Histological observation of Vitis rotundifolia ‘Carlos’ × V. rotundifolia ‘Carlos’ (CC) and V. rotundifolia ‘Carlos’ × V. vinifera ‘Zijinqiunong’ (CZ). Self-pollen germination and pollen tube at 2 h (A) and 8 h (B) after pollination. Cross-pollen germination and the pollen tube at 2 h (C) and 8 h (D) after pollination. Arrows indicate the pollen tube.
Heatmap with hierarchical clustering of differentially expressed proteins (DEPs) in Vitis rotundifolia ‘Carlos’ × V. rotundifolia ‘Carlos’ (CC) and V. rotundifolia ‘Carlos’ × V. vinifera ‘Zijinqiunong’ (CZ). Two independent biological replicates were collected from CC (CC-1, CC-2) and CZ (CZ-1, CZ-2). Red and orange indicate high expression and low expression according to the color bar. The DEPs clustered into four groups (groups I, II, III, and IV).
Gene ontology (GO) terms (top 10) of upregulated differentially expressed proteins in group I (or group II) between Vitis rotundifolia ‘Carlos’ × V. rotundifolia ‘Carlos’ (CC) and V. rotundifolia ‘Carlos’ × V. vinifera ‘Zijinqiunong’ (CZ). Group I and group II were clustered according to the results of heatmap.
Gene ontology (GO) terms (top 10) of downregulated differentially expressed proteins in group I (or group II) between Vitis rotundifolia ‘Carlos’ × V. rotundifolia ‘Carlos’ (CC) and V. rotundifolia ‘Carlos’ × V. vinifera ‘Zijinqiunong’ (CZ). Group III and group IV were clustered according to the results of heatmap.
Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway of differentially expressed proteins in Vitis rotundifolia ‘Carlos’ × V. rotundifolia ‘Carlos’ (CC) and V. rotundifolia ‘Carlos’ × V. vinifera ‘Zijinqiunong’ (CZ).
Expression level of differentially expressed proteins genes in Vitis rotundifolia ‘Carlos’ × V. rotundifolia ‘Carlos’ (CC) and V. rotundifolia ‘Carlos’ × V. vinifera ‘Zijinqiunong’ (CZ). *Significant difference at P < 0.05.
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Muscadine grape (Vitis rotundifolia) is highly resistant to many diseases and insects that attack european grape (Vitis vinifera). However, distant hybridization incompatibility between V. rotundifolia (female) and V. vinifera (male) impedes the utilization of V. rotundifolia in grape breeding. This study used fourth-dimension label-free protein quantitation to detect the key genes and pathways in the V. rotundifolia stigma after self-pollination (V. rotundifolia × V. rotundifolia) and cross-pollination (V. rotundifolia × V. vinifera). A histological analysis showed that pollen tube growth in the stigma of V. rotundifolia was arrested 8 hours after cross-pollination, but not after self-pollination. A proteomic analysis identified 32 differentially expressed proteins (DEPs) in the stigma of V. rotundifolia between self-pollination and cross-pollination. A heatmap analysis grouped these DEPs into four clusters. The top gene ontology terms were ATPase-coupled transmembrane transporter activity, extracellular region, DNA replication, and cellular carbohydrate biosynthetic process. A Kyoto Encyclopedia of Genes and Genomes analysis revealed that these DEPs participated in DNA replication and starch and sucrose metabolism pathways. The downregulated A5AY88, D7TJ35, D7SU26, F6HJI1, and F6GUE7 may have a role in cross incompatibility. This study revealed the cross incompatibility of grapes at histological and proteomic levels.
Distant hybridization is an important means of expanding genetic variation and creating new species (Chen et al., 2018; Sharma and Gill, 1983). As a valuable method of plant breeding, distant hybridization has been widely used for various crops (An et al., 2019; Batra et al., 1990; Chung et al., 2013; Kamstra et al., 1999). However, reproductive barriers, including prefertilization barriers (cross incompatibility) and postfertilization barriers (embryo abortion, hybrid sterility), that commonly exist in distant hybridization of plants lead to low hybridization efficiency (Widmer et al., 2009).
Grapes (Vitis sp.) are a valuable fruit crop. European grapevine (V. vinifera) is a major grape cultivated worldwide that is used as table grape, processed, or dried to produce raisins. However, european grape cultivars are susceptible to various diseases, such as downy mildew (Plasmopara viticola), powdery mildew (Uncinula necator), and phylloxera (Daktulosphaira vitifoliae) (Cui et al., 2021; Powell, 2012). Grape resistance to biotic stress and abiotic stress has been confirmed in Asian (V. amurensis) and American wild species (V. labrusca, V. riparia, V. rupestris) (Dubrovina et al., 2015; Zhao et al., 2010). Various resistance genes have been introgressed from wild species of grapes into cultivated grapes (Divilov et al., 2018; Venuti et al., 2013). However, the level of resistance in interspecific table grape hybrids is often lower than that of the wild parent, whereas interspecific hybrids with high resistance exhibit quality traits of lesser value compared with those of european grape.
Vitis rotundifolia is a native American species with high resistance to fungal diseases that attack european grapevine (Lu et al., 2000; Patel and Olmo, 1955). The berries are black or bronze and have unique fruit aroma (Brown et al., 2016). The chromosome number of V. rotundifolia is 2n = 2x = 40, and that of V. vinifera is 2n = 2x = 38 (Patel and Olmo, 1955). Because of chromosomal differences between V. rotundifolia and V. vinifera, cross incompatibility and embryo abortion often occur in distant hybridization of V. rotundifolia and V. vinifera. First hybrids between V. vinifera and V. rotundifolia were produced by A.P. Wylie in 1859, but they bore no fruit. Detjen (1919) produced the first documented V. vinifera–V. rotundifolia hybrid seedling, and fertile hybrids were successfully produced by a French breeding program (Bouquet, 1980). Later, resistance genes from V. rotundifolia were introgressed into V. vinifera through backcrossing with the use of valuable hybrids as parents (Bouquet, 1986; Pauquet et al., 2001; Rubio et al., 2020). Although some resistance genes, such as those conferring downy mildew and powdery mildew resistance, were introduced into V. vinifera, the low cross efficiency and partial sterility of the hybrid progeny persisted and seriously restrict the use of V. rotundifolia in grape breeding. Delame et al. (2019) studied the recombination rate of homologous chromosome pairs in interspecific hybrids of V. vinifera and V. rotundifolia and found that meiotic recombination is suppressed in homeologous regions and enhanced in homologous regions of recombined chromosomes, whereas the crossover rate remains unchanged when chromosome pairs are entirely homeologous. In general, interspecific hybrids are obtained using V. vinifera as the female parent and V. rotundifolia as the male parent, because the reciprocal cross is difficult to achieve (Lu et al., 2000; Patel and Olmo, 1955). The molecular mechanism of grape distant cross-incompatibility remains unknown.
This study aimed to explore the molecular mechanisms of hybridization incompatibility in grape. In the present study, we used V. rotundifolia ‘Carlos’ as the female parent and V. vinifera ‘Zijinqiunong’ as the pollinizer and conducted histological observations of stigmas at different time points after pollination. Differentially expressed proteins (DEPs) in the stigmas of V. rotundifolia cross-pollinated with V. vinifera (‘Carlos’ × ‘Zijinqiunong’) or self-pollinated (‘Carlos’ × ‘Carlos’) were analyzed after pollination. The key pathway involved in grape distant hybridization incompatibility is also discussed. Our results provide useful molecular information for future research of cross incompatibility between V. rotundifolia and V. vinifera.
The vineyard was established at the Jiangsu Academy of Agricultural Sciences, Nanjing City, Jiangsu Province, China (lat. 32°02′N, long. 118°52′E). All plant material was maintained under a rain shelter and cultivated with root restriction. Vitis rotundifolia ‘Carlos’ was used as the maternal cultivar and cultivated in rectangular concrete containers at a density of seven vines per container. Vitis vinifera ‘Zijinqiunong’ was used as a pollinizer. It was also cultivated in square concrete containers with one vine per container.
All the clusters of ‘Zijinqiunong’ and ‘Carlos’ were covered with single-layer white bags to prevent pollen contamination from foreign sources. Pollen collection and artificial emasculation were initiated at the beginning of flowering. The flowering date of ‘Zijinqiunong’ was 5 May 2020. Pollen was collected from four healthy vines, dried with silica gel for 24 h, and stored at 4 °C. Flowering of ‘Carlos’ began on 28 May.
A total of 60 clusters were emasculated and covered with bags. Pollination treatment was conducted after 2 d. Two replicates of 15 clusters each were prepared per treatment. Two cross-combination treatments were performed: V. rotundifolia × V. vinifera [‘Carlos’ × ‘Zijinqiunong’ (CZ)] and V. rotundifolia × V. rotundifolia [‘Carlos’ × ‘Carlos’ (CC)]. Pollination was executed during the morning between 0700 and 0900 hr by gently touching the stigma with a fine brush dipped in pollen grains. Stigmas from 10 clusters per replicate were cut with a scalpel 8 h after pollination, immediately frozen in liquid nitrogen, and stored at −80 °C for proteome analysis. The entire processes of self-pollination and cross-pollination were repeated in May 2021 to collect stigmas for quantitative real-time polymerase chain reaction (PCR).
Stigmas were randomly harvested 2 and 8 h after pollination in each treatment, immediately fixed in a solution of formaldehyde, acetic acid, and 70% ethanol (1:1:18 v/v/v), and stored at 4 °C. The stigmas were treated with a decolorizing solution (ultrapure water: glycerinum: lactic acid: phenol: 75% ethanol, 1:1:1:1:8 v/v/v/v/v) (Servicebio, Wuhan, China). After decolorizing, the stigmas were embedded in paraffin, dewaxed, and stained with 0.01% aniline blue. The samples were observed under a microscope (Eclipse E100; Nikon, Tokyo, Japan) equipped with a 4′,6-diamidino-2-phenylindole filter block.
Stigmas (100 mg) were ground into a powder in liquid nitrogen for protein extraction. Powdered samples were added to four volumes of lysis buffer [8 m urea, 1% protease inhibitor, 50 μm (PR-619; Selleck, Shanghai, China)] and lysed with ultrasound. Debris was removed by centrifugation at 12,000 gn at 4 °C for 10 min. The supernatant was collected to measure protein concentration using a bicinchoninic acid kit (Beyotime Biotechnology, Shanghai, China). For digestion, the protein solution was reduced with 5 mm dithiothreitol for 30 min at 56 °C and alkylated for 15 min at room temperature in the dark. The protein samples were diluted by adding 200 mm tetraethylammonium bromide. Trypsin was added initially in a ratio of 1:50 to the total protein mass for overnight digestion, and then in a ratio of 1:100 to protein mass and digested for 4 h.
The mobile phase was a mixture of 0.1% formic acid and 2% acetonitrile in water (eluent A) and 0.1% formic acid in acetonitrile (eluent B). Liquid chromatography tandem mass spectrometry (MS/MS) was conducted using an ultra-high-performance liquid chromatography system (nanto Elute; Bruker Daltonics, Bremen, Germany) with the following gradient program: 6% to 22% B (0–43 min), 22% to 30% B (13 min), 30% to 80% B (2 min), and 80% B (2 min) at a flow rate of 300 nL⋅min−1. The peptides were determined by four-dimensional (4D) label-free quantitative protein. The 4D label-free quantitative method is highly sensitive and adds 4D-ion mobility (Meier et al., 2018). The peptides were subjected to a capillary source, followed by MS/MS (timsTOF Pro; Bruker Daltonics).
The resulting MS/MS data were processed using MaxQuant version 1.6.6.0 (Max Planck Institute of Biochemistry, Munich, Germany). Trypsin/P was specified as a cleavage enzyme, allowing up to two missing cleavages. The mass tolerance for precursor ions was set as 0.002% in the first search, and as 0.002% in the main search. Carbamidomethyl on cysteine was specified as a fixed modification, and acetyl (protein N-term), oxidation, and deamidation were specified as variable modifications. The quantitative method was set as label-free quantitation. The false discovery rate of protein identification and peptide-spectrum matches were set at 1%.
Fold differences (CZ:CC ratio) of more than 1.5 was as the threshold for significant up-accumulation, less than 1/1.5 was as the threshold for significant down-accumulation. The coefficient of variation values of the proteins in the two samples indicated the significance of DEPs.
A heatmap of DEPs was created using the BMK cloud. Gene ontology (GO) annotations were classified into three categories: biological process, cellular compartment, and molecular function. Statistical significance was set at P < 0.05. The GO and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed using TBtools (Chen et al., 2020).
Total RNA was extracted using a Plant RNA Kit (Omega Bio-tek, Norcross, GA) and reverse-transcribed with RTIII All-in-One Mix with dsDNase (Monad, Wuhan, China). Gene expression patterns were analyzed using the CFX Connect Real-Time PCR Detection System (Bio-Rad, Hercules, CA). SYBR Green qPCR mix (Monad) was used for the quantitative PCR. The actin gene was used as the reference gene. Primer sequences for candidate genes are listed in Supplemental Table 1. The relative expression was calculated using the 2−ΔΔCt method (Livak and Schmittgen, 2001).
At 2 h after pollination, there were no differences between the CC and CZ treatments (Fig. 1A and 1C). At 8 h after pollination, the pollen tube elongated and grew vigorously in the CC treatment (Fig. 1B). In contrast, pollen tube elongation ended, and no obvious pollen tube was found in the CZ treatment (Fig. 1D).
Citation: Journal of the American Society for Horticultural Science 147, 3; 10.21273/JASHS05153-21
A total of 937,664 spectra were identified by MS, of which 168,111 spectra matched known spectra and 39,964 were unique peptides. A total of 7032 proteins were obtained and 5516 proteins were quantified (Supplemental Table 2).
The expression of 32 proteins was significantly different between the CZ and CC treatments. Sixteen proteins were upregulated (Table 1) and 16 proteins were downregulated (Table 2). The DEPs between the CC and CZ treatment 8 h after pollination clustered into four groups in the heatmap (Fig. 2). Group I consisted of one protein. Group II consisted of 15 proteins. Group III included five proteins. Group IV comprised 11 proteins. Compared with CC, the protein expression in groups I and II was upregulated, and that in groups III and IV was downregulated in CZ.
Citation: Journal of the American Society for Horticultural Science 147, 3; 10.21273/JASHS05153-21
The GO analysis was performed separately for each cluster (groups I, II, III, and IV). Significant differences observed in the top 10 DEPs of each group are displayed in Figs. 3 and 4. In group I, the most significantly enriched GO term was ATPase-coupled transmembrane transporter activity, followed by primary active transmembrane transporter activity and active transmembrane transporter activity. All the terms were related to transmembrane transporter activity (Fig. 3). In group II, the most significantly enriched GO term was extracellular region, followed by calcium-dependent phospholipase A2 activity. Another GO term was related to eicosanoid transport and secretion, arachidonic transport and secretion, and fatty acid transport (Fig. 3). In group III, the most significantly enriched GO term was DNA unwinding involved in DNA replication. Most of the terms in this group were related to DNA and included single-strand DNA binding, double-strand break repair via homologous recombination, and others (Fig. 4). In group IV, the most significantly enriched GO term was cellular carbohydrate biosynthetic process, followed by carbohydrate biosynthetic process, hexosyltransferase activity, trehalose-phosphatase activity, and 1,4-alpha-glucan branching enzyme activity (Fig. 4).
Citation: Journal of the American Society for Horticultural Science 147, 3; 10.21273/JASHS05153-21
Citation: Journal of the American Society for Horticultural Science 147, 3; 10.21273/JASHS05153-21
The KEGG pathway analysis of the 32 DEPs demonstrated that these proteins were significantly enriched in two pathways, namely, DNA replication (D7SU26, F6HJI1) and starch and sucrose metabolism (D7TJ35, A5AY88) (Fig. 5).
Citation: Journal of the American Society for Horticultural Science 147, 3; 10.21273/JASHS05153-21
Five downregulated proteins were selected to verify their transcript levels using quantitative reverse-transcription PCR (qRT-PCR). Among them, four proteins (A5AY88, D7TJ35, D7SU26, and F6HJI1) were correlated with the KEGG pathway. The relative expression of selected proteins was consistent with the results of the proteome analysis (Fig. 6).
Citation: Journal of the American Society for Horticultural Science 147, 3; 10.21273/JASHS05153-21
In studies on breeding compatibility, a histological analysis can provide key clues to determine the reason for reproductive obstacles. Previous studies have shown that incompatibility reactions are caused by prevention of pollen germination, pollen tube growth arrest, or pollen tube navigation during its growth (He et al., 2019; Kacar et al., 2015; Okamoto and Ureshino, 2015; Varotto et al., 1995). In a recent study, callus tissue that developed in the stigma and style blocked the development of the pollen tube and led to incompatibility of Passiflora species (P. alata, P. cincinnata, P. edulis, P. gibertii, P. mucronata) and changed the direction of crossings (Soares et al., 2020). Lu and Lamikanra (1996) showed that the failure of V. rotundifolia × V. vinifera was caused by the abortion of pollen tubes. In the present study, a histological analysis of V. rotundifolia ‘Carlos’ showed that pollen tube growth was inhibited after cross-pollination with V. vinifera ‘Zijinqiunong’. This result is in line with that of Lu and Lamikanra (1996).
Carbohydrates are an important energy source and have a crucial role in the reproductive process. Pollen grains and pollen tubes are responsible for delivering sperm cells to the embryo (Malhó et al., 2006). Pollen grain germination uses stored carbohydrates (mainly starch), whereas pollen tube growth is rapid and requires the intake of external carbohydrates (mainly sucrose) (Ruan, 2014). Various studies have demonstrated the role of sucrose in pollen tube growth. Dinesh et al. (2007) showed that sucrose can break the intergeneric barrier in papaya (Carica papaya) by enhancing pollen germination and pollen tube growth. Apart from its major role as an energy source, sucrose is also an osmotic solute and a signaling molecule in pollen grain germination and pollen tube growth (Ruan, 2014). Any factor that affects sucrose metabolism including the distribution of sucrose synthase (Çetinbaş-Genç et al., 2020) or the synthesis and regulation of sugar transporter proteins (Li et al., 2020; Rottmann et al., 2018) can alter pollen tube growth. In the present study, the downregulation of D7TJ35 [glycogen branching enzyme 1 (GBE1)] in the cross-pollinated (CZ) stigma was identified and confirmed by qRT-PCR. According to the result of the KEGG analysis, D7TJ35 (GBE1) is involved in the starch and sucrose metabolism. The pollen tube arrest observed in our study may be regulated by D7TJ35 through sucrose metabolism.
Trehalose and sucrose metabolism are tightly correlated (Hirsche et al., 2017). Trehalose 6-phosphate (T6P), which is indispensable for the development of Arabidopsis thaliana, acts as an indicator of sucrose availability (Schluepmann et al., 2003). It can inhibit sucrose nonfermenting-1-related kinase1 (SnRK1), which regulates key genes involved in the allocation (sugars will eventually be exported transporters proteins) and use (starch biosynthesis) of sucrose, and affects the regulation of carbon allocation and utilization in plants (Chen and Wang, 2008; Chen et al., 2019). A feedback loop likely operates where SnRK1 phosphorylates bZIP11, which regulates the expression of T6P phosphatase (TPPs), and other trehalose-6-phosphate synthases (TPSs) are regulated by SnRK1 as established by SnRK1 marker genes (Paul et al., 2018). TPS generates T6P from glucose-6-phosphate and UDP-glucose, with subsequent dephosphorylation of T6P to trehalose by TPP. In the stigma of the CZ treatment, the expression of A5AY88 (TPS) was downregulated. Downregulation of TPS may affect pollen tube growth via sucrose metabolism, which is regulated by the trehalose pathway. Trehalose can also directly affect pollen development. Chiang (1974) showed that trehalose supported raffinose pollen germination and pollen tube growth, but the germination percentage and tube length were lower than those induced by sucrose. Another study demonstrated similar effects of trehalose on pollen germination, but it was a poor substitute for sucrose in pollen germination media (Hirsche et al., 2017). We conclude that the effect of A5AY88 (TPS) on pollen tube growth may be correlated with the sucrose pathway.
Previous studies have shown that DNA replication has a role in pollen development. The DNA-binding protein WHIRLY2 (AtWHY2) affects the mitochondrial DNA copy number in pollen vegetative cells, resulting in decreased mitochondrial respiration and pollen tube growth (Cai et al., 2015). A mutant bicellular pollen1 can prolong DNA synthesis in the generative cell and result in arrested pollen development (Long et al., 2018). In the present study, we identified two DNA replication-related proteins, namely, D7SU26 (RFA2) and F6HJI1 (MCM6), that were downregulated in the CZ treatment when compared with that in CC, which was grouped in map03030 DNA replication (Fig. 5). RFA2 is a single-strand DNA-binding protein that is regulated by phosphorylation and has a key role in DNA replication (Wang et al., 2013). MCM6 is a key DNA replication regulator that provides a DNA unwinding function (Dang et al., 2011). ZmmCM6 is strongly induced after fertilization but present at low levels in vegetative tissues; its downregulation seems to affect pollen development (Dresselhaus et al., 2006). Therefore, the downregulation of D7SU26 and F6HJI1 may affect pollen tube growth by participating in DNA replication.
Callose is the major component of the pollen tube wall that strengthens the cell wall (Williams, 2008; Winship et al., 2011). The precursor of callose is UDP-glucose, which can be obtained from glucose and is also synthesized through the action of sucrose synthase (Persia et al., 2008). Proteomic analysis and qRT-PCR results showed that F6GUE7 was downregulated in the CZ cross-pollinated stigma compared with that of the CC cross. As discussed, sucrose and starch pathways were also downregulated in the CZ treatment. We conclude that the downregulation of callose may be correlated with sucrose metabolism.
The results of the present study indicate that arrested pollen tube growth was the main cause of cross incompatibility between V. rotundifolia and V. vinifera. DNA replication and the sucrose metabolic pathway may have key roles in cross-incompatibility reactions, with the related proteins (A5AY88, D7TJ35, D7SU26, F6HJI1, and F6GUE7) potentially contributing to pollen tube arrest.
Compared with transcriptome and metabolomic methods, proteomics has an advantage in terms of the sampling amount. Proteomics is a suitable method of studying grape cross incompatibility because of the small sample size. However, the combined analysis of multiple omics could provide more insights into the distant hybridization between V. rotundifolia and V. vinifera. The regulation or mechanisms of related proteins should be studied using multiomics methods.
Histological observation of Vitis rotundifolia ‘Carlos’ × V. rotundifolia ‘Carlos’ (CC) and V. rotundifolia ‘Carlos’ × V. vinifera ‘Zijinqiunong’ (CZ). Self-pollen germination and pollen tube at 2 h (A) and 8 h (B) after pollination. Cross-pollen germination and the pollen tube at 2 h (C) and 8 h (D) after pollination. Arrows indicate the pollen tube.
Heatmap with hierarchical clustering of differentially expressed proteins (DEPs) in Vitis rotundifolia ‘Carlos’ × V. rotundifolia ‘Carlos’ (CC) and V. rotundifolia ‘Carlos’ × V. vinifera ‘Zijinqiunong’ (CZ). Two independent biological replicates were collected from CC (CC-1, CC-2) and CZ (CZ-1, CZ-2). Red and orange indicate high expression and low expression according to the color bar. The DEPs clustered into four groups (groups I, II, III, and IV).
Gene ontology (GO) terms (top 10) of upregulated differentially expressed proteins in group I (or group II) between Vitis rotundifolia ‘Carlos’ × V. rotundifolia ‘Carlos’ (CC) and V. rotundifolia ‘Carlos’ × V. vinifera ‘Zijinqiunong’ (CZ). Group I and group II were clustered according to the results of heatmap.
Gene ontology (GO) terms (top 10) of downregulated differentially expressed proteins in group I (or group II) between Vitis rotundifolia ‘Carlos’ × V. rotundifolia ‘Carlos’ (CC) and V. rotundifolia ‘Carlos’ × V. vinifera ‘Zijinqiunong’ (CZ). Group III and group IV were clustered according to the results of heatmap.
Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway of differentially expressed proteins in Vitis rotundifolia ‘Carlos’ × V. rotundifolia ‘Carlos’ (CC) and V. rotundifolia ‘Carlos’ × V. vinifera ‘Zijinqiunong’ (CZ).
Expression level of differentially expressed proteins genes in Vitis rotundifolia ‘Carlos’ × V. rotundifolia ‘Carlos’ (CC) and V. rotundifolia ‘Carlos’ × V. vinifera ‘Zijinqiunong’ (CZ). *Significant difference at P < 0.05.
Contributor Notes
This research was supported by Exploring and Overturning Innovation Programs of Jiangsu Academy of Agricultural Sciences (ZX(21)1208), Jiangsu Province Breeding Project of Agricultural Leading New Varieties (PZCZ201722), and China Agriculture Research System of MOF and MARA.
W.W. is the corresponding author. E-mail: 5wm@163.com.
Histological observation of Vitis rotundifolia ‘Carlos’ × V. rotundifolia ‘Carlos’ (CC) and V. rotundifolia ‘Carlos’ × V. vinifera ‘Zijinqiunong’ (CZ). Self-pollen germination and pollen tube at 2 h (A) and 8 h (B) after pollination. Cross-pollen germination and the pollen tube at 2 h (C) and 8 h (D) after pollination. Arrows indicate the pollen tube.
Heatmap with hierarchical clustering of differentially expressed proteins (DEPs) in Vitis rotundifolia ‘Carlos’ × V. rotundifolia ‘Carlos’ (CC) and V. rotundifolia ‘Carlos’ × V. vinifera ‘Zijinqiunong’ (CZ). Two independent biological replicates were collected from CC (CC-1, CC-2) and CZ (CZ-1, CZ-2). Red and orange indicate high expression and low expression according to the color bar. The DEPs clustered into four groups (groups I, II, III, and IV).
Gene ontology (GO) terms (top 10) of upregulated differentially expressed proteins in group I (or group II) between Vitis rotundifolia ‘Carlos’ × V. rotundifolia ‘Carlos’ (CC) and V. rotundifolia ‘Carlos’ × V. vinifera ‘Zijinqiunong’ (CZ). Group I and group II were clustered according to the results of heatmap.
Gene ontology (GO) terms (top 10) of downregulated differentially expressed proteins in group I (or group II) between Vitis rotundifolia ‘Carlos’ × V. rotundifolia ‘Carlos’ (CC) and V. rotundifolia ‘Carlos’ × V. vinifera ‘Zijinqiunong’ (CZ). Group III and group IV were clustered according to the results of heatmap.
Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway of differentially expressed proteins in Vitis rotundifolia ‘Carlos’ × V. rotundifolia ‘Carlos’ (CC) and V. rotundifolia ‘Carlos’ × V. vinifera ‘Zijinqiunong’ (CZ).
Expression level of differentially expressed proteins genes in Vitis rotundifolia ‘Carlos’ × V. rotundifolia ‘Carlos’ (CC) and V. rotundifolia ‘Carlos’ × V. vinifera ‘Zijinqiunong’ (CZ). *Significant difference at P < 0.05.
