarate metabolism’ and
‘Oxidative stress response’). In contrast, enriched categories
were more similar between CB-downregulated genes at both
studied stages. Indeed, several categories related to vesicular
or transmembrane transport (‘Vesicle-mediated trafficking’,
‘Proton transport’, ‘Electron transport’, ‘Phagosome’, and
‘Triose-phosphate transport’) were over-represented among
genes that were downregulated in CB at both 10 and 1 dba.
Moreover, genes downregulated at each stage were specifically
enriched in different cellular (‘Oil body organization and biogenesis’ at 10 dba, and ‘Storage proteins’ and cytoskeleton
and cell wall biogenesis-related at 1 dba), metabolic (terpenoid
266 | Royo et al.
Fig. 4. (A, B) Transcriptome analysis. PCA of RMA-normalized NimbleGen microarray expression data from 10 dba flower bud samples (A) and TMMnormalized RNA-seq read counts from 1 dba flower bud samples (B). (C) Venn diagram comparing DEGs at 10 and 1 dba. The number of transcripts
within each group is indicated. (D, E) Enrichment of functional categories (D) and Corvina expression atlas (Fasoli et al., 2012) organ-specific transcripts
(E) in groups of CB versus PX DEGs. (This figure is available in colour at JXB online.)
and pyruvate metabolism-related at 10 dba and monosaccharide and starch metabolism-related at 1 dba) and regulatory
processes (‘PseudoARR-B family transcription factor’ at 10
dba and ‘KIP1 SANTA family transcription factor’, together
with two distinct secondary signalling processes at 1 dba).
Similarly, ‘Desiccation stress response’ and ‘Drought stress
response’ were enriched in 10 and 1 dba downregulated transcripts, respectively.
chr8:19689156
chr8:20446425
chr19:3532149
-chr19:3532150
CB specific
CB specific
CB specific
a
chr11:4634655
chr13_
random:2434505
chr13:192216
chr16:20771586
chr18:4166072
VIT_13s0067g00270
VIT_16s0098g00350
VIT_18s0001g05110
VIT_11s0016g05300
VIT_13s0019g00350
VIT_05s0020g02350
VIT_06s0061g01340
VIT_19s0014g03380
VIT_08s0007g05750
VIT_08s0007g06780
VIT_02s0025g01660
VIT_05s0020g01400
VIT_07s0104g01290
Gene ID
Ribosomal protein 60S
SDG29 (SET domain group 29)
DNA2-NAM7 helicase
Heme oxygenase 1
ARF GTPase activator
SAC3/GANP
Phosphoglycerate mutase
PAP phosphatase
Ankyrin
Unknown
Seven-in-absentia SINA5
SRC2 kinase
TUBBY TF
Functional annotation
C:C
T:T
G:G
C:C
A:A
G:G
C:C
AG:AG
G:G
A:A
T:T
C:C
C:C
PN40024
Genotype
C:T
A:T
A:G
C:T
A:T
G:A
C:T
AG:AG
G:G
A:A
T:T
C:C
C:C
PX
C:C
T:T
G:G
C:C
A:A
G:G
C:C
AG:CT
G:A
A:G
T:C
C:T
C:T
CB
A281T
N920I
Q443Stop
A9T
S260R
A610T
R263C
5′UTR
L274S
3′UTR
A264V
I42V
P53P
Variant allele effect
on protein sequence
Deleterious
Neutral
Deleterious
Neutral
Deleterious
Neutral
Deleterious
Deleterious
Neutral
–
Neutral
Synonym
–
Variant amino acid effect on
protein function
–2.56*
–0.15
–1468*
–1.90
–4.78*
0.20
–3.33*
–4.32*
0.88
–
–0.11
–
–
PROVEAN scorea
A PROVEAN score for the effect of variant alleles in the protein function of less than or equal to –2.5 was considered ‘deleterious’ and greater than –2.5 was considered ‘neutral’.
PX specific
PX specific
PX specific
PX specific
PX specific
chr5:4050256
chr6:19176704
chr2:1600341
chr5:3126511
chr7:2328801
CB specific
CB specific
CB specific
CB specific
PX specific
PX specific
Variant position(s)
Variant type
Somatic line specificity for the variant allele in comparison with the PN40024 reference genome, locus affected, locus functional annotation, genotype of each cultivar, and predicted effect of
the variant allele in the protein sequence and function are shown. Variant alleles are highlighted in bold.
Table 3. RNA-seq polymorphisms distinguishing CB and PX
Alterations associated with parthenocarpy in grapevine | 267
268 | Royo et al.
To elucidate the floral organs that might be affected by
the identified gene expression differences, organ-specific
genes and genes expressed in floral organs identified in a
whole transcriptome atlas of the Corvina grapevine cultivar
(Fasoli et al., 2012) were compared for CB versus PX DEGs
(Supplementary Table S4). Enrichment meta-analysis of
DEGs in Corvina organ-specific genes identified that genes
specifically expressed in sporophytic organs, including vegetative organs, were over-represented among CB-upregulated
genes (‘Stamen and petal’ and ‘Stamen’ at 10 dba, ‘Leaf’ and
‘Rachis’ at 1 dba). In contrast, only genes expressed specifically
in floral organs, including genes expressed specifically in the
microgametophyte (pollen) and genes expressed specifically in
carpel (the organ bearing the macrogametophyte, which was
not analysed separately in Corvina), were over-represented
among CB-downregulated genes (Fig. 4E and Supplementary
Table S5). Similar results were obtained when DEGs were
meta-analysed for enrichment in genes detected in Corvina
floral organs, which showed that CB-upregulated genes were
not enriched in genes expressed in floral organs with the exception of petal, while CB-downregulated genes were enriched
mainly in pollen-expressed genes (Supplementary Table S5).
Sequence polymorphisms between CB and PX,
including parthenocarpy-candidate mutations,
identified by RNA-seq
Polymorphisms between CB and PX were searched for among
expressed transcripts resulting from 10 dba flower bud RNAseq alignments. Such polymorphisms might help to discriminate between the CB parthenocarpic variant and PX seeded
ancestor, regardless of the fruit phenotype, and furthermore
might involve the mutation responsible for the phenotypic
variation.
A total of 14 polymorphisms were identified between CB
and PX. All were heterozygous SNPs in the cultivar carrying the variant allele in comparison with the PN40024 reference genome and were homozygous with the same genotype
as the reference genome in the other cultivar (Table 3). Only
polymorphisms involving CB-specific variant alleles (in comparison with PX and the reference genome) were considered
as potentially responsible for its parthenocarpic phenotype.
Seven SNPs involving CB-specific variant alleles were identified, and all were heterozygous SNPs located in exonic
regions. The seven SNPs comprised six genes, because two of
them were located consecutively on the same gene (Table 3).
Capillary electrophoresis sequencing of genomic DNA validated all five CB-specific SNPs tested (Supplementary Fig.
S3, available at JXB online). Three CB-specific SNPs involved
synonymous changes in the respective predicted proteins,
while the other four SNPs involved non-synonymous changes
in three genes. Among these, predictive in silico analysis
indicated that VIT_19s0014g03380, encoding a HAL2-like
3′-phosphoadenosine-5′-phosphate (PAP) phosphatase, presented the only non-neutral change, which resulted from two
consecutive SNPs causing a leucine-to-serine substitution in
the PAP phosphatase-like domain (Table 3, Supplementary
Fig. S4A, available at JXB online). Although the mutated
leucine was conserved in homologous proteins from other
plant species, it was not present in the originally described
Saccharomyces cerevisiae HAL2 protein (Supplementary Fig.
S4B). This leucine is located in the context of a predicted
cyclin recognition site only detected in the grapevine protein
(Supplementary Fig. S4B).
On the other hand, although genomic deletions have been
involved in gamete sterility and grapevine somatic variation (Page et al., 2004; Vezzulli et al., 2012), PX-specific
heterozygous SNPs did not concentrate in chromosomal
regions (Table 3), and thus they do not support the presence
of CB-specific hemizygous regions underlying its variant
phenotype.
Several grapevine somatic variants resulted from dominant heterozygous cis-acting mutations causing upregulated gene expression (Fernandez et al., 2010, 2013). As
hints for these issues, differential allelic frequency between
CB and PX in heterozygous positions of CB-upregulated
transcripts was also searched for in RNA-seq alignments.
No clear evidence of allelic imbalance was identified (data
not shown). However, given its homozygosity (confirmed
in exonic fragments by capillary electrophoresis sequencing
of genomic DNA; data not shown), this phenomenon cannot be ruled out for the putative ERD6 sugar transporterencoding gene VIT_14s0030g00240 (Supplementary Fig.
S5, available at JXB online), which was among the most
overexpressed genes in both 10 and 1 dba CB flower buds
(Supplementary Table S4). In the Corvina expression atlas
(Fasoli et al., 2012), VIT_14s0030g00240 was shown to be
expressed preferentially in young vegetative tissues, showing its lowest expression in pollen (Supplementary Fig.
S5), which may be consistent with ectopic expression in CB
flowers.
Discussion
In order to understand the genetic origin of grapevine parthenocarpy, this study took advantage of recent findings demonstrating that CB is a parthenocarpic somatic variant of the
seeded cultivar PX (Vargas et al., 2007). Here, it was shown
that the absence of a mature embryo sac is the ultimate cause
preventing seed formation in CB. Nevertheless, fruit setting
and ripening took place in the absence of fertilization or seed
formation, although fruit growth was considerably diminished (Fig. 1). Similarly, a certain amount of small seedless
fruits can develop and ripen in grapevine bunches of seeded
cultivars, a phenomenon that intensifies when proper pollination is prevented by emasculation or by adverse environmental conditions (Dry et al., 2010; Dauelsberg et al., 2011;
Kidman et al., 2014). Indeed, some degree of natural facultative parthenocarpy is extended to other species including the
model plant Arabidopsis, whose unfertilized pistils also show
partial fruit development with reduced growth (CarbonellBejerano et al., 2010; Sotelo-Silveira et al., 2014). Hence, it
is suggested here that CB’s somatic mutation turns the grapevine characteristic facultative parthenocarpy into an obligatory parthenocarpy due to sexual sterility.
Alterations associated with parthenocarpy in grapevine | 269
Given the regular development of sporophytic tissues in
CB ovules (Fig. 2) and considering the presumable arrest of
female meiosis observed at the four-macrospore stage, parthenocarpic CB is more likely to be a meiotic than a gametophytic mutant. Since meiotic defects depend on the diploid
sporophyte genotype (Liu and Qu, 2008), a recessive homozygous mutation or, more probably in a somatic variant, a dominant heterozygous mutation, could be the cause for the CB
sterile phenotype.
The genetic analyses of seeds produced occasionally by
CB fruits revealed that CB infrequent functional male and
female gametes are mostly unreduced gametes (Table 2 and
Supplementary Table S2). These gametes should result from
apomeiosis (omission of meiotic reduction), considered as
the first step of apomixis (Schmidt et al., 2015). However, the
absence of diploid seedlings displaying the CB genotype indicated that apomixis did not take place in CB. The ontogeny
of diplogametes can be elucidated by analysing the segregation of chromosomes according to microsatellite genotypes
(Supplementary Table S2). In tetraploid CB offspring, chromosome duplication might mask losses of heterozygosity
produced by meiotic segregation events. In contrast, triploid
seedlings, derived from fertilization of CB female diplogametes by non-CB haploid sperm cells, provide an opportunity
to study the segregation in CB diplogametes. Segregation of
CB alleles was found in 25.7% of the analysed triploids (type
3 in Supplementary Table S2), indicating that the meiotic process was initiated and therefore at least these diplogametes
were probably formed by meiotic diplospory, i.e. unreduced
megaspores arising from a meiosis modified by nuclear restitution (Koltunow, 1993). By considering in triploid plants
those microsatellite loci that are heterozygous in CB and
carry a non-CB allele (Supplementary Table S2), a heterozygosity loss rate of 3.2% could be estimated for CB’s female
diplogametes. Such a low rate is typically produced by firstdivision nuclear restitution (gametes containing two chromosomes of non-sister chromatids) due to abnormal meiosis
I (Bretagnolle and Thompson, 1995).
During microgametogenesis of diploid grapevine cultivars,
spontaneous formation of unreduced gametes has been estimated at rates between 0 and of 0.37% (Zhang et al., 1998).
The level of female unreduced gametes in CB might be above
this range considering that, despite that fact that effective fertilization of generally unreduced gametes is required for seed
development in CB, 1.6% of setting berries produced one
seed, and each berry involved four independent macrogametogenesis events. Consequently, and taking into account the
fact that viable unreduced gametes are frequently produced
in meiotic mutants (Schmidt et al., 2015), their production by
diplospory in CB seems more likely to be a consequence of
the CB parthenocarpic syndrome than a grapevine infrequent
process unmasked by the sterility of CB plants.
When combined with genetic analysis, transcriptomic comparisons have previously been demonstrated to be effective
in discovering misexpressed genes harbouring the mutation
responsible for the mutant phenotype observed in different
grapevine somatic variants (Fernandez et al., 2010, 2013).
Unfortunately, CB male and female sterility restricts the
possibilities of using genetic analysis in identification of the
mutation causing its parthenocarpy, although triploid and
diploid generated plants could be useful in the future. As an
alternative, this study used RNA-seq to search for candidate
mutations by analysing the sequence of expressed transcripts.
Moreover, transcriptomic comparisons allowed characterization of the molecular syndrome associated with the somatic
variation in CB. Homologues of Arabidopsis microgametophyte development genes were among the most highly
downregulated genes in 10 dba CB flowers (Supplementary
Table S4). They included the VIT_15s0021g02040 and
VIT_17s0000g05490 genes homologous to DUO POLLEN1
and SIDECAR POLLEN, respectively, both required in
Arabidopsis for post-meiotic cell division and differentiation of the male germline (Chen and McCormick, 1996;
Rotman et al., 2005). Accordingly, CB-downregulated genes
were enriched in vesicular trafficking, cell wall biogenesis,
cytoskeleton biogenesis, phosphatidylinositol signalling, and
G-protein signalling functions (Fig. 4D and Supplementary
Table S5); processes that are are involved in cell plate formation and cytokinesis (De Storme and Geelen, 2013;
McMichael and Bednarek, 2013). Collectively, downregulation of these genes indicates a lack of post-meiotic mitosis in
CB microspores, suggesting that the CB male germline development is also arrested during meiosis. Later, putative pollen
maturation and function genes were downregulated at 10 and
1 dba or only at 1 dba, including homologues of AtWRKY2
(VIT_04s0023g00470), AtAGL66 (VIT_07s0031g01140), and
AtMYB101 (VIT_12s0059g00700) transcription factors, and
RESPIRATORY BURST OXIDASE HOMOLOG J (RBOHJ,
VIT_06s0004g03790), CROLIN1 (VIT_14s0066g02480),
POLLEN-SPECIFIC LIM PROTEIN 2C (PLIM2C,
VIT_15s0046g03370, and VIT_02s0025g03980), AGC
KINASE 1.7 (VIT_19s0085g01140), and FERONIAlike receptor kinase ANX1 (VIT_05s0077g00970 and
VIT_07s0005g01640). (Supplementary Table S4). All of these
are required in Arabidopsis for pollen maturation and/or tube
growth differentiation (Adamczyk and Fernandez, 2009;
Boisson-Dernier et al., 2009; Zhang et al., 2009; Ye and Xu,
2012; Jia et al., 2013; Leydon et al., 2013; Guan et al., 2014;
Kaya et al., 2014). Similarly, the observed over-representation
of starch metabolism, sugar transport, and proton pumprelated functional categories among CB-downregulated genes
resembles gene expression defects in maize pollen sterile lines
lacking the post-meiotic pollen starch-filling pathway (Datta
et al., 2002). These expression profiles agree with a lack of
regular post-meiotic microgametophyte development, which
yields sterile pollen grains in CB flowers.
In contrast, genes upregulated in 10 dba CB flowers were
enriched in stamen-specific genes (Fig. 4E and Supplementary
Table S5), which comprised VIT_01s0011g06390, a homologue of the Arabidopsis PHD-type TF MALE STERILITY1
(MS1) required for tapetal pollen wall biosynthesis (Ito et al.,
2007; Yang et al., 2007). Expression of MS1 in Arabidopsis is
confined to the tapetum, while maximum expression is seen
during microspore release and no expression is seen by microspore mitosis I. Highly upregulated genes coding for putative tapetum preferential genes also included homologues
270 | Royo et al.
of Arabidopsis sporopollenin biosynthetic enzymes or
transporters such as LESS ADHESIVE POLLEN 5 and 6
(VIT_03s0038g01460 and VIT_15s0021g02170), ACYLCOA SYNTHETASE 5 (VIT_01s0010g03720), CYP703A2
(VIT_15s0046g00330), CYP704B1 (VIT_01s0026g02700),
MS2 (VIT_08s0007g07100), TETRAKETIDE ALPHAPYRONE REDUCTASE 2 (VIT_01s0011g03480), HYDRO­
FLAVONOL 4-REDUCTASE-LIKE 1 (VIT_03s0038g04220),
and ABCG26 (VIT_19s0015g00960) (Liu and Fan, 2013).
In Arabidopsis, most of these genes are regulated downstream of MYB35 and MYB80 (Phan et al., 2011; Liu and
Fan, 2013), while homologues of these transcription factors
(VIT_17s0000g05400 and VIT_19s0015g01280) along with
other MYBs were also CB upregulated in 10 dba flower buds
(Fig. 4D and Supplementary Tables S4 and S5). These findings suggest that the exine biosynthetic genetic machinery
described in Arabidopsis is conserved in grapevine. Since this
machinery generally acts in the tapetum prior to pollen grain
post-meiotic mitosis, the overexpression of these genes in 10
dba CB flowers might relate to an arrest of the CB male germline in a pre-mitotic microsporocyte state.
Regarding female macrogametophyte development genes,
only a few homologues of Arabidopsis genes (Pagnussat
et al., 2005; Johnston et al., 2007; Jones-Rhoades et al., 2007;
Sanchez-Leon et al., 2012) were detected among DEGs, and
were generally downregulated in CB (data not shown). A high
number of macrogametophyte-related CB-downregulated
genes could be expected given the absence of mature macrogametophytes in CB. The low detection of these genes might
be explained by the dilution of macrogametophyte transcripts in the wild-type PX flower given the low proportion
of female gametophyte cells in the grapevine flower with only
four ovules.
Degeneration of the nucellus, dying macrospores, and unviable pollen grains were observed in mature CB flower buds
(Figs 2 and 3). Probably connected to these processes, biotic
stress, salicylic acid signalling, oxidative stress, and cell death
genes were prominently overexpressed in 1 dba CB flowers
(Fig. 4D and Supplementary Tables S4 and S5). These genes
comprise several EDS1-like defence genes including VvEDS1
(VIT_17s0000g07560) reported as salicylic acid and pathogen responsive in grapevine (Gao et al., 2014). Thus, these
CB-upregulated genes are likely to operate in the degeneration of aborted sexual organs.
Considering that our results pointed to meiosis abortion as
the likely cause of CB’s sterility and impeded seed formation,
genes involved in meiosis and/or the cell cycle were screened
among the detected DEGs. Few meiosis-specific genes have
been reported in plants (Yang et al., 2011; Liu and Qu, 2008).
Among them, this study identified that VIT_02s0025g00410
was downregulated in 1 dba CB flowers (Supplementary Table
S4), encoding a homologue of the SPO11-2 toposiomerase
VIA protein required in Arabidopsis for proper meiotic recombination (Stacey et al., 2006). Such reduced expression might
be related to meiosis defects in CB. Nonetheless, the lack of
differential expression at 10 dba does not support the presence
of a causal mutation involving this gene. Additionally, several
putative mitotic cyclins were also downregulated, including a
CYCB1;2 (VIT_06s0009g02090), which was the most downregulated gene at 1 dba (Supplementary Table S4). However,
genetic variation between CB and PX was not identified by
sequencing of this gene plus 1 kb upstream from its predicted
start codon (data not shown). Similarly, two putative kinases
homologous to AtAurora1 and AtAurora3 were downregulated at 1 dba (VIT_04s0008g00530 and VIT_15s0048g01730,
respectively). Reduced activity of these kinases inhibits meiotic chromosome segregation and leads to sexual sterility and
the formation of unreduced gametes in Arabidopsis (Demidov
et al., 2014), resembling the variant syndrome observed in
CB. Nevertheless, read sequences and abundance identified by
RNA-seq did not indicate the presence of CB-specific mutations in these genes, suggesting that their misregulation is a
consequence of a causal mutation(s) in other loci.
RNA-seq sequence data was explored specifically for
CB-specific genetic variation that could be responsible for the
CB variant phenotype. Seven CB-specific SNPs were identified (Table 3) and considered as parthenocarpy-responsible
candidate mutations. Among these, a deleterious effect was
predicted for two consecutive CB-specific SNPs producing the substitution of a leucine, with a hydrophobic sidechain, by serine, with a polar side-chain, in the predicted
catalytic domain of a putative HAL2-like PAP phosphatase
(VIT_19s0014g03380; Supplementary Figs S3 and S4).
Considering that the same amino acid substitution produces
dominant-negative effects in other proteins (Geller et al.,
1998), it is conceivable that this mutation could be involved
in the variant phenotype of CB in spite of its heterozygous
state. The substituted leucine is integrated in a cyclin recognition site specifically predicted for the grapevine protein
(Supplementary Fig. S4B), which might suggest grapevinespecific functions for this gene. Cyclins recognize these motifs
and thereby increase the specificity of phosphorylation by
cyclin/cyclin-dependent kinase complexes. Nevertheless, cyclin-dependent kinase phosphorylation sites were not detected
in the HAL2-like protein. The HAL2-like gene family groups
together cation-sensitive phosphatases and AtAHL, the closest Arabidopsis homologue, catalyses the conversion of PAP
to AMP, preventing the toxicity of PAP and facilitating the
sulphur assimilation pathway. Other family members have
been proposed to participate in PAP, phosphoinositol, and
abiotic stress signalling (Quintero et al., 1996; Gil-Mascarell
et al., 1999; Xiong et al., 2001; Estavillo et al., 2011). Single
amino acid substitutions were reported to alter the activity
of AtAHL (Cheong et al., 2007), which might support an
effect for the mutation identified in VIT_19s0014g03380.
Deleterious protein activity of the CB variant allele might
alter any of the abovementioned pathways in male and female
CB meiocytes, which might in turn impair the meiosis process, resulting in the absence of germline development and,
exceptionally, in diplosporic meiosis. Following the grapevine
gene expression atlas developed for cultivar Corvina (Fasoli
et al., 2012), VIT_19s0014g03380 was preferentially expressed
in reproductive organs and showed its maximum expression
in pollen (Supplementary Fig. S6, available at JXB online),
which might be consistent with a novel sexual reproductionrelated function for the HAL2 gene family.
Alterations associated with parthenocarpy in grapevine | 271
Currently, other detected SNPs, unidentified mutations,
or epigenetic variation cannot be discarded as possibly being
responsible for the CB parthenocarpic phenotype. For example, gene expression and SNP profiles detected in the ERD6
sugar transporter-like gene VIT_14s0030g00240 suggest that
it might carry a mutation in regulatory regions causing its
ectopic overexpression in CB flowers (Supplementary Fig.
S5). Grapevine anthers and ovules are considered highly sensitive organs to sugar deprivation, and particularly at the stage
of female meiosis (Lebon et al., 2008). Thus, altered sugar
status related to misregulation of this gene might be involved
in the CB variant phenotype. Further functional work will be
required to confirm these possibilities and demonstrate any
connection between candidate genes and the CB phenotype.
Conclusions
This study represents an integrative approach towards understanding the origin of parthenocarpy in grapevine. The results
show that the absence of a mature microgametophyte turns CB
into an obligatory parthenocarpic variant. CB is also pollen
sterile, while seeds occasionally formed in this variant generally
involve polyploid embryos resulting from the fertilization of
unreduced gametes produced by meiotic diplospory. Our findings suggest that impaired meiosis is the cellular origin of the
altered sexual reproduction and fruit development displayed
by CB. Accordingly, transcriptomic analyses revealed a set of
grapevine genes likely to be involved in post-meiotic gametophyte division and maturation processes that were downregulated in CB. Transcriptomic hints suggesting that pathogen-like
responses operate in the degeneration of aborted sexual organs
in CB were also found. Despite the fact that the identification of
meiotic mutations causing sterility is generally difficult because
of the unfeasibility of genetic analysis, our RNA-seq experiment detected SNP polymorphisms distinguishing CB and PX
variant lines and revealed several parthenocarpy-responsible
candidate genes. Among these, a putative causal mutation was
detected in a HAL2-like gene that has not previously been related
to meiosis or sexual reproduction. This study provides a basis
for further cytological and molecular assays focusing on cells
undergoing meiosis, and shows how genomic approaches can
shed light on the origin of grapevine somatic variants including
the genetic variation responsible for seedlessness traits.
Supplementary data
Supplementary data are available at JXB online.
Supplementary Fig. S1. Embryos developed within seeds
of occasional CB-seeded berries.
Supplementary Fig. S2. Representative ploidy profiles
obtained by cytometry.
Supplementary Fig. S3. Capillary electrophoresis sequencing validation of RNA-seq-identified SNPs.
Supplementary Fig. S4. HAL2-like variant protein analysis.
Supplementary Fig. S5. ERD6-like VIT_14s0030g00240
candidate gene expression.
Supplementary Fig. S6. HAL2-like VIT_19s0014g03380
candidate gene expression.
Supplementary Table S1. List of primers used for PCR and
sequencing.
Supplementary Table S2. Full list of Corinto bianco
offspring analysed for microsatellite genotyping and/or
ploidy level.
Supplementary Table S3. RNA-seq library size and normalization factors.
Supplementary Table S4. List of DEGs between CB and
PX developing flowers.
Supplementary Table S5. Functional and organ enrichment analyses of CB versus PX DEGs.
Supplementary File S1. Bash shell and scripts generated to
search for sequence polymorphisms.
Acknowledgements
This study was funded by the Spanish MINECO grant BIO2011-026229.
COST (European Cooperation in Science and Technology) Actions FA1003
and FA1106 supported the exchange of researchers and the presentation of
preliminary results. Microarray hybridizations and histological preparations
were performed at the Genomics and Histology services of the National
Centre for Biotechnology, CNB-CSIC, Madrid, Spain, respectively. RNAseq was carried out at the Centre for Genomic Regulation (CRG), Barcelona,
Spain. We are grateful to Silvia Hernáiz, Elisabet Vaquero, and José Luis
Pérez-Sotés at the ICVV and to the Servicio de Investigación y Desarrollo
Tecnológico Agroalimentario (SIDTA) and IMIDRA for technical assistance and/or plant maintenance.
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