Identification of MicroRNAs Related to Phytohormone Signal Transduction and Self-incompatibility of Rabbiteye Blueberry Pollen

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Qin YangCollege of Life and Health Science, Kaili University, Kaili 556000, China

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Yan FuQiandongnan National Polytechnic, Kaili 556000, China

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Tingting ZhangCollege of Life and Health Science, Kaili University, Kaili 556000, China

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Shu PengCollege of Life and Health Science, Kaili University, Kaili 556000, China

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Jie DengCollege of Life and Health Science, Kaili University, Kaili 556000, China

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MicroRNAs (miRNAs) related to phytohormone signal transduction and self-incompatibility may play an important role in the xenia effect. However, associated research in this area is still lacking in rabbiteye blueberry (Vaccinium ashei). In this study, we identified miRNAs, predicted their target genes, performed functional enrichment analysis of the target genes, and screened for miRNAs related to phytohormone signaling and self-incompatibility. A total of 491 miRNAs were identified, of which 27 and 67 known miRNAs as well as 274 and 416 new miRNAs were found in the rabbiteye blueberry cultivars Brightwell and Premier, respectively. Compared with ‘Premier’, 31 miRNAs were upregulated and 62 miRNAs were downregulated in ‘Brightwell’. Kyoto Encyclopedia of Genes and Genomes (KEGG) functional enrichment analysis indicated that the 4985 target genes predicted were involved in biosynthesis of amino acids, plant–pathogen interaction, and spliceosome pathways. A total of 10, one, one, five, two, five, and two candidate miRNAs related to auxin, cytokinin, gibberellin, abscisic acid, ethylene, brassinosteroid, and salicylic acid signaling, respectively, in rabbiteye blueberry pollen were identified. Further analysis indicated that novel_miR_49 was a candidate miRNA related to self-incompatibility, and their target gene was maker-VaccDscaff21-snap-gene-21.37. In addition, the KEGG enrichment analysis of the target genes of novel_miR_49 showed that they were involved in the ribosome, aminoacyl-tRNA biosynthesis, and glycosylphosphatidylinositol-anchor biosynthesis pathways. The results revealed that the microRNAs of rabbiteye blueberry pollen regulated to phytohormone signal transduction and self-incompatibility signal transduction based on related to auxin, cytokinin, gibberellin, abscisic acid, ethylene, brassinosteroid, and salicylic acid signaling. Results suggest that more research of the effects of miRNAs on regulation of hormone signal transduction and self-incompatibility is necessary for elucidating the molecular mechanism of the xenia effect.

Abstract

MicroRNAs (miRNAs) related to phytohormone signal transduction and self-incompatibility may play an important role in the xenia effect. However, associated research in this area is still lacking in rabbiteye blueberry (Vaccinium ashei). In this study, we identified miRNAs, predicted their target genes, performed functional enrichment analysis of the target genes, and screened for miRNAs related to phytohormone signaling and self-incompatibility. A total of 491 miRNAs were identified, of which 27 and 67 known miRNAs as well as 274 and 416 new miRNAs were found in the rabbiteye blueberry cultivars Brightwell and Premier, respectively. Compared with ‘Premier’, 31 miRNAs were upregulated and 62 miRNAs were downregulated in ‘Brightwell’. Kyoto Encyclopedia of Genes and Genomes (KEGG) functional enrichment analysis indicated that the 4985 target genes predicted were involved in biosynthesis of amino acids, plant–pathogen interaction, and spliceosome pathways. A total of 10, one, one, five, two, five, and two candidate miRNAs related to auxin, cytokinin, gibberellin, abscisic acid, ethylene, brassinosteroid, and salicylic acid signaling, respectively, in rabbiteye blueberry pollen were identified. Further analysis indicated that novel_miR_49 was a candidate miRNA related to self-incompatibility, and their target gene was maker-VaccDscaff21-snap-gene-21.37. In addition, the KEGG enrichment analysis of the target genes of novel_miR_49 showed that they were involved in the ribosome, aminoacyl-tRNA biosynthesis, and glycosylphosphatidylinositol-anchor biosynthesis pathways. The results revealed that the microRNAs of rabbiteye blueberry pollen regulated to phytohormone signal transduction and self-incompatibility signal transduction based on related to auxin, cytokinin, gibberellin, abscisic acid, ethylene, brassinosteroid, and salicylic acid signaling. Results suggest that more research of the effects of miRNAs on regulation of hormone signal transduction and self-incompatibility is necessary for elucidating the molecular mechanism of the xenia effect.

Blueberry (Vaccinium sp.) fruit are rich in physiologically active substances such as mineral elements, folic acid, vitamins, flavonoids, anthocyanins, and catechin, which are beneficial to human health as dietary antioxidants (Yang et al., 2019). Its fruit extracts have been developed as components of functional foods and dietary supplements (Dróżdż et al., 2018). The rich nutrients and unique flavor make blueberries popular whether consumed as a fresh fruit or as a functional health product (Yang et al., 2019). In the past decade, due to longer growing seasons and high economic returns, the cultivation area of blueberry in China has increased significantly, and China has become a major producer of blueberries worldwide (Yu et al., 2016). At present, commercially cultivated types of blueberry are mainly tetraploid northern highbush blueberry (Vaccinium corymbosum) and southern highbush blueberry (V. corymbosum interspecific hybrids), diploid and tetraploid lowbush blueberry (Vaccinium angustifolium), and hexaploid rabbiteye blueberry [Vaccinium ashei (Rowland et al., 2012)]. Most cultivars are self-incompatible (Chavez and Lyrene, 2009; Ehlenfeldt and Kramer, 2012; Miller et al., 2011; Yang et al., 2017).

Self-incompatibility is an important mechanism by which higher plants achieve genetic recombination and maintain genetic variation (Yang et al., 2015a). According to the difference in genetic determination, self-incompatibility is divided into sporophytic self-incompatibility (SSI) and gametophytic self-incompatibility (GSI) (Yang and Fu, 2015). The phenotype of SSI is determined by the S genotype of the sporophyte, showing either germination failure of pollen on the surface of stigma papillary cells or abnormal growth of germinated pollen, with the abnormal pollen failing to enter the style tissue. The phenotype of GSI is determined by the haploid genes of the pollen itself, and the pollen tube usually stops growing in the style tissue or between the pollen tube and the embryo sac tissue (Sassa, 2016). Previous studies have shown that GSI exists in many fruit trees including European pear [Pyrus communis (Quinet et al., 2014)], apple [Malus pumila (Li et al., 2011)], plum [Prunus salicina (Nantongo et al., 2016)], loquat [Eriobotrya japonica (Yang et al., 2018)], strawberry [Fragaria ×ananassa (Du et al., 2021)], and pummelo [Citrus maxima (Hu et al., 2021)]. If the S allele of pollen is identical to one of the S alleles in the style, the growth of the pollen tube in the style is inhibited and fertilization fails, which seriously reduces fruit yield (Yang et al., 2015a). Either artificial pollination during flowering or planting trees of an appropriate cultivar to provide pollen is required in commercial production (Zhang et al., 2006).

Therefore, for a long time, multiple cultivars were used in blueberry cultivation to guarantee a high fruit setting rate and yield (Chavez and Lyrene, 2009; Miller et al., 2011; Yang et al., 2015a). Several studies have shown that pollination with different genotypes of pollen significantly affects blueberry fruit ripening time (Miller et al., 2011), fruit size (Yang et al., 2015b, 2015c), and fruit flavor and nutritional quality, showing an evident xenia effect (Yang et al., 2017). Xenia refers to the phenomenon that the pollen genotype directly affects seed and fruit development during the period from fertilization to seed germination, which leads to different characteristics in phenotypic traits, such as the fruit-ripening period; fruit shape, size, and color; the flavor quality, such as sugars and acids; as well as the nutrient quality, such as anthocyanins (Yang et al., 2020a, 2020b). Therefore, the study of the xenia effect in blueberry can inform production by providing theoretical guidance for the selection of pollen-supplying cultivars, increasing yield, and improving the internal and external quality of the fruit. It is also valuable for theoretical research regarding the genetics, physiology, and breeding of blueberry (Liu, 2008; Yang et al., 2020b). However, research on the blueberry xenia effect has mainly focused on the observation of phenotypes, and the mechanism of the xenia effect has not been studied. The pollen genotype is the cause of the xenia effect. The function of pollen is to produce and carry sperm cells into the embryo sac to achieve double fertilization. In this process, the differences in the vigor and endogenous hormones of pollen—which may cause differences in endogenous hormone content in the pollination styles of different cultivars and different degrees of affinity, in turn leading to differences in the number of seeds and endogenous hormones in young fruit pulp—may eventually account for xenia effects (Yang et al., 2020b). Therefore, the difference in vigor and endogenous hormone content of pollen may be one of the key factors that cause the xenia effect.

Previous studies showed that the pollen viability in the rabbiteye blueberry cultivar Premier was significantly lower than that of the rabbiteye blueberry cultivar Brightwell, and in the pollen of Premier, the levels of indole-3-acetic acid (IAA), gibberellic acid (GA3), and zeatin (ZT) were significantly lower than in the pollen of Brightwell. The abscisic acid (ABA) content in the pollen of ‘Premier’ was significantly higher than that in the pollen of ‘Brightwell’ (Yang et al., 2021).

MicroRNAs (miRNAs) are small noncoding, evolutionarily conserved RNAs that are ubiquitous in plants (Chen et al., 2018a) and are highly specific posttranscriptional regulators of eukaryotic genes, participating in the regulation of various biological processes (Chen et al., 2018b; Voinnet, 2009), such as plant hormone signal transduction (Shen et al., 2019), and causing phenotypic changes in developing tissues (Kim et al., 2001; Piotto et al., 2013). miRNAs and their target genes are related to auxin, ABA, and gibberellin signal transduction. They play an important role in the process of apple flower bud differentiation (Xing et al., 2016) and grape (Vitis vinifera) ripening (Zhao et al., 2017) and also participate in hormone pathways during litchi (Litchi chinensis) fruit senescence (Yao et al., 2015).

However, high-throughput sequencing of blueberry pollen, the identification and differential expression analysis of miRNAs, the mining and identification of miRNAs related to phytohormone signal transduction, miRNA target gene prediction, and functional enrichment analysis of the target genes have not been reported. Therefore, in this study, we performed high-throughput sequencing of miRNAs of the pollen of the rabbiteye blueberry cultivars Brightwell and Premier; conducted bioinformatics analysis; carried out miRNA identification, target gene prediction, and functional enrichment analysis; and screened phytohormone signal transduction and self-incompatibility-related miRNAs. The study provides a basis for the future elucidation of the molecular mechanism of the xenia effect and the self-incompatibility of rabbiteye blueberry pollen.

Materials and Methods

Materials and sampling.

The two rabbiteye blueberry cultivars Brightwell and Premier are derived from the common female parent Tifblue (Sakhanokho et al., 2018), and the xenia effect of the two cultivars was significant after cross-pollination. This led to different characteristics in phenotypic traits, such as fruit longitudinal diameter, fruit transverse diameter, single fruit weight, and soluble solid content (Yang et al., 2015b). Therefore, 6-year-old trees of the rabbiteye blueberry cultivars Brightwell and Premier planted in the Agricultural and Forestry Training Base of Kaili University, Kaili, Guizhou, China (lat. 26°31′N, long. 107°53′E) were used in this study. Sixty healthy, disease-free plants of the same size were randomly selected from each cultivar. During the full blooming period in 2019, pollen was collected from the two cultivars with three biological replicates according to a method reported previously (Yang et al., 2015a), and the collected pollen was immediately frozen in liquid nitrogen and then stored at –80 °C for later use.

RNA library construction and sequencing.

The collected pollen of ‘Brightwell’ and ‘Premier’ was sent to Biomarker Technologies (Beijing, China) for RNA extraction using the TRIzol method (Tiangen Biotech, Beijing, China) and treated with RNase-free DNase I (TaKaRa, Dalian, China). RNA degradation and contamination was monitored on 1% agarose gels. RNA was quantified using a bioanalyzer (model 2100; Agilent Technologies, Santa Clara, CA, USA), the quality and integrity were assessed by a spectrophotometer (NanoDrop 2000; Thermo Fisher Scientific, Waltham, MA, USA). The library preparations were sequenced on a sequencing platform (NovaSEq. 6000; Illumina, San Diego, CA, USA) by Biomarker Technologies and paired-end 150-bp reads were generated.

Analysis of miRNA sequences.

Raw reads of each sample were obtained by sequencing, of which the low-quality reads (reads with quality values ≤20, with the number of bases exceeding one, and containing ambiguous base N) were removed to obtain high-quality reads. Reads without the 3' adapter sequence, the adapter sequence on the 5'and 3' ends of the reads, contaminated sequences, and reads shorter than 18 nt or longer than 30 nt were removed to obtain clean reads. The distribution of the length of the obtained tags (18–25 nt) was analyzed. Using Bowtie software [ver. 1.0.0 (Langmead et al., 2009)] with the parameters of –v 0, the clean reads were submitted to the SILVA (Pruesse et al., 2007), GtRNAdb (Chan and Lowe, 2009), Rfam (Griffiths-Jones et al., 2003), and Repbase (Jurka et al., 2005) databases to perform a sequence alignment search. Ribosomal RNA (rRNA), transfer RNA (tRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), and repetitive sequences were removed to obtain unannotated reads containing miRNAs. V. corymbosum from Colle et al. (2019) was used as the reference genome for sequence alignment and subsequent analysis. Bowtie software (ver. 1.0.0) with the parameters of –v 0 was used to align the unannotated reads with the reference genome to obtain mapped reads with location information on the reference genome.

Identification and differential expression analysis of miRNAs.

The reads mapped to the reference genome were aligned (at most one mismatch was allowed) with the mature sequence and the sequence from 2 nt upstream to 5 nt downstream of known miRNAs in the miRBase [ver. 22 (Li et al., 2021)] database to identify known miRNAs. The unidentified miRNAs were predicted using miRDeep2 software [ver. 2.0.5 (Friedländer et al., 2012)] through parameter adjustment (Zhang et al., 2014). The miRNA expression levels were estimated by transcript per million [TPM (Li et al., 2009)]. The miRNAs with |log2(FC)| ≥1.00 [fold change (FC)] and false discovery rate (FDR) ≤0.05 were defined as being differentially expressed using DESeq2 software [ver. 1.6.3 (Love et al., 2014)].

Quantitative real-time polymerase chain reaction validation.

To confirm the sequencing results, quantitative real-time polymerase chain reaction (qRT-PCR) was used to test eight randomly selected miRNAs (novel_miR_268, novel_miR_343, novel_miR_137, novel_miR_117, novel_miR_391, novel_miR_359, novel_miR_76, novel_miR_43) according to previous research methods (Wu et al., 2014). Based on the full miRNA sequences, the forward miRNA primers listed in Table 1 for qRT-PCR were designed using universal reverse primers for miRNA.

Table 1.

Primer sequences of eight microRNAs (miRNAs) randomly screened in the pollen of the rabbiteye blueberry cultivars Brightwell and Premier for quantitative real-time polymerase chain reaction.

Table 1.

Prediction and functional enrichment analysis of miRNA target genes.

TargetFinder software [ver. 1.6 (Allen et al., 2005)] was used to predict the target genes, which were then annotated with the reference genome (Colle et al., 2019) and National Center of Biotechnology Information (Bethesda, MD, USA) nonredundant (Nr) database. After that, Gene Ontology [GO (Ashburner et al., 2000)] and Kyoto Encyclopedia of Genes and Genomes [KEGG (Kanehisa et al., 2004)] analyses of the target genes were performed using clusterProfiler [ver. 3.10.1 (Yu et al., 2012)] with Parameter of pAdjustMethod=fdr firstSigNodes=10.

Screening of miRNAs related to phytohormone signaling.

On the basis of the sequencing result, the differences in the expression level of each miRNA and target gene was calculated by comparing their TPM value between ‘Brightwell’ and ‘Premier’, and the results together with the KEGG metabolic pathway of the target genes, were used to screen for miRNAs related to phytohormone signal transduction and self-incompatibility of rabbiteye blueberry pollen.

Results and Analysis

Analysis of miRNA sequencing data.

Through Illumina high-throughput sequencing, we obtained 23,419,205 and 16,197,862 raw reads from the pollen of ‘Brightwell’ and ‘Premier’, respectively. After the removal of adapter sequences from the 5' and 3' ends, contaminated sequences, and low-quality reads, we obtained 23,408,242 and 16,183,339 clean reads from ‘Brightwell’ and ‘Premier’, respectively (Table 2). We estimated the distribution of the lengths of tag sequences ranging from 18 to 25 nt and found that in both ‘Brightwell’ and ‘Premier’, the tag sequences were mostly 21 and 24 nt but with different ratios. The ratios of the 21 and 24 nt sequences in ‘Brightwell’ were 29.63% and 40.48%, respectively, while the ratios of the 21 and 24 nt sequences in ‘Premier’ were 38.81% and 36.54%, respectively (Fig. 1).

Fig. 1.
Fig. 1.

Distribution of microRNAs with different sequence lengths according to their total reads and unique tags in the pollen of the rabbiteye blueberry cultivars Brightwell and Premier.

Citation: Journal of the American Society for Horticultural Science 147, 6; 10.21273/JASHS05143-21

Table 2.

Sequencing data statistics information table of microRNAs in the pollen of the rabbiteye blueberry cultivars Brightwell and Premier.

Table 2.

Identification and differential expression of miRNA.

As shown in Table 3, the 23,408,242 clean reads from ‘Brightwell’ and the 16,183,339 clean reads from ‘Premier’ were used for alignment analysis with the SILVA, GtRNAdb, Rfam, and Repbase databases. We found that although the reads obtained from both cultivars were mainly rRNAs, the amount and the ratio of rRNAs differed between the two cultivars. The ratio and number of rRNA reads in ‘Brightwell’ and ‘Premier’ were 49.21% (11,519,196) and 45.12% (7,301,922), respectively. In addition, 411,985 (1.76%) and 163,452 (1.01%) tRNAs as well as 18,727 and 8,092 repetitive sequences were found among the clean reads of ‘Brightwell’ and ‘Premier’, respectively. Moreover, we found 11,451,312 and 8,687,216 unannotated reads in ‘Brightwell’ and ‘Premier’, respectively. The unannotated reads were aligned to the V. corymbosum reference genome using Bowtie software, and 1,301,784 (34.88%) and 2,342,682 (43.01%) reads from ‘Brightwell’ and ‘Premier’, respectively, were aligned to the V. corymbosum reference genome. We identified 491 miRNAs in total, of which 301 and 483 miRNAs were from ‘Brightwell’ and ‘Premier’, respectively. Those miRNAs were compared with the V. corymbosum miRNAs in the miRBase (ver. 22), and 27 and 67 known miRNAs were identified in ‘Brightwell’ and ‘Premier’, respectively. Using miRDeep2 (ver. 2.0.5) software, through hairpin structure prediction, we found 274 and 416 new miRNAs in ‘Brightwell’ and ‘Premier’, respectively.

Table 3.

Statistics on microRNAs (miRNAs) classification in the pollen of the rabbiteye blueberry cultivars Brightwell and Premier.

Table 3.

Differential expression analysis of miRNAs was performed based on the threshold |log2(FC)| ≥1.00 and FDR ≤0.05, and the results are shown in Supplemental Table 1. Compared with ‘Brightwell’, ‘Premier’ presented 93 differentially expressed miRNAs, of which 31 were downregulated and 62 were upregulated. The bar chart in Fig. 2 indicates that the eight randomly selected miRNAs (novel_miR_268, novel_miR_343, novel_miR_137, novel_miR_117, novel_miR_391, novel_miR_359, novel_miR_76, and novel_miR_43) were highly expressed, and they showed similar patterns of expression between the qRT-PCR and TPM results, although the values of miRNA expression measured by the two methods varied to some extent. Thus, the qRT-PCR results confirmed the reliability and differences in expression of the miRNAs involved in blueberry pollen identified through high-throughput sequencing.

Fig. 2.
Fig. 2.

Expression differences confirmed by quantitative real-time polymerase chain reaction (qRT-PCR) and comparison with log10 of transcripts per million (TPM). Relative expression differences of eight different conserved microRNAs were verified by qRT-PCR using the 2-ΔΔCt method. For visualization, log10 was applied to compute the TPM data of sequencing results. The paragraph A to H represent the expression profiles of novel_miR_268, novel_miR_343, novel_miR_137, novel_miR_117, novel_miR_391, novel_miR_359, novel_miR_76, and novel_miR_43 in sequence.

Citation: Journal of the American Society for Horticultural Science 147, 6; 10.21273/JASHS05143-21

GO enrichment analysis of differentially expressed miRNA target genes.

A total of 4985 target genes of the differentially expressed miRNAs were predicted using TargetFinder software (ver. 1.6). Through GO annotation, they were classified into three categories: biological process, cellular component, and molecular function (Fig. 3). The target genes involved 20, 16, and 15 GO terms in the biological process, cellular component, and molecular function categories, respectively. In the biological process category, the GO terms were mainly concentrated in RNA secondary structure unwinding, regulation of gene expression, Golgi organization, response to biotic stimulus, and regulation of protein serine/threonine phosphatase activity based on an FDR ≤0.01. In the cellular component category, the GO terms were mainly concentrated in chloroplast, trans-Golgi network, endosome, Golgi transport complex, and small nucleolar ribonucleoprotein complex based on an FDR ≤0.05. In the molecular function category, the GO terms were mainly concentrated in ATP-dependent RNA helicase activity, ABA binding, protein phosphatase inhibitor activity, receptor activity, and transcription regulatory region DNA binding based on an FDR ≤0.01. The KEGG analysis of the target genes showed that the metabolic pathways were enriched mainly in biosynthesis of amino acids, ABC transporters, glycosylphosphatidylinositol (GPI)-anchor biosynthesis, galactose metabolism, and base excision repair based on an FDR ≤0.1.

Fig. 3.
Fig. 3.

Gene Ontology annotation of the predicted target genes of differentially expressed microRNAs in the pollen of the rabbiteye blueberry cultivars Brightwell and Premier using TargetFinder software [ver. 1.6 (Allen et al., 2005)], which were then annotated with the reference genome from Colle et al. (2019) and National Center of Biotechnology Information (Bethesda, MD, USA) nonredundant (Nr) database.

Citation: Journal of the American Society for Horticultural Science 147, 6; 10.21273/JASHS05143-21

Differentially expressed miRNAs and KEGG pathways related to the metabolism of phytohormone signal transduction.

The annotated target genes of the differentially expressed miRNAs were subjected to KEGG analysis, and the pathways related to phytohormone signal transduction metabolism were obtained (Fig. 4), which indicated that the following genes were differentially expressed: auxin resistant 1 (AUX1), auxin/indole acetic acid (AUX/IAA), auxin response factor (ARF), Gretchen Hagen 3 (GH3), and small auxin up RNA (SAUR) in the auxin signaling pathway; type-B Arabidopsis thaliana response regulators (B-ARR) involved in the cytokinin signaling pathway; gibberellin insensitive dwarf 1 (GID1) involved in the gibberellin signaling pathway; pyrabactin resistance/PYR1-like proteins (PYR/PYL), protein phosphatase 2C (PP2C), and ABA response element binding factor (ABF) involved in the ABA signaling pathway; Ein3-binding F-box1/F-box2 (EBF1/2) involved in the ethylene signaling pathway; BR-signaling kinases (BSK), brassinazole resistant 1/2 (BZR1/2), and cyclins D3 (CYCD3) involved in the brassinolide signaling pathway; and nonexpresser of PR genes1 (NPR1) involved in the salicylic acid (SA) signaling pathway.

Fig. 4.
Fig. 4.

Differentially expressed microRNAs (miRNAs) and Kyoto Encyclopedia of Genes and Genomes pathway annotations of phytohormone signal transduction–related metabolism in the pollen of rabbiteye blueberry cultivars Brightwell and Premier using clusterProfiler [ver. 3.10.1 (Yu et al., 2012)] with Parameter of pAdjustMethod=fdr firstSigNodes=10. The purple boxes in the figure indicate that the regulatory factors denoted by letters within the box are associated with the target genes of the differential miRNA. AUX1 = auxin resistant 1; TIR1 = transport inhibitor response 1; AUX/IAA = auxin/indole acetic acid; ARF = auxin response factor; GH3 = Gretchen hagen 3; SAUR = small auxin up RNA; CRE1 = cytokinin response 1; AHP = Arabidopsis thaliana histidine phosphotransfer proteins; B-ARR = type-B A. thaliana response regulators; A-ARR = type-A A. thaliana response regulators; GID1 = gibberellin insensitive dwarf 1; GID2 = gibberellin insensitive dwarf 2; DELLA = aspartic acid–glutamic acid–leucine–leucine–alanine is a group of the GIBBERELIC ACID INSENSITIVE REPRESSOR OF ga1–3 SCARECROW family of plant-specific transcriptional regulators that act as master negative regulators of gibberellin signaling; TF = transcription factor; PYR/PYL = pyrabactin resistance/PYR1-like proteins; PP2C = protein phosphatase 2C; SnRK2 = sucrose non-fermenting-1 related protein kinase 2; ABF = ABA response element binding factor; ETR = ethylene receptor; CTR1 = constitutive triple-response 1; SIMKK = mitogen-activated protein kinases kinases; MPK6 = mitogen-activated protein kinase 6; EIN2 = ethylene-insensitive protein 2; EIN3 = ethylene-insensitive protein 3; EBF1/2 = Ein3-binding F-box1/F-box2; ERF1/2 = ethylene response factor 1/2; BAK1 = brassinosteroid insensitive 1 associated kinase receptor 1; BRI1 = brassinosteroid-insensitive 1; BKI1 = brassinosteroid-insensitive 1 kinase inhibitor 1; BSK = BR-signaling kinases; BSU1 = bri1 suppressor 1; BIN2 = brassinosteroid-insensitive 2; BZR1/2 = brassinazole resistant 1/2; TCH4 = Touch 4, a xyloglucan endotransglucosylase; CYCD3 = cyclins D3; JAR1 = jasmonate-resistant 1; JA-Ile = jasmonyl-isoleucine; COI1 = coronatine-insensitive 1; JAZ = jasmonate zim-domin; MYC2 = myelocytomatosis proteins 2; ORCA3 = octadecanoid-derivative responsive catharanthus AP2-domain; NPR1 = nonexpressor of pathogenesisrelated genes 1; TGA = TGACG motif-binding factor; PR1 = pathogenesis related genes 1.

Citation: Journal of the American Society for Horticultural Science 147, 6; 10.21273/JASHS05143-21

As shown in Supplemental Table 2, analysis of the miRNAs and target genes related to the differentially expressed transcription factors of auxin signal transduction showed that the transcription factor AUX1 was regulated by 10 novel miRNAs. Additionally, the KEGG and Pfam annotations showed that the 10 target genes of these five novel miRNAs belong to the AUX1 LAX family and are associated with auxin transport. Meanwhile, the transcription factor AUX/IAA was regulated by four novel miRNAs. The KEGG and Pfam annotations showed that their 15 target genes belong to the AUX/IAA family and are associated with auxin response factor. In addition, the transcription factor GH3 was regulated by novel_miR_343, and novel_miR_49. The KEGG and Pfam annotations showed that their two target genes belong to the auxin responsive GH3 gene family and are associated with auxin-responsive promoter. The transcription factor SAUR was regulated by novel_miR_76. The KEGG and Pfam annotations showed that their target genes belong to the SAUR family protein and are associated with auxin-responsive.

As shown in Supplemental Table 3, analysis of the miRNAs and target genes related to the differentially expressed transcription factors of cytokinin signal transduction showed that the transcription factor B-ARR was regulated by the 18-nt novel_miR_49. The KEGG and Pfam annotations showed that their four target genes belong to the B-ARR family and are associated with the response regulator receiver domain.

As shown in Supplemental Table 4, analysis of the miRNAs and target genes related to the differentially expressed transcription factors of gibberellin signal transduction showed that the transcription factor GID1 was regulated by the 19-nt novel_miR_137. Furthermore, the KEGG and Pfam annotations showed that their two target genes belong to the carboxylesterase family and are associated with gibberellin receptor GID1.

As shown in Supplemental Table 5, analysis of the miRNAs and target genes related to the differentially expressed transcription factors of ABA signal transduction showed that the transcription factor PYR/PYL was regulated by the 21-nt novel_miR_343 and novel_miR_391. The KEGG and Pfam annotations showed that their two target genes belong to the ABA receptor PYR/PYLx family and are associated with polyketide cyclase/dehydrase and lipid transport.

The transcription factor PP2C was regulated by the 21-nt novel_miR_392, and the KEGG and Pfam annotations showed that its target gene was associated with protein phosphatase 2C. The transcription factor ABF was regulated by novel_miR_137, and novel_miR_374. Additionally, the KEGG and Pfam annotations showed that their target genes are associated with ABA-responsive element binding factor.

As shown in Supplemental Table 6, analysis of the miRNAs and target genes related to the differentially expressed transcription factors of ethylene signal transduction showed that the transcription factor EBF1/2 was regulated by the novel_miR_374 and novel_miR_117. The KEGG and Pfam annotation showed that their target genes are associated with EIN3-binding F-box protein and leucine-rich repeat.

As shown in Supplemental Table 7, analysis of the miRNAs and target genes related to the differentially expressed transcription factors of brassinosteroid signal transduction showed that the transcription factor BSK was regulated by novel_miR_268 and novel_miR_391, and the KEGG and Swiss-Prot (Apweiler et al., 2004) annotations showed that their target genes are associated with BR-signaling kinase and serine/threonine-protein kinase BSKx7. The transcription factor BZR1/2 was regulated by novel_miR_76. The KEGG and Pfam annotations showed that their target gene is associated with brassinosteroid resistant 1/2 and BES1/BZR1q plant transcription factor. The transcription factor CYCD3 was regulated by the 22-nt novel_miR_43, and novel_miR_181, and the KEGG and Pfam annotations showed that their target genes are associated with cyclin D3 of plants.

As shown in Supplemental Table 8, analysis of the miRNAs and target genes related to the differentially expressed transcription factors of SA signal transduction showed that the transcription factor NPR1 was regulated by novel_miR_359 and novel_miR_312. Additionally, the KEGG and Pfam annotations showed that their target genes are associated with regulatory protein NPR1.

Screening for miRNAs of target genes related to self-incompatibility of rabbiteye blueberry pollen.

By comparing the TPM value of each miRNA between ‘Brightwell’ and ‘Premier’, we screened the differentially expressed miRNAs that were then analyzed together with its target genes and found that both of the 18-nt miRNAs (novel_miR_49) had the target gene maker-VaccDscaff21-snap-gene-21.37, which is related to the self-incompatibility of rabbiteye blueberry pollen. In addition, KEGG analysis of the target genes of novel_miR_49 (Fig. 5) showed that the enriched metabolic pathways were ribosome, aminoacyl-tRNA biosynthesis, GPI-anchor biosynthesis, arachidonic acid metabolism, and galactose metabolism.

Fig. 5.
Fig. 5.

Kyoto Encyclopedia of Genes and Genomes enrichment of the predicted target genes of novel_miR_49 in the pollen of the rabbiteye blueberry cultivars Brightwell and Premier using clusterProfiler [ver. 3.10.1 (Yu et al., 2012)] with Parameter of pAdjustMethod=fdr firstSigNodes=10.

Citation: Journal of the American Society for Horticultural Science 147, 6; 10.21273/JASHS05143-21

Discussion

miRNAs are a form of noncoding small RNAs that exist widely in eukaryotes to regulate the posttranscriptional expression of genes (Cai et al., 2013). miRNAs perform regulatory functions mainly by inhibiting the translation of target genes or by specifically cutting them (Ma et al., 2019). Most of the plant miRNA targets are transcription factors, enzymes, and signal proteins, which are involved in physiological and biochemical processes, including the growth and development of roots, stems, leaves, and flowers; phytohormone (auxin, gibberellin, and abscisic acid) regulation and signal transduction; biotic stress; stress response; and their own negative feedback regulation (Li et al., 2016). miRNAs have multiple effects on crop traits and play a key role in regulating plant shape, flowering time, fertility, yield, and the stress resistance of crops (Jiang et al., 2019). Since the discovery that the lin-4 gene in the nematode Caenorhabditis elegans encodes a 22-nt non-coding RNA (Zheng and Wang, 2014), miRNAs and their role in regulating target genes have become of particular interest in pomological research, which has attracted the attention of many researchers, and a number of studies have been conducted on fruit trees including grape, peach (Prunus persica), pear, apple, and mandarin orange (Citrus reticulata) (Shen et al., 2019). In our study, high-throughput sequencing data of the pollen of two rabbiteye blueberry cultivars, Brightwell and Premier, were analyzed, and a total of 491 miRNAs were obtained, of which 27 and 67 known miRNAs as well as 274 and 416 new miRNAs were identified in Brightwell and Premier, respectively. These miRNAs may be closely related to the differences in endogenous hormones and pollen vitality in the pollen of Brightwell and Premier (Yang et al., 2021).

The level of IAA in the pollen of ‘Premier’ was significantly lower than in the pollen of ‘Brightwell’ (Yang et al., 2021), which may be attributed to 10 differentially expressed miRNAs related to auxin signal transduction in this study. The four upregulated miRNAs and one downregulated miRNAs coregulated the expression of AUX1 and may inhibit auxin transport in the auxin signal transduction pathway (Zhang et al., 2010), and the two upregulated miRNAs and one downregulated miRNA in ‘Brightwell’ may coregulate the expression of ARF in the auxin signal transduction pathway through the transport inhibitor response 1/auxin-binding F-box protein (TIR1/AFB)-auxin/indole-3-acetic acid (Aux/IAA)/topless (TPL)-ARFs and transmembrane kinases (TMK) 1-IAA32/34-ARFs pathways (Kepinski and Leyser, 2005; Zhu et al., 2014). When the concentration of auxin is low, free Aux/IAA and ARF form heterodimers to inhibit the expression of auxin-responsive genes; when the concentration of auxin increases, auxin binds to TIR1/AFB, leading to the ubiquitination and degradation of Aux/IAA and the release of ARF, thereby promoting the expression of the downstream SAUR and GH3 genes (Kepinski and Leyser, 2005; Li and Li, 2019; Zhu et al., 2014). In addition, the SAUR gene was also coregulated by one upregulated miRNA, and the GH3 gene was coregulated by two downregulated miRNAs, causing significant differences in the auxin content between the pollen of ‘Brightwell’ and ‘Premier’ (Yang et al., 2021).

In this study, we found that the positive transcription factor B-ARR in the cytokinin signal transduction pathway (Li and Li, 2019) was regulated by the downregulated novel_miR_49 in ‘Premier’ compared with ‘Brightwell’, which may result in the content of ZT in the pollen of ‘Premier’ being significantly decreased compared with the content of ZT in the pollen of ‘Brightwell’ (Yang et al., 2021). The receptor GID1 in the gibberellin signal transduction pathway (Li and Li, 2019) was regulated by the downregulated novel_miR_137, which may explain the significantly decreased content of GA3 in the pollen of ‘Premier’ compared with the pollen of ‘Brightwell’ (Yang et al., 2021). ABA receptors PYR/PYLs/RCARs are coregulated by novel_miR_343 and novel_miR_391 and exist as dimers in the resting state, but they bind to phosphatase PP2Cs in the monomer form after ABA binding. The inhibition of PP2Cs on SnRK2s is relieved due to the inhibition of PP2Cs by novel_miR_392; as a result, ABI5 and RAV1 are phosphorylated, and the downstream response gene ABF regulated by the novel_miR_137 and novel_miR_374 is activated in the ABA signal transduction pathway (Li and Li, 2019). This may result in the content of ABA in the pollen of ‘Premier’ being significantly greater than in the pollen of ‘Brightwell’ (Yang et al., 2021).

Furthermore, in this study, we also found that the kinase BSK was coregulated by novel_miR_268 and novel_miR_391; the transcription factor BZR1/2 was regulated by novel_miR_76; and CYCD3 was regulated by novel_miR_43, and novel_miR_181. This might promote the transcription of the D-type plant cyclin gene CYCD3, thereby promoting brassinolide (BR) signal transduction and cell differentiation (Chu et al., 2006). BR exhibits strong biological activity even at a very low concentration (nmol or pmol level). BR plays an important regulatory role in cell elongation and division, vascular bundle differentiation, leaf morphogenesis, plant fertility, senescence, and resistance (Zheng and Wang, 2014) and, in particular, plays an irreplaceable regulatory role in the development of male organs. BR defects cause dysplasia or infertility of the male organs (Hewitt et al., 1985), resulting in the abnormal development of spores; a significantly reduced number of spore mother cells, spores, and pollen; and slow pollen tube growth (Ye et al., 2010). Therefore, the five differentially expressed miRNAs and their target genes in the pollen of ‘Premier’ may cause dysplasia of its male organs and a reduced amount and vigor of pollen (Xu and Wang, 2011; Yang et al., 2015b, 2015c, 2017, 2021).

In addition, SA plays an important role not only in inducing the plant response to stress caused by salt, drought, low temperature, ultraviolet radiation, and heavy metals, but also in regulating growth, development, maturation, and senescence (Zou et al., 2020). In our study, we found that two upregulated miRNAs (novel_miR_359, novel_miR_312) in ‘Premier’ (compared with ‘Brightwell’) coregulated the expression of NPR1 in the SA signaling pathway. NPR1 is a key regulator of the SA signaling pathway, and the transcriptional activity of NPR1 plays an extremely important role in regulating downstream TGACG motif-binding factor (TGA) expression (Gu et al., 2020). TGA negatively regulates the expression of downstream SA genes and plays a key role in plant responses to SA signals. Under normal growth conditions, nonexpresser of pathogenesis-related genes 3/4 and TGA interact to inhibit the transcription of downstream SA genes, and this inhibition is relieved when SA accumulates (Gu et al., 2020; Zou et al., 2020). In our study, the two upregulated miRNAs might regulate the expression of NPR1 in the SA signaling pathway, changing the concentration of SA and the expression intensity of SA downstream genes to regulate growth and development and inhibit the growth of the pollen tubes (Liu et al., 2019), causing dysplasia of the male organs of ‘Premier’ (Yang et al., 2015b, 2015c, 2017, 2021).

We verified the reliability and differences in expression of miRNAs involved in blueberry pollen identified through high-throughput sequencing by qRT-PCR. Furthermore, 10, one, one, five, two, five, and two candidate miRNAs related to auxin, cytokinin, gibberellin, ABA, ethylene, brassinosteroid, and SA signaling in rabbiteye blueberry pollen, respectively, were found. These miRNAs may cause the content of hormones in the pollen and pollen viability of ‘Premier’ to be significantly lower than that of ‘Brightwell’ (Yang et al., 2021), which may inhibit pollen tube growth (Liu et al., 2019), causing dysplasia of the male organs of ‘Premier’ and self-incompatibility (Yang et al., 2015b, 2015c, 2017, 2021). This finding provides a foundation for understanding the differences in the seed number of fruits as well as the xenia effect. Our results help elucidate the molecular mechanism of the xenia effect and the self-incompatibility of rabbiteye blueberry pollen. The regulation of miRNA on the levels of hormones in pollen and pollen vitality is not limited to the maturation stage but should occur during the entire pollen development process. Therefore, an analysis of the expression differences in miRNA and changes in endogenous hormone content during pollen development should be the focus of future research.

Several studies have shown that RNA also acts as a noncell-autonomous signal molecule that travels intercellularly to regulate gene expression in remote target tissues (Bayraktar et al., 2017). Additionally, miRNAs regulate the development, size, color, and maturation time of the fruit of horticultural plants (Chen et al., 2018b) and regulate the fertility and fertilization ability of pollen in a dose-dependent way (Akagi et al., 2014). Therefore, miRNAs in the pollen may enter unfertilized maternal cells through the double fertilization process to regulate gene expression—such as miRNAs in the pollen entering the style cells during pollen tube growth and regulating endogenous hormone levels in the styles, thus affecting pollination compatibility—or in the process of fertilization, whereby miRNAs in the pollen not only enter the egg cell and polar nucleus but also enter nonfertilized tissues such as the integument, ovary wall, and receptacle and regulate the expression of traits, thus producing the xenia effect. This is an interesting and important research direction for elucidating the molecular mechanism of the xenia effect in the future.

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Supplemental Table 1.

Analysis of differentially expressed microRNAs (miRNAs) in the pollen of the rabbiteye blueberry cultivars Brightwell and Premier.

Supplemental Table 1.
Supplemental Table 1.
Supplemental Table 2.

Selected auxin signal transduction-related microRNAs (miRNAs) in the pollen of the rabbiteye blueberry cultivars Brightwell and Premier.

Supplemental Table 2.
Supplemental Table 2.
Supplemental Table 2.
Supplemental Table 2.
Supplemental Table 2.
Supplemental Table 2.
Supplemental Table 2.
Supplemental Table 2.
Supplemental Table 3.

Selected cytokinin signal transduction-related microRNAs (miRNAs) in the pollen of the rabbiteye blueberry cultivars Brightwell and Premier.

Supplemental Table 3.
Supplemental Table 4.

Selected gibberellin signal transduction-related microRNAs (miRNAs) in the pollen of the rabbiteye blueberry cultivars Brightwell and Premier.

Supplemental Table 4.
Supplemental Table 5.

Selected abscisic acid signal transduction-related microRNAs (miRNAs) in the pollen of the rabbiteye blueberry cultivars Brightwell and Premier.

Supplemental Table 5.
Supplemental Table 5.
Supplemental Table 6.

Selected ethylene signal transduction-related microRNAs (miRNAs) in the pollen of the rabbiteye blueberry cultivars Brightwell and Premier.

Supplemental Table 6.
Supplemental Table 7.

Selected brassinosteroid signal transduction-related microRNAs (miRNAs) in the pollen of the rabbiteye blueberry cultivars Brightwell and Premier.

Supplemental Table 7.
Supplemental Table 8.

Selected salicylic acid signal transduction-related microRNAs (miRNAs) in the pollen of the rabbiteye blueberry cultivars Brightwell and Premier.

Supplemental Table 8.

Contributor Notes

This research was funded by the Key Projects of Science and Technology Research Scheme of Guizhou Province (Project No. Guizhou Science Basic [2019]1443); the National Natural Science Foundation of China (Project No. 31860546); and the Supporting Scheme of Guizhou Provincial Department of Education for the Top-notch Talents (Project No. Guizhou Education [KY 2018]076); First-class Discipline of Kaili University (Horticulture).

Q.Y. is the corresponding author. E-mail: yangqin1028518@126.com

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    Fig. 1.

    Distribution of microRNAs with different sequence lengths according to their total reads and unique tags in the pollen of the rabbiteye blueberry cultivars Brightwell and Premier.

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    Fig. 2.

    Expression differences confirmed by quantitative real-time polymerase chain reaction (qRT-PCR) and comparison with log10 of transcripts per million (TPM). Relative expression differences of eight different conserved microRNAs were verified by qRT-PCR using the 2-ΔΔCt method. For visualization, log10 was applied to compute the TPM data of sequencing results. The paragraph A to H represent the expression profiles of novel_miR_268, novel_miR_343, novel_miR_137, novel_miR_117, novel_miR_391, novel_miR_359, novel_miR_76, and novel_miR_43 in sequence.

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    Fig. 3.

    Gene Ontology annotation of the predicted target genes of differentially expressed microRNAs in the pollen of the rabbiteye blueberry cultivars Brightwell and Premier using TargetFinder software [ver. 1.6 (Allen et al., 2005)], which were then annotated with the reference genome from Colle et al. (2019) and National Center of Biotechnology Information (Bethesda, MD, USA) nonredundant (Nr) database.

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    Fig. 4.

    Differentially expressed microRNAs (miRNAs) and Kyoto Encyclopedia of Genes and Genomes pathway annotations of phytohormone signal transduction–related metabolism in the pollen of rabbiteye blueberry cultivars Brightwell and Premier using clusterProfiler [ver. 3.10.1 (Yu et al., 2012)] with Parameter of pAdjustMethod=fdr firstSigNodes=10. The purple boxes in the figure indicate that the regulatory factors denoted by letters within the box are associated with the target genes of the differential miRNA. AUX1 = auxin resistant 1; TIR1 = transport inhibitor response 1; AUX/IAA = auxin/indole acetic acid; ARF = auxin response factor; GH3 = Gretchen hagen 3; SAUR = small auxin up RNA; CRE1 = cytokinin response 1; AHP = Arabidopsis thaliana histidine phosphotransfer proteins; B-ARR = type-B A. thaliana response regulators; A-ARR = type-A A. thaliana response regulators; GID1 = gibberellin insensitive dwarf 1; GID2 = gibberellin insensitive dwarf 2; DELLA = aspartic acid–glutamic acid–leucine–leucine–alanine is a group of the GIBBERELIC ACID INSENSITIVE REPRESSOR OF ga1–3 SCARECROW family of plant-specific transcriptional regulators that act as master negative regulators of gibberellin signaling; TF = transcription factor; PYR/PYL = pyrabactin resistance/PYR1-like proteins; PP2C = protein phosphatase 2C; SnRK2 = sucrose non-fermenting-1 related protein kinase 2; ABF = ABA response element binding factor; ETR = ethylene receptor; CTR1 = constitutive triple-response 1; SIMKK = mitogen-activated protein kinases kinases; MPK6 = mitogen-activated protein kinase 6; EIN2 = ethylene-insensitive protein 2; EIN3 = ethylene-insensitive protein 3; EBF1/2 = Ein3-binding F-box1/F-box2; ERF1/2 = ethylene response factor 1/2; BAK1 = brassinosteroid insensitive 1 associated kinase receptor 1; BRI1 = brassinosteroid-insensitive 1; BKI1 = brassinosteroid-insensitive 1 kinase inhibitor 1; BSK = BR-signaling kinases; BSU1 = bri1 suppressor 1; BIN2 = brassinosteroid-insensitive 2; BZR1/2 = brassinazole resistant 1/2; TCH4 = Touch 4, a xyloglucan endotransglucosylase; CYCD3 = cyclins D3; JAR1 = jasmonate-resistant 1; JA-Ile = jasmonyl-isoleucine; COI1 = coronatine-insensitive 1; JAZ = jasmonate zim-domin; MYC2 = myelocytomatosis proteins 2; ORCA3 = octadecanoid-derivative responsive catharanthus AP2-domain; NPR1 = nonexpressor of pathogenesisrelated genes 1; TGA = TGACG motif-binding factor; PR1 = pathogenesis related genes 1.

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    Fig. 5.

    Kyoto Encyclopedia of Genes and Genomes enrichment of the predicted target genes of novel_miR_49 in the pollen of the rabbiteye blueberry cultivars Brightwell and Premier using clusterProfiler [ver. 3.10.1 (Yu et al., 2012)] with Parameter of pAdjustMethod=fdr firstSigNodes=10.

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