Pollen germination
Pollen germination tests were carried out on pollen on the in vitro germination medium, and pollen germination rate was counted and expressed as a percentage. The results showed the pollen germination rate of ZB pollen increased from 4.30% after 3 h to 10.70% after 12 h (Figure 1 and Table 1). In general, the germination rate of ZB pollen is low, which presents a huge obstacle to sexual reproduction in this species.
Figure 1. In vitro germination and pollen morphology of Zanthoxylum bungeanum pollen. (a) Pollen germination In vitro for 3 h, (b) Pollen germination In vitro for 6 h, (c) Pollen germination In vitro for 12 h, (d) anther, (e) Pollen grain, (f) Pollen grain.
Table 1. Germination rate of Zanthoxylum pollen in vitro.
Time (h)
|
3
|
6
|
12
|
Germination rate (%)
|
4.30±0.30
|
6.20±0.50
|
10.70±1.40
|
Figure 2. Germination of pollen on the stigma of Zanthoxylum bungeanum. ca: callosum, ce:cellulose, ow: ovary wall,po:pollen, pt:pollen tube, sdp: stigma drop position.
Fluorescence microscopy shows pollen germinates on the stigma of ZB. Pollen tube growth commenced by 6 h (Figure 2). After 12 h pollen tube began to extend. After 2 d, the pollen tube penetrated the ovary wall. By 3 d after pollination the stigma begins to wither. Stigma fluorescence gradually dims during the withering process. After 4 d, the stigma falls off but the pollen tube in the ovary wall can still extend further downwards. After 5 d, the pollen tube is broken down into callosum so the fertilization process cannot be completed. We observed 300 samples and found no successful fertilization in any of them. It is thus preliminarily concluded that the reproductive mode of ZB cv. ‘Hancheng Dahongpao’ is obligate apomixis.
Path enrichment analysis
The pathway enrichment analysis of differential genes reveals changes before and after pollination. It can be seen from Figure 3 that pollination has a significant effect on the various pathways of ZB than unpollinated. After pollination, hormone signal transduction is most active, as are metabolic pathways including starch and sucrose metabolism, glycolysis/gluconeogenesis and phenylpropanoid synthesis. Figure 3b shows the pathway of increased expression after pollination, and Figure 3c shows the pathway of decreased expression after pollination. More than 100 genes were enriched and show elevated expression levels after pollination in the hormone signal transduction pathway. Meanwhile, starch and sucrose metabolism, glycolysis/gluconeogenesis, amino sugar and nucleotide metabolism all showed significant downward trends.
Figure 3. Path enrichment analysis. (a) Total pathway enrichment analysis. (b) Active pathway after pollination. (c) Inactive pathway after pollination.
Figure 4. Differential gene Venn diagram and volcano map.
Transcriptome sequencing of pollinated and unpollinated fruits showed that a total of 69,131 reads were detected. Of these 4,431 were unique to unpollinated material and 4,078 were unique to pollination material (Figure 4). As indicated in the volcano map 7,102 genes were up-regulated and 6491 genes were down-regulated. From this it can be inferred that pollination has a strong influence on transcriptome level in ZB fruits.
miRNAs and target genes expression patterns
By analyzing interactions between miRNAs and mRNA in pollinated and non-pollinated fruit, it was found that pollinated fruits were more active in a range of hormone signaling pathways. Pollination can activate ABA, IAA, GA3, jasmonic acid synthesis genes and genes associated with asexual development, such as somatic embryogenesis receptor kinase 2 (SERK2), agamous-like MADS-box protein (AGL9), also activating a large number of flower development genes and transcription factors. To further determine the relationship of miRNAs to target genes, RT-qPCR was used to detect relative expression levels of miRNAs and their target genes.
According to the results of the RT-qPCR, the reliability of transcriptome and miRNA sequencing results can be determined and the relative expression levels of some miRNAs and their target genes after pollination were confirmed (Figure 5). The results show the expression levels of the ABA, IAA, GA3 and JA synthesis genes and some transcription factors and their corresponding miRNAs were negatively correlated after pollination. This indicates miRNAs and mRNAs are widely involved in the regulation of hormones after pollination of this apomictic Chinese prickly ash cultivar.
Figure 5. Relative expression levels of miRNAs and their target genes.
Hormone content analysis
The transcriptome and miRNA sequencing shows many hormone pathways became active after pollination. We recorded the levels of four hormones ABA, IAA, GA3 and JA were for pollinated and unpollinated materials. The results show the four hormones were significantly higher in the pollination material than in the unpollinated (Figure 6). This indicates pollination can activate the synthetic pathway of these four hormones, it also suggests these four hormones participate in the development of embryos and fruits of ZB and, ultimately, in fruit set.
Figure 6. Hormone content of unpollinated and pollinated material of Zanthoxylum bungeanum.
Comparing the pollinated with the unpollinated ZB material (Table 2), it is clear that pollination significantly increased fruit set rate but had little effect on fruit size.
Table 2. Pollination and non-pollination fruit set rate and fruit size.
Samples
|
Non-pollination
|
Pollination
|
Fruit set rate
|
74.12% b
|
89.31% a
|
Fruit length (axial)
|
5.83±0.72 a
|
5.85±0.56 a
|
Fruit diameter (transverse)
|
5.59±0.81 a
|
5.60±0.88 a
|
Conclusion
Pollen of ZB was cultured in vitro on a solid culture medium for 12 h. At this stage germination percentage was only 10.77%. Fluorescence microscopy showed that the pollen does germinate on the ZB stigma and that the pollen tube extended to the ovary after 2 d but then degenerated to callosum after 5 d without fertilization. The reproductive mode of ZB (Hancheng Dahongpao) is identified as obligate apomixis. Enrichment analysis of differentially expressed genes in the pollination and non-pollination materials indicate plant hormone signaling pathways were activated by fertilization. The relative expressions of ABA, IAA, GA3 and JA were up-regulated. At the same time, genes related to asexual development were also activated, including somatic embryogenesis receptor kinase 2 (SERK2) and agamous-like MADS-box protein (AGL9). A large number of other genes and transcription factors related to flower development were also activated. RT-qPCR showed the relative expression levels of mRNAs and miRNAs related to the hormone signaling pathway and to flower development were negatively correlated, suggesting miRNA is an important factor influencing hormone regulation after pollination of apomictic ZB. Comparison of pollination and non-pollination materials showed increases in the contents of ABA, IAA, GA3 and JA. Nevertheless, although pollination did not result in fertilization, it did increase fruit set significantly.