CircRNA identification
Rice circRNAs had been identified in multiple tissues, including leaves, anthers, pistils, seeds, shoots, and roots, implying the roles of circRNAs in different rice growth and development stages (Lu et al, 2015; Ye et al, 2015; Chu et al, 2017; Ye et al, 2017). However, it is still much unclear how circRNAs participate in the response of rice to salt stress. Previously, Wang et al, (2017c) developed salt-tolerant introgression line 9L136 using wild Oryza rufipogon accession as the donor, and Oryza sativa indica cultivar 93-11 as the recipient. RNA-seq profiling revealed more salt stress-induced differentially expressed genes (1,391) in introgression line 9L136 than recipient 93-11. In this study, this RNA-seq profiling dataset was used to explore candidate salt tolerance-related circRNAs. In total, 190 circRNAs supported by at least two unique back-spliced reads were detected, including 118 in 93-11 and 130 in 9L136 (Fig. 1B).
circRNAs related to salt stress
In plants, many circRNAs have been reported to exhibit stress-inducible expression patterns (Zhao et al, 2019). For example, Zhu et al, (2019) identified 2,787 circRNAs in cucumber, with 1,934 in root and 44 in leaf being differentially regulated under salt stress. Annotation and enrichment analysis of both parental genes and target mRNAs of salt-induced circRNAs suggested that circRNAs may paly roles in salt stress response by regulating abiotic stress associated genes. In Arabidopsis, Pan et al, (2018) uncovered 1,583 heat-specific circRNAs, suggesting these circRNAs may participate in heat stress response. Plant salt stress tolerance is a very complex regulating progress involving in signal transduction, substance and energy metabolism (Zhu et al, 2019b, c). In this study, Wilcoxon rank-sum test indicated that salt stress significantly decreased the average expression level of circRNAs in salt-susceptible cultivar 93-11 (p-value < 0.05. Fig. 3A), but slightly increased it in salt tolerant introgression line 9L136.
In order to reveal rice circRNAs that may have biological functions in response to salt stress, we compared the expression profiles of differentially expressed circRNAs between control and salt stress samples. Among the 190 circRNAs, 96 circRNAs were significantly differentially induced by salt stress, including 93 in recipient parent 93-11 (36 up- and 57 down-regulated, Supplementary table 2) and 95 in introgression line 9L136 (46 up- and 49 down-regulated, Supplementary table 3) (Fig. 3B). Since circRNAs have been proposed to be positive or negative regulators on their parent coding genes (Lu et al, 2015; Cheng et al, 2018), we predicted and annotated the parent genes of these differentially expressed circRNAs. GO enrichment analysis implied that the parent genes of differentially expressed circRNAs in 9L136 were involved in more biological and biochemical processes than in salt-susceptible cultivar 93-11 (Fig. 4A, Supplementary table 2; Fig. 4B, Supplementary table 3). Considering the fact that 9L136 is much more tolerant to salt stress than 93-11, it seems that circRNAs contributed to the salt-tolerant phenotype in introgression line 9L136.
CircRNA-miRNA-mRNA regulating network prediction
Besides regulating parent genes, previous studies suggested that circRNAs could bind specific miRNAs to repress the regulating ability of miRNAs (Liu et al, 2017). In this study, 27 miRNAs and 37 miRNAs were predicted to be competitively bound by 19 and 26 differentially expressed circRNAs in 9L136 and 93-11, respectively (Supplementary tables S4-7). Among those miRNAs, some were reported to be closely related to plant stress responses in rice. For example, osa-mir2925 was reported to be up-regulated by salt stress in rice, and osa-mir2925 potentially targets stress-response-associated genes, such as LOC_Os05g24780 (calcium-binding protein CML21), LOC_Os09g28200 (Heat stress transcription factor B-4c) in 9L136, and LOC_Os01g43320 (GABA transporter) in 93-11 (Fig. 5) (Wang et al, 2017c). Another osa-miR2925 was competitively bound by circular RNA Chr10:18818949|18958248 in 9L136 and by Chr6:2046190|2059120 in 93-11. Interestingly, Chr10:18818949|18958248 and Chr6:2046190|2059120 showed distinct regulation patterns in 9L136 and 93-11 under salt stress. In salt-tolerant line 9L136, Chr10:18818949|18958248 was down-regulated, which may decrease its binding effect to osa-mir2925. Meanwhile, in 93-11, Chr6:2046190|2059120 was up-regulated, which may promote the sponge effect of Chr6:2046190|2059120 to osa-mir2925. It seems that in this regulation network, the distinct regulation patterns of circular RNAs in salt-resistant line 9L136 and salt-susceptible cultivar 93-11 result in the opposite regulating effect on miRNA, which further leads to the differential expression of stress-response-associated mRNAs, and may contribute to the salt-resistant phenotype of 9L136 and salt-susceptible phenotype of 93-11.
Additionally, it should be noticed that several miRNAs competitively bound by circRNAs were markedly different between the two rice cultivars, thus indicating the different regulating networks of circRNAs in the two rice cultivars in response to salt stress (Fig. 5. Supplementary tables 4-7). For example, the salt-tolerant introgression line 9L136 showed several unique regulation networks. In 9L136, osa-miR2102-5p and osa-mir5809 were predicted to be competitively bound by circRNAs Chr10:18818949|18958248 and Chr7:14201482|14303571, respectively. The osa-miR2102-5p has been widely reported to be involved in salt and drought stresses in Gramineae plants, such as barley (Zare et al, 2019), maize (Wang et al, 2014), and Spartina alterniflora (Qin et al, 2015). In rice, osa-mir5809 was reported to be involved in salt (Huang et al, 2019) and heat stresses (Mangrauthia et al, 2017), as well as leaf senescence (Xu et al, 2014). Both circRNA Chr10:18818949|18958248 and Chr7:14201482|14303571 were down-regulated by salt stress in 9L136, which could decrease the competitive binding of circRNAs to miRNA osa-miR2102-5p, and further enhance the suppression of osa-miR2102-5p to target mRNAs. These findings implied the involvement of circRNAs in the response of rice to salt stress. CircRNAs may also have various biological functions during the growth and stress responses of rice. However, the function of circRNAs in rice needs further experimental validation. Further analysis demonstrated that 426 mRNAs were predicted to be the targets of 27 miRNAs in 9L136, and 327 mRNA were predicted to be the targets of 37 miRNAs in 93-11 (Supplementary tables 4-7). EggNOG class annotation analysis revealed that these mRNAs participated in the regulation of physiological and biochemical processes, including transcription, signal transduction, and secondary metabolism (Fig. 5, Supplementary figure 1, Supplementary tables 4-7). Among those miRNA targets, more mRNAs were differentially expressed in 9L136 (108) than in 93-11 (83), suggesting a more complex regulation network in the salt stress tolerance introgression line 9L136.
Expression patterns of circRNAs and their corresponding parental genes in different cultivars
Previous studies in several species have suggested that most circRNAs regulate the expression level of their corresponding parental genes. In rice, Lu et al, (2015) proposed that circRNA and its linear form may act as a negative regulator of its parental gene. However, expression profiles of circRNAs in tea and Arabidopsis showed a positive correlation between circRNAs and their parental genes (Tong et al, 2018; Cheng et al, 2018). In cucumber, both opposite and positive trends between the expression levels of circRNAs and parental mRNAs have been reported (Zhu et al, 2019a).
In salt-sensitive cultivar 93-11, circRNA Chr1:30513415|30521330 was down-regulated and its parental gene LOC_Os01g53090, a pathogen-related protein, was up-regulated by NaCl stress (Table 2). Previous studies reported that LOC_Os01g53090 was up-regulated by both biotic and abiotic stresses such as Magnaporthe oryzae and aluminum stresses (Vijayan et al, 2013; Arbelaez et al, 2017). Another circRNA, Chr2:18305592|18305907, and its parental gene LOC_Os02g30714, a putative 11-β-hydroxysteroid dehydrogenase that has been proposed to take part in perception and transduction for many environmental stimuli (Wang et al, 2014), were up-regulated (Table 2, Table 3). In rice, LOC_Os02g30714, which was up-regulated by OsWRKY13, was an activator of rice in resistance to both bacterial and fungal pathogens, and was up-regulated during subsequent recovery after cold stress (Qiu et al, 2008; Yang et al, 2015). Chr4:18170122|18170775 was down-regulated and its parental gene LOC_Os04g30420, a zinc-binding dehydrogenase, was down-regulated. A previous study showed that LOC_Os04g30420 participated in the response to heat and drought stresses (Wilkins et al, 2016). These analyses reveal that many stress related genes in rice are the potential targets of circRNAs and could be differentially regulated by associated circRNAs in salt-sensitive cultivars.
Table 2. Differentially expressed circRNAs and corresponding parent genes in 93-11, and function annotation of parental genes.
circRNA
|
circRNA
regulated
|
Parent gene
|
Parental
log2(FC)
|
Parental function
|
Chr1:30513415|30521330
|
down
|
LOC_Os01g53090
|
1.42
|
Pathogen-related protein
|
Chr1:38521288|38521427
|
up
|
LOC_Os01g66330
|
1.98
|
CLP protease
|
Chr10:2483373|2483520
|
up
|
LOC_Os10g05069
|
1.49
|
α-mannosidase
|
Chr11:4106581|4106802
|
down
|
LOC_Os11g07940
|
-1.77
|
Centromere protein
|
Chr12:14488613|14488960
|
down
|
LOC_Os12g25200
|
2.92
|
Chloride channel protein
|
Chr2:16542324|16543258
|
down
|
LOC_Os02g27950
|
1.35
|
Polyadenylate-binding protein-interacting protein
|
Chr2:18305592|18305907
|
up
|
LOC_Os02g30714
|
2.66
|
11-β-hydroxysteroid dehydrogenase
|
Chr2:23868290|23871119
|
up
|
LOC_Os02g39550
|
-1.59
|
Calcium ion binding protein
|
Chr2:30459474|30459790
|
up
|
LOC_Os02g49840
|
2.03
|
MADS-box transcription factor
|
Chr3:269113|269568
|
down
|
LOC_Os03g01360
|
-1.66
|
Actin binding protein
|
Chr3:35103079|35103219
|
up
|
LOC_Os03g61920
|
2.12
|
Electron transfer flavoprotein
|
Chr3:8623603|8623803
|
down
|
LOC_Os03g15630
|
-1.59
|
Component of membrane
|
Chr4:18170122|18170775
|
down
|
LOC_Os04g30420
|
-2.09
|
Zinc-binding dehydrogenase
|
Chr6:3470974|3474679
|
down
|
LOC_Os06g07250
|
-2.07
|
Jacalin-like lectin
|
Table 3 Differentially expressed circRNAs and corresponding parent genes in 9L-136, and function annotation of parental genes.
circRNA
|
circRNA
regulated
|
Parent gene
|
Parental
log2(FC)
|
Parental function
|
Chr1:26607355|26607630
|
up
|
LOC_Os01g46710
|
1.46
|
Translation initiation factor
|
Chr1:30512877|30520821
|
down
|
LOC_Os01g53090
|
4.00
|
Pathogen-related protein
|
Chr2:18305592|18305907
|
up
|
LOC_Os02g30714
|
4.50
|
11-β-hydroxysteroid dehydrogenase
|
Chr2:23868553|23868970
|
up
|
LOC_Os02g39550
|
-1.65
|
Calcium ion binding protein
|
Chr2:4198743|4199218
|
up
|
LOC_Os02g08010
|
-2.07
|
Ca2+-transporting ATPase
|
Chr3:12838424|12838608
|
down
|
LOC_Os03g22420
|
1.36
|
AAA-ATPase FIGL-1
|
Chr3:30454536|30454982
|
up
|
LOC_Os03g53110
|
-1.43
|
Mg2+ transporter
|
Chr3:9760244|9760659
|
down
|
LOC_Os03g17570
|
4.83
|
Signal transduction regulator
|
Chr4:18170122|18170775
|
up
|
LOC_Os04g30420
|
-1.43
|
Zinc-binding dehydrogenase
|
Chr4:30172735|30173954
|
down
|
LOC_Os04g50970
|
4.00
|
Component of membrane
|
Chr5:21655167|21655378
|
up
|
LOC_Os05g37060
|
4.79
|
Myb-like transcription factor
|
Chr8:18479857|18480021
|
up
|
LOC_Os08g30060
|
1.31
|
Proton pump-interactor
|
Chr8:3226424|3226681
|
up
|
LOC_Os08g05940
|
1.37
|
RNA polymerase Rpb1
|
Chr9:4354645|4354999
|
down
|
LOC_Os09g08390
|
1.47
|
SEC14 cytosolic factor
|
Chr10:20345844|20346873
|
down
|
LOC_Os10g37980
|
2.10
|
Prephenate dehydratase
|
Chr11:23233410|23233648
|
up
|
LOC_Os11g39020
|
2.13
|
ABC transporter
|
Chr11:8467507|8467683
|
down
|
LOC_Os11g15040
|
-0.594708
|
Anthranilate O-methyltransferase
|
Chr12:9356168|9356432
|
down
|
LOC_Os12g16350
|
0.686881
|
Enoyl-CoA hydratase
|
Similarly, in introgression line 93-11, LOC_Os01g53090, LOC_Os02g30714, LOC_Os02g39550, and LOC_Os04g30420, the four parent genes of corresponding differentially regulated circRNAs, were also differentially expressed under NaCl treatment, implying the roles of these parent genes and circRNAs in salt stress response in both salt-susceptible and salt-tolerant rice cultivars (Table 3). Furthermore, many genes that function in ion transportation, such as LOC_Os02g08010 (Ca2+-transporting ATPase, parental gene of Chr2:4198743|4199218), LOC_Os03g53110 (Mg2+ transporter, parental gene of Chr3:30454536|30454982), LOC_Os08g30060 (proton pump-interactor, parental gene of Chr8:18479857|18480021), LOC_Os02g39550 (a calcium ion binding protein, and parental gene of Chr2:23868290|23871119), and genes that function in hydrolase activity, such as LOC_Os10g37980 (prephenate dehydratase, parental gene of Chr10:20345844|20346873), and LOC_Os12g16350 (Enoyl-CoA hydratase, parental gene of Chr12:9356168|9356432), were differentially expressed (Table 3).
Furthermore, LOC_Os05g31254 (calmodulin-related calcium sensor protein gene), a candidate gene for salt tolerance in rice (Wang et al, 2017c), was predicted to be the parent gene of circRNA Chr6:2046190|2059120 in 93-11, and the parent gene of circRNA Chr10:18818949|18958248 in 9L-163. In Arabidopsis, the calmodulin-related calcium sensor protein gene has been reported to modulate stress responses. The functions of LOC_Os05g31254 and Chr10:18818949|18958248 need to be further studied, since they will be valuable for determining salt stress mechanisms and conducting salt-resistance breeding.
In summary, our study reveals the possible roles of rice circRNA in response to salt stress, which will expand our understanding of the characteristics of plant circRNAs and facilitate the determination of salt stress regulatory mechanisms in rice. Furthermore, the complicated relationships between the abundances of circRNAs and their parent genes need to be further explored through molecular biology approaches.