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. 2021 Feb 23;186(1):519–533. doi: 10.1093/plphys/kiab084

OsmiR396/growth regulating factor modulate rice grain size through direct regulation of embryo-specific miR408

Xiaofang Yang 1,2,#, Xiaoling Zhao 3,#, Zhengyan Dai 1,#, Feilong Ma 1,2, Xuexia Miao 1,2, Zhenying Shi 1,✉,2
PMCID: PMC8154042  PMID: 33620493

Abstract

microRNAs (miRNAs) are promising targets for crop improvement of complex agricultural traits. Coordinated activity between/among different miRNAs may fine-tune specific developmental processes in diverse organisms. Grain size is a main factor determining rice (Oryza sativa L.) crop yield, but the network of miRNAs influencing this trait remains uncharacterized. Here we show that sequestering OsmiR396 through target mimicry (MIM396) can substantially increase grain size in several japonica and indica rice subspecies and in plants with excessive tillers and a high panicle density. Thus, OsmiR396 has a major role related to the regulation of rice grain size. The grain shape of Growth Regulating Factor8 (OsGRF8)-overexpressing transgenic plants was most similar to that of MIM396 plants, suggesting OsGRF8 is a major mediator of OsmiR396 in grain size regulation. A miRNA microarray analysis revealed changes to the expression of many miRNAs, including OsmiR408, in the MIM396 plants. Analyses of gene expression patterns and functions indicated OsmiR408 is an embryo-specific miRNA that positively regulates grain size. Silencing OsmiR408 expression (miR408KO) using CRISPR technology resulted in small grains. Moreover, we revealed the direct regulatory effects of OsGRF8 on OsMIR408 expression. A genetic analysis further showed that the large-grain phenotype of MIM396 plants could be complemented by miR408KO. Also, several hormone signaling pathways might be involved in the OsmiR396/GRF-meditated grain size regulation. Our findings suggest that genetic regulatory networks comprising various miRNAs, such as OsmiR396 and OsmiR408, may be crucial for controlling rice grain size. Furthermore, the OsmiR396/GRF module may be important for breeding new high-yielding rice varieties.

The OsmiR396/Growth Regulating Factor module plays a pivotal role in rice grain size regulation and genetically regulates OsmiR408, which acts as an embryo-specific grain size regulator.

Introduction

Rice is a source of food for more than half of the global population, but its sustainable production is increasingly being challenged by a growing population worldwide, fluctuating climate conditions, and decreases in the arable land area and the availability of water for irrigation (Wang et al., 2018). Rice grain yield is determined by the number of panicles per plants, number of grains per panicle, and grain weight. As the major determinant of grain weight, grain size is one of the most important yield-related traits in cereal plants, influencing both yield potential and grain quality, with implications for the commercial value of the grain (Xing and Zhang, 2010).

MicroRNAs (miRNAs) are promising targets for crop improvement because of their potential effects on complex agricultural traits (Tang and Chu, 2017; Zhang et al., 2017). The pairing of plant miRNAs with their mRNA targets through base complementarity triggers posttranscriptional repression of gene expression (Bartel, 2004). Many miRNAs target regulatory factor genes, including those encoding transcription factors and F-box proteins, suggesting they may influence diverse plant developmental processes and physiological plasticity (Rubio-Somoza and Weigel, 2011). Quite a few miRNAs regulate rice grain size, such as OsmiR156 (together with several of its target SQUAMOSA Promoter Binding Protein-like (SPL) genes; Jiao et al., 2010; Wang et al., 2012; Zhang et al., 2017), OsmiR397 (Zhang et al., 2013), OsmiR398 (Zhang et al., 2017), OsmiR408 (Zhang et al., 2017), OsmiR159 (Gao et al., 2018), OsmiR1432 (Zhao et al., 2019), and OsmiR530 (Sun et al., 2020), most of which are expressed in rice grain (Xue et al., 2009).

The coordination among various small RNAs helps modulate specific developmental processes. The first identified miRNA, lin-4 in Caenorhabditis elegans, regulates developmental transition by interacting with another miRNA, let-7 (Pasquinelli and Ruvkun, 2002). In plants, the miR156-SPL9-miR172 regulatory pathway controls the transition from vegetative growth to reproductive growth (Wang, 2014). Specifically, TAS3 trans-acting small interfering RNA (tasiRNA) mediate vegetative phase transitions and plant polarity development (Fahlgren et al., 2006; Garcia et al., 2006). The biogenesis of TAS3 tasiRNA is regulated by miR390, whereas its functionality is coordinated by miR166 (Nogueira et al., 2007; Wang et al., 2010). The miR319-TCP4-miR396b pathway regulates leaf size in Arabidopsis (Schommer et al., 2014). In rice, a network formed by miR156, miR172, miR529, and their target genes coordinately regulates rice tillering and panicle branching (Wang et al., 2015). Embryogenesis patterning involves many miRNAs, although the coordination among them remains unclear (Armenta-Medina et al., 2017). Similarly, although several miRNAs contribute to the regulation of rice grain size, the coordinated activities among them remains largely elusive.

Agriculturally important traits, including grain size, are usually genetically controlled by quantitative trait loci (QTLs). Over the past 2 decades, the molecular and genetic basis for grain size modulations in multiple rice genetic resources has been extensively investigated, resulting in the isolation and analysis of many relevant QTLs from different genetic resources(Xing and Zhang, 2010; Huang et al., 2013; Zuo and Li, 2014). Several of these QTLs were identified as miRNA targets. For example, Grain Weight 8 (GW8) is one target of miR156, OsSPL16, and the encoded protein controls grain size by directly regulating another QTL (GW7; Wang et al., 2012, 2015). Additionally, GLW7 (OsSPL13), which is another a target of miR156, positively regulates grain size by modulating the cell size (Si et al., 2016). Moreover, Grain Size 2 (GS2), Grain Length 2 (GL2), and Grain Length and Width 2 (GLW2), which are synonymous with Growth Regulating Factor 4 (OsGRF4), are targeted by OsmiR396 (Che et al., 2015; Hu et al., 2015; Li and Li, 2016). These findings indicate that miRNAs are important genetic factors regulating grain size. Several signaling pathways controlling grain size were clarified on the basis of mechanistic studies of these cloned QTLs (Li and Li, 2016), and new factors are increasingly being identified. For example, the functional characterization of DEP1 and GS3 further established the regulatory effects of G-protein signaling on grain size (Liu et al., 2018; Sun et al., 2018). Furthermore, Grain Size and Number1 GSN1 influences grain size through the OsMKKK10–OsMKK4–OsMPK6 cascade, confirming that the MAPK pathway affects grain size (Guo et al., 2018). The modulatory effects of GS9 on grain size are mediated by the brassinosteroids (BR) signaling pathway (Zhao et al., 2018).

Although many QTLs and miRNAs functioning in grain size regulation have been isolated, still new factors might be involved in this comprehensive process. Moreover, the regulatory networks between different factors and the underlying mechanism are still fragmentary, the elucidation of which should further broaden our understanding of the molecular mechanism of grain development and provide guidance for crop breeding practice. In rice, the target gene of OsmiR396, OsGRF4 has been identified as QTLs that positively regulate grain size (Che et al., 2015; Duan et al., 2015; Hu et al., 2015; Li and Li, 2016). Additionally, OsmiR396 controls other reproductive processes, including panicle development (Gao et al., 2015) and floral organ development (Liu et al., 2014). Furthermore, MIR396e and MIR396f negatively regulate grain size, especially under nitrogen-deficient condition (Miao et al., 2019). Increasing evidence reputes the role for OsmiR396 in rice grain size regulation. However, the underlying molecular mechanism remains to be comprehensively determined, including the associated network involving multiple factors.

In this study, we revealed the extensive and conserved role of OsmiR396 as a negative regulator of grain size as well as the underlying signal transduction pathways interweaved with other miRNAs. Sequestering OsmiR396 by target mimicry (MIM396) in several rice genetic backgrounds (japonica and indica subspecies) increased grain size to various degrees. Moreover, MIM396 could increase grain size under more branches conditions at both vegetative (more tillers) and reproductive stages (dense panicle). Although several OsmiR396 target genes positively regulated rice grain size, the greatest similarity in grain shape was observed between the OsGRF8-overexpressing plants and the MIM396 plants. Furthermore, miRNA microarray data revealed changes to several miRNAs in MIM396 plants. Biochemical and genetic analyses proved that the OsmiR396/GRF module directly regulates other miRNAs, such as OsmiR408, to modulate grain size via several hormone signaling pathways. Therefore, OsmiR396 might be a crucial regulator of rice grain size development, which interweave with other miRNAs and signal transduction pathways.

Results

OsmiR396 negatively regulated grain size in different rice varieties

We previously determined that sequestering OsmiR396 influences the panicle architecture of rice variety YuetaiB, an indica subspecies (Gao et al., 2015). Furthermore, the grains of the transgenic plants (hereafter referred to as YM396) were enlarged (Supplemental Figure S1A), with an increased grain length (Supplemental Figure S1B) and 1,000-grain weight (KGW; Supplemental Figure S1D). To further investigate the influence of sequestering OsmiR396 on grain size, we transformed the MIM396 plasmid into more genetic backgrounds, namely ZH11, R8015 and Xiushui134 (XS134), with the resulting transgenic plants named MIM396, RM396 and XM396 respectively. There are eight genes encoding OsmiR396 in rice (if there is 5p and 3p distinction, we refer to 5p here; http://structuralbiology.cau.edu.cn/PNRD). Mature OsmiR396a and OsmiR396b have identical sequences, whereas mature OsmiR396d, OsmiR396g, and OsmiR396h share same sequences. In MIM396 plants, all OsmiR396 members were down-regulated as expected (Supplemental Figure S2A). The grains of the MIM396 plants were much larger than those of the wild-type (WT) ZH11 plants (Figure 1A), with an increased grain length and decreased grain width (Figure 1B) as well as an increased KGW (Figure 1C). The XM396 plants also produced enlarged grains (Figure 1D), with increased grain length (Figure 1E) and KGW (Figure 1F). Similarly, the grains of the RM396 plants were larger than those of the corresponding WT plants (Figure 1G), with an increased grain length (Figure 1H) and KGW (Figure 1I).

Figure 1.

Figure 1

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Grain characteristics due to the down regulation of OsmiR396 in several japonica subspecies. A, Grain morphology of the MIM396 and corresponding WT ZH11 plants. B, Statistical analysis of the grain length and width of the MIM396 and ZH11 plants (n = 30). C, Statistical analysis of the 1,000 KGW of the MIM396 and ZH11 plants (n = 100). D, Grain morphology of the XM396 plant and the corresponding WT XS134 plants. E, Statistical analysis of the grain length and width of the XM396 plants and XS134 plants (n = 30). F, Statistical analysis of the KGW of the XM396 plants and XS134 plants (n = 100). G, Grain morphology of the RM396 plant and the corresponding WT R8015 plants. H, Statistical analysis of the grain length and width of the RM396 and R8015 plants (n = 30). I, Statistical analysis of the KGW of the RM396 and R8015 plants (n = 100). Comparisons were made compared to the respective WT unless otherwise indicated. Error bars represent ± sd, single and double asterisks in the statistical analyses represent significant difference determined by the Student’s t test at *P < 0.05 and **P < 0.01.

The grains of the japonica and indica subspecies differ regarding the grain length-to-width (L/W) ratio, with the L/W of indica rice much greater than that of japonica rice. Specifically, in the japonica background, the grain length of MIM396, XM396, and RM396 increased by 15.68, 21, and 12.3%, respectively, and KGW increased by 30.64, 23.8, and 7.24, respectively, relative to the corresponding values for the respective WT controls. In the indica background, the grain length and KGW of YM396 plants increased by 21.6 and 6.9%, respectively. Therefore, down-regulating OsmiR396 increased grain length in both japonica and indica subspecies, despite their respective intrinsic difference in grain L/W ratio, and thereby increasing grain weight.

Along with the enlarged grains, the husk of the MIM396 plants was also enlarged in association. In SEM analysis, the length of the epidermal cells in the husk obviously increased as compared with that of the WT (Supplemental Figure S2, B and C). And the starch granules of the MIM396 plants were enlarged (Supplemental Figure S2D).

We also investigated the effects of OsmiR396 overexpression on grain size. There were no grain size differences between the transgenic plants overexpressing OsMIR396b and the WT ZH11 plants (Supplemental Figure S3, A and C). The overexpression of OsMIR396d also did not lead to grain size changes (Supplemental Figure S3, B and D; Liu et al., 2014). Therefore, the overexpression of OsmiR396 does not appear to influence rice grain size.

Down-regulating OsmiR396 increased grain size in plants with a high panicle density and excessive tillers

Large grains are generally accompanied by sparse panicles. Regarding the MIM396 plants, the panicle length increased (Supplemental Figures S4, A and S5, A), but the number of grains per panicle was unaffected by the decrease in the number of secondary branches (Supplemental Figures S4, A, S5, B and C). The panicle was sparse because of the increased panicle length. To determine if downregulation of OsmiR396 can increase grain weight in a rice line with a high panicle density, we crossed MIM396 plants with the A989 mutant, in which RCN2 is overexpressed to substantially increase the number of grains per panicle (Li et al., 2010). In addition to an increase in the number of grains per panicle, the grains of the hybrid plants were larger than those of the WT plants, but were a little smaller than those of the MIM396 plants (Supplemental Figure S4B). The grain length and KGW of the hybrid plants were also greater than the corresponding values of the WT plants (Supplemental Figure S4, C and D). Therefore, downregulating OsmiR396 increased grain size even in dense-panicle plants.

Increasing the number of tillers may also positively affect grain yield. We wonder that if downregulating OsmiR396 could also increase grain size in plants exhibiting excessive tillers. To assess this possibility, we crossed the MIM396 plant with the cd mutant, in which miR156f is upregulated to produce multiple tillers (Dai et al., 2018). A comparison with the WT plants revealed that the MIM396/cd plants had more tillers (Supplemental Figures S4, E and S5, D) and produced larger and longer grains (Supplemental Figure S4, F and G) with a higher KGW (Supplemental Figure S4H). These results indicated that downregulating OsmiR396 can increase grain size, even in plants with excessive tillers.

OsGRFs positively regulated grain size

Twelve OsGRF genes are targeted by OsmiR396 (Gao et al., 2015). We previously proved that in the MIM396 plants, the expression levels of most of the OsGRF genes are upregulated (Dai et al., 2019). To determine which OsGRF genes encode regulators of grain size, we analyzed their expression patterns during the process of grain development. The expression of most of the OsGRF genes gradually increased over time, with high transcript levels in embryos at 8–12 d after fertilization (DAF; Figure 2A), implying these genes may influence grain filling. Among these genes, OsGRF9 had the highest expression level, and OsGRF4 has been widely demonstrated to positively affect grain size (Che et al., 2015; Hu et al., 2015). We then selected OsGRF9, OsGRF1, and OsGRF8 for further functional analyses via genetic transformation.

Figure 2.

Figure 2

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Analysis of the OsGRF1, OsGRF8, and OsGRF9 expression levels and functions. A, Expression profiles of 12 OsGRFs genes after fertilization (n = 3). B, Expression profiles of OsGRF1, OsGRF8, and OsGRF9 in various tissues (n = 3). C, Northern blot analysis of OsmiR396b and OsmiR396d in various tissues. D, Grain morphology of the GRF1OE and WT plants. E, Statistical analysis of the grain length and width of the GRF1OE and WT plants (n = 30). F, Statistical analysis of the KGW of the GRF1OE and WT plants (n = 100). G, Grain morphology of the GRF8OE and WT plants. (H) Statistical analysis of the grain length and width of the GRF8OE and WT plants (n = 30). I, Statistical analysis of the KGW of the GRF8OE and WT plants (n = 100). Comparisons were made compared to the respective WT unless otherwise indicated. Error bars represent ± sd, single and double asterisks in the statistical analyses represent significant difference determined by the Student’s t test at *P <0.05 and **P <0.01.

First, we analyzed the expression profile of these three OsGRFs in various tissues, including the roots, stems, leaves, leaf sheaths, seedlings, 6-cm young panicles, and 10 DAF embryos. It was revealed that OsGRF1, OsGRF8, and OsGRF9 showed the highest expression in 6-cm young panicles and embryos at 10 DAF (Figure 2B), providing evidence of their major roles in reproductive development. In contrast, the OsmiR396s were mainly expressed in the leaves and the seedlings (Figure 2C), where the OsGRF gene expression were lowest (Figure 2B). The high OsGRF expression in the embryos at 8–12 DAF might inhibit the expression of OsmiR396 to a level undetectable by a miRNA northern blot assay.

Then we analyzed the phenotypes of the transgenic lines overexpressing OsGRF1, OsGRF8, or OsGRF9 (named as GRF1OE, GRF8OE, and GRF9OE respectively). Both GRF1OE and GRF8OE produced enlarged grains (Figure 2, D–I). Specifically, we detected an increase in the GRF1OE grain length and KGW (Figure 2, E and F), but no decrease in the grain width as that in the MIM396 grains (Figure 2E). The GRF8OE grain length and KGW also increased (Figure 2, H and I), but the grain width decreased (Figure 2H), similar to the grains of the MIM396 plants. Moreover, the epidermal cells in the husk of the GRF8OE plants were elongated (Supplemental Figure S2, B and C), and the starch granules in the grains were enlarged (Supplemental Figure S2D), similar to those in MIM396 plants. Further, OsGRF4 is identified as QTLs that positively regulate grain size (Che et al., 2015; Hu et al., 2015), so that targets of OsmiR396 might collectively take a great part in the regulation of grain size. Out of our expectation, the GRF8KO lines we constructed using CRISPR technology (Supplemental Figure S6A) also produced enlarged grains (Supplemental Figure S6B). The reason for this might be the upregulated expression of several other OsGRF gene targets of OsmiR396 in these GRF8KO lines (Supplemental Figure S6C), possibly as part of a compensatory mechanism.

The grains of the MIM396 plants were longer and narrower than those of the ZH11 plants (Supplemental Figure S7, A and B). The GRF1OE grains were also longer, but there were no obvious changes to the grain width (Supplemental Figure S7, A and B). Additionally, both the grain length and width increased in the GS2ZH11 plants, in which OsGRF4 is over-expressed (Supplemental Figure S7, A and B; Hu et al., 2015). An increase in the length and a decrease in the width of GRF8OE grains resulted in a grain shape that was most similar to that of MIM396 grains. Consistently, the largest increases in the L/W ratio were detected for the MIM396 and GRF8OE grains (Supplemental Figure S7C). Thus, we speculated that the OsGRF8 genes might be the mediator of OsmiR396 in grain size regulation.

Expression of many miRNAs were affected in MIM396 plants

Since miRNAs function variously in plant development, including grain size regulation, we wondered whether OsmiR396 regulates grain size by modulating other miRNAs. Therefore, we completed miRNA microarray assays to compare the MIM396 and WT plants. A volcano plot was used to illustrate the distribution of the miRNA expression in the MIM396 and WT plants. The combined results from three biological replicates revealed that the expression of many miRNAs differed between the MIM396 and WT plants (Figure 3A). With a fold-change of ≥2.0 and a P ≤0.05 as the threshold, 7 downregulated and 38 upregulated miRNAs were identified (Figure 3A;  Supplemental Table S1). A heat map for the expression levels of the differentially expressed miRNAs between MIM396 and WT plants clearly presented the data for the 7 downregulated and 38 upregulated miRNAs in the MIM396 plants (Figure 3B;  Supplemental Table S1). The downregulated miRNAs included OsmiR396e and OsmiR396f. Additionally, OsmiR398 (Zhang et al., 2017), OsmiR397 (Zhang et al., 2013), OsmiR408 (Zhang et al., 2017), which regulate grain size, were also filtered out. We used RT-qPCR assay and verified the upregulation of OsmiR408 in the 10 DAF embryos not only of MIM396 plant, but also of GRF8OE plants (Figure 3C). These analyses suggested that OsmiR396 might regulate grain size by regulating several other miRNAs.

Figure 3.

Figure 3

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miRNA microarray analysis of the young panicles of the MIM396 and WT plants as well as verification of OsmiR408. A, Volcano plot of the overall expression of the miRNAs in the MIM396 and WT plants. Green dots indicated the differentially down-regulated miRNAs, whereas red dots represent the differentially up-regulated miRNAs in the MIM396 plants compared with the WT plants. B, Expression of the differentially expressed miRNAs in the MIM396 and WT plants. The bar represents the scale of relative miRNA expression levels on the basis of Log2 value. C, RT-qPCR analysis of OsmiR408 in GRF8OE, MIM396, and WT plants at 10 DAF. Comparisons were made compared to the respective WT unless otherwise indicated. Error bars represent ± sd, double asterisks in the statistical analyses represent significant difference determined by the Student’s t test at **P < 0.01.

The OsmiR408 expression profile revealed an embryo-specific expression profile

The MIM396 versus WT miRNA microarray data revealed an upregulation of OsmiR408, which was previously indicated to be an embryo-specific miRNA (http://structuralbiology.cau.edu.cn/PNRD). Accordingly, we completed an miRNA northern blot assay to detect the OsmiR408 transcript in embryos at different developmental stages. When the embryos at 1, 2, 3, 5, and 10 DAF were examined, OsmiR408 was detected only in the embryos at 10 DAF (Figure 4A). When more detailed stages around 10 DAF were checked, it was revealed that OsmiR408 was most highly expressed in embryos at 8 and 10 DAF (Figure 4B).

Figure 4.

Figure 4

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Expression profile of OsmiR408. A, miRNA northern blot analysis of OsmiR408 in young embryos at 1–10 DAF. B, miRNA northern blot analysis of OsmiR408 in young embryos at 6–14 DAF. C, The OsmiR408 LNA antisense probe detected OsmiR408 in the embryos at 8 DAF. D, The OsmiR408 LNA antisense probe detected OsmiR408 in the embryos at 12 DAF. E, Analysis with the OsmiR408 LNA sense probe. F–M, GUS staining of the following p408::GUS parts: the root (F), the shoot apical meristem (G), the leaf (H), the spikelet (I), the germinating seeds (J), the isolated young embryo (K), the seed (cross section) (L), and the enlarged embryo (M). Abbreviations: ra, radicle; pa, plumular axis; pl, plumule; co, coleoptile. Bars in C–E were 100 µm, and those in F–M were 1 mm.

We next checked the spatial expression of OsmiR408 via in situ hybridization. When the embryos of 8 and 12 DAF were analyzed, obvious signals for the antisense OsmiR408-LNA probe were detected in the radicle and plumule, but not in the plumular axis and the coleoptile (Figure 4, C and D). No signal was detected for the corresponding sense probe (Figure 4E). These results implied that OsmiR408 may contribute to the development of young roots and shoots in young embryos.

We also investigated the activity of the OsMIR408 promoter by fusing a GUS reporter gene to it. In the p408::GUS transgenic plants, staining was undetectable in the roots, stems, young panicles, leaves, and spikelets (Figure 4, F–I), whereas GUS signals were obvious in the embryos (Figure 4, J and K), specifically in the scutella. In contrast, there was no signal in the endosperm (Figure 4, L and M). These results demonstrated that the OsMIR408 promoter is functional and specifically active in the embryos.

OsmiR408 positively regulated grain size

The embryo-specific expression profile of OsmiR408 strongly suggests its contribution to late-embryo development. During this process, the most obvious developmental events are starch content enrichment and grain enlargement. To functionally characterize OsmiR408, the OsMIR408 gene under the control of the 35S promoter (35S::miR408) was genetically transformed into ZH11 plants. We obtained 35 transgenic lines, but only one survived till the reproductive stage. This line was considerably shorter and produced more tillers than the WT plants (Figure 5A). A miRNA northern blot assay revealed that OsmiR408 accumulated in the leaves of 35S::miR408 plants, but was undetectable in WT leaves, confirming the ectopic expression of OsmiR408 (Figure 5B). Furthermore, an increase in the leaf angle was detected in the 35S::miR408 line, suggestive of possible BR signaling disruption (Figure 5C). However, this transgenic line was infertile, implying that ectopic expression of the late-embryo specific OsmiR408, seriously disturbed seed development.

Figure 5.

Figure 5

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Functional analysis of OsmiR408 in rice. A, Overall morphology of the 35S::miR408 transgenic and WT plants. Bar was 10 cm. B, miRNA northern blot analysis of OsmiR408 in the 35S::miR408 and WT plants. C, Leaf angle of the 35S::miR408 and WT plants. Bar was 1 cm. D, Sequence deletions in homozygous miR408KO-1 and miR408KO-2 plants. Red bases represent the mature OsmiR408 sequences, whereas red dashes indicate the deletions in the 408KO-1 and 408KO-2 lines. E, miRNA northern blot analysis of OsmiR408 transcripts in the miR408KO-1 and miR408KO-2 plants. F, Grain morphology of the WT, miR408KO-1, and miR408KO-2 lines. G, Statistical analysis of the grain length and width of the WT and the 408KO lines (n = 3). H, Statistical analysis of the KGW of the WT and 408KO lines (n = 3). Comparisons were made compared to the respective WT unless otherwise indicated. Error bars represent ± sd, single and double asterisks in the statistical analyses represent significant difference determined by the Student’s t test at *P < 0.05 and **P < 0.01.

We subsequently constructed OsmiR408 knockout lines using CRISPR technology. Guide DNA was designed on the basis of the genomic sequence downstream of the mature OsmiR408, with three nucleotides overlapping with the mature OsmiR408 sequence. The T0 generation included more than 30 transgenic lines, with the homozygous deletion of the sequence surrounding the mature OsmiR408 region confirmed for two lines (Figure 5D). The deletion completely “knocked out” OsmiR408 (Figure 5E). The elimination of OsmiR408 resulted in various developmental defects, as indicated by the phenotypes of the 408KO-1 and 408KO-2 lines. Specifically, the grains were smaller than those of the WT plants (Figure 5F), with a decreased grain length, grain width, and KGW (Figure 5, G and H). In contrast to the MIM396 and GRF8OE plants, the cells in the husk of the 408KO-1 and 408KO-2 lines were shortened (Supplemental Figure S2, E and F). Accordingly, OsmiR408 positively regulated grain size.

Genetic interaction between OsmiR396/GRF8 and OsmiR408

Because OsmiR396 negatively regulates grain size, while OsGRF genes and OsmiR408 positively regulated grain size, and both OsGRF genes and OsmiR408 could express in the embryo around 10 DAFs, we wondered whether there is any genetic interaction between OsmiR396 and OsmiR408. To figure out, we crossed the MIM396 plants with 408KO-2 plants. In the resulting F2 generation, the cross plants homozygous for the OsmiR408 deletion site were verified by sequencing, and the overexpression of the mimicry miR396 was verified by upregulation of the backbone ips gene (Figure 6A). The cross plants showed a medium grain size between that of MIM396 and 408KO-2 (Figure 6B), with the grain length and 1,000 KGW characters showed medium values (Figure 6, C and D), while grain width is similar to that of the WT (Figure 6C), indicating that 408KO-2 could partially complement the large-grain phenotype of MIM396 through grain length.

Figure 6.

Figure 6.

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Morphological analysis of the cross between MIM396 and 408KO-2 as well as biochemical analysis of the binding of OsGRF8 to the OsMIR408 promoter. A, Expression analysis of the IPS gene in the ZH11, MIM396, 408KO-2, and cross plants (n = 3). B, Grain morphology of the ZH11, MIM396, 408KO-2, and cross plants. C, Grain length and width of the ZH11, MIM396, 408KO-2, and cross plants (n = 3). D, Statistical analysis of the KGW of the ZH11, MIM396, 408KO-2, and cross plants (n = 3). E, Schematic representation of the 2-kb OsMIR408 promoter including the positions of the putative GRF-binding motifs as well as the relative sites for the YOH, EMSA, and ChIP assays. The black line represents the OsMIR408 promoter, with the gene coding direction indicated by the arrowhead. The red bar indicates the mature OsmiR408 region. The blue bars indicate the predicted GRF-binding motifs. The pink and blue squares, respectively, indicate the normal and truncated regions used for the EMSA. The orange squares indicate the regions used for the YOH. The green squares indicate the regions used for the ChIP assay. F, The YOH assay of OsGRF8 and the OsMIR408 promoter. The fragments used for the YOH assay (YOHP1 and YOHP2) are indicated in (E). Mutations were introduced into the respective primers (Supplemental Table S2). G, EMSA assay of the OsGRF8 WRC domain and the normal OsMIR408 promoter fragments containing the putative motifs and the truncated fragments lacking the putative motifs. H, ChIP analysis of the GRF8OE and WT plants. The ChIP1, 2 and 3 sites ware indicated in (E) (n = 3). ChIP0 refers to a site upstream of the OsMIR408 promoter where there is no putative GRF-binding motif (internal reference). Comparisons were made compared to the respective WT unless otherwise indicated. Error bars represent ± sd, single and double asterisks in (A), (C), and (D) represent significant difference determined by the Student’s t test at *P < 0.05 and **P < 0.01.

Direct regulatory effect of OsGRF8 on OsmiR408

We hypothesized that OsGRF8 might directly regulate OsmiR408. Additionally, the molecular basis for the genetic interaction between OsmiR396 and OsmiR408 might involve the transcriptional regulation of OsMIR408 by OsGRFs. Therefore, we analyzed the possible direct regulatory effects of OsGRF on OsmiR408. The 2-kb OsMIR408 promoter contains 14 putative GRF-binding motifs (Figure 6E). We first performed a yeast one hybrid (YOH) assay to detect the possible binding of OsGRF8 to these motifs. The YOHP1 fragment containing the first GRF-binding motif and the YOHP2 fragment containing the fourth and fifth GRF-binding motifs in the OsMIR408 promoter were able to be bound by OsGRF8 in yeast cells. The interactions were not detected when the motifs were mutated (Figure 6F).

The DNA-binding domain of GRFs is the conserved WRC (zinc finger; Kim et al., 2003). We cloned the WRC domain and completed an electrophoretic mobility shift assay (EMSA). The WRC domain of OsGRF8 was able to bind to the EMSAP1 and EMSAP2 fragments in the OsMIR408 promoter in vitro, but not to the respective truncated fragments lacking the GRF-binding motifs (Figure 6G).

Finally, we performed a chromatin immunoprecipitation (ChIP) assay using the anti-GFP antibody to analyze the GRF8OE plants. The GRF8–GFP fusion protein was enriched in the motif-containing regions of the OsMIR408 promoter (Figure 6H).

Hormone pathways might contribute to the OsmiR396-mediated regulation of rice grain size

Studies on the mechanism controlling seed sizes revealed that the plant hormone signaling pathways have important regulatory effects (Li and Li, 2016; Li et al., 2019). To identify the hormones that may be involved in the OsmiR396-mediated grain size regulation, we analyzed the expression of OsmiR396 members following BR, NAA, and GA treatments, which are considered to regulate grain size. The BR treatment substantially downregulated the expression of OsmiR396b and OsmiR396f, but upregulated that of OsmiR396d as well as OsmiR396e at some time points (Supplemental Figure S8A). However, the OsGRF1, OsGRF8, and OsGRF9 expressions were considerably up-regulated by BR treatment (Supplemental Figure S8B). In response to NAA treatment, the OsmiR396d, OsmiR396e, and OsmiR396f expressions were increased, but OsmiR396b expression was decreased (Supplemental Figure S8C). Moreover, these OsmiR396s were most obviously responsive to NAA after 4 h. In contrast, OsGRF1 and OsGRF9 expression levels exhibited a general downregulated trend, whereas OsGRF8 expression was slightly upregulated (Supplemental Figure S8D). In plants treated with GA, the expression of all OsmiR396s was upregulated before the 4-h time point, after which it decreased (Supplemental Figure S8E). Compared with the effects of NAA treatment, the OsmiR396s responded to GA treatment varied before the 4-h time point, indicating that GA can induce rapid changes to OsmiR396 expression. Accordingly, the OsGRF8 and OsGRF9 expressions were obviously downregulated (Supplemental Figure S8F). These findings revealed that the expression of the OsmiR396/GRF module was extensively influenced by BR, NAA, and GA, and coordination among them may contribute greatly to rice grain size regulation.

Discussion

Grain size is comprehensively characterized by grain length, grain width, L/W ratio, thickness. Among which, the grain length and so that the L/W ratio is most variable and might considerably affect the grain weight (Huang et al., 2013). In this study, we revealed clear increases in the grain weight of MIM396 plants (Figure 1C). This increase was mainly due to increases in the grain length, but not the grain width (Figure 1, A and B). Consistently, both the starch granule and the husk cells were enlarged (Supplemental Figure S2, B–D), indicating that miR396 influence grain size through regulating cell elongation/expansion, consistent with the study of Zhang et al. in rice (Zhang et al., 2020), and in Arabidopsis, miR396 function to regulate cell proliferation (Rodriguez et al., 2016). Furthermore, most of the targets of OsmiR396, including OsGRF1 (Figure 2) and OsGRF4 (Che et al., 2015; Hu et al., 2015) regulate grain size by increasing the grain length and width. However, we observed that OsGRF8 positively regulates grain length, but has the opposite effect on grain width (Figure 2H), resulting in grains similar to those of the MIM396 plants (Supplemental Figure S7). Thus, OsGRF8 might be a major factor influencing grain size. More specifically, it may be involved in OsmiR396-mediated regulation of the grain length. Considering the function conservation of the miR396/GRF modules, our study does not obviate the participation of other OsGRFs in the miR396 mediated grain size regulation, as illustrated by Zhang et al., besides OsGRF8, OsGRF4, and OsGRF6 might mediate the miR396e and miR396f mediated grain size regulation (Zhang et al. 2020). At the same time, OsGRF4 is identified as GS2 QTLs in regulating grain size (Hu et al., 2015), and OsGRF6 is the major mediator in miR396 regulation of panicle development (Gao et al., 2015).

Several signaling pathways have been revealed to control grain size (Li and Li, 2016). Additionally, a number of genes/QTLs related to grain size regulation have been identified (Zuo and Li, 2014). However, the crosstalk between these pathways and genetic factors, as well as the underlying regulatory network remains unclear. Therefore, to further our understanding of the genetic and molecular mechanism that determines rice grain size, the functions of the underlying factors must be elucidated. Doing so may enable researchers to breed new rice varieties with yields that can satisfy the increasing demand for rice by a growing global population (Sakamoto and Matsuoka, 2008; Xing and Zhang, 2010; Miura et al., 2011; Tilman et al., 2011). miRNAs are crucial mediators of various gene expression programs underlying plant development and physiological changes, including phase transitions and alterations to plant architecture (Rubio-Somoza and Weigel, 2011). Sequestering OsmiR396 leads to substantial increases in grain weight, with the KGW of some lines increased by more than 30%. Moreover, overexpression of OsGRF4 also increases grain weight (Che et al., 2015; Hu et al., 2015). We revealed that sequestering OsmiR396 positively affects grain size in japonica and indica subspecies. Moreover, the increased grain weight of MIM396 plants is accompanied by a high panicle density and an increase in the number of tillers (Supplemental Figure S4), which are two other important factors that contribute to yield promotion. Therefore, OsmiR396 is a mighty and stable regulator of grain size that not easily influenced by other factors.

miRNA networks regulate plant development. For example, developmental stage transitions are regulated by miR156 and miR172 (Wang, 2014), whereas leaf size is controlled by miR319 and miR396b (Schommer et al., 2014). These networks may be established at the transcriptional level (Wu et al., 2009; Schommer et al., 2014) or through interactions between the miRNA-targeted transcription factors (Rubio-Somoza et al., 2014). Some important aspects of these networks need to be clarified to elucidate the functional complexity of miRNAs, and the coordinated activities of miRNAs in specific processes need to be determined (Rubio-Somoza and Weigel, 2011). In this study, we verified that OsmiR396 and OsmiR408 could genetically interact with each other and form pathway bridged by OsGRF8, to coordinately modulate grain size (Figure 6). Furthermore, our miRNA microarray analysis revealed that many miRNAs were influenced by OsmiR396 (Figure 3), several of which have already been identified as grain size modulators, it is highly likely that the OsmiR396/GRF module is a central component of the mechanism regulating grain size. Accordingly, OsmiR396 and other miRNAs might regulate grain size via coordinated activities, but the functions of these miRNAs remain to be investigated.

We identified OsmiR408 as an embryo-specific miRNA, with peak expression levels at 8–12 DAF (Figure 4, A and B), which coincides with the starch development and grain filling period. Accordingly, OsmiR408 positively regulates rice grain size (Figure 5). In an in situ hybridization assay, the OsmiR408 signal was concentrated in the radicle and plumular axis of young embryos (Figure 4, C and D), but the GUS protein produced by the OsMIR408 promoter-driven expression of the GUS gene was concentrated in the scutella (Figure 4, L and M). This inconsistency might reflect the differences in the promoter activities for protein coding and noncoding genes. Earlier research confirmed that OsmiR408 helps regulate grain size, with the overexpression of it resulting in larger grains (Zhang et al., 2017). However, we determined that the overexpression of OsmiR408 leads to dwarfism, an increase in the leaf angle, and infertility. This discrepancy may be associated with differences in the extent of the OsmiR408 upregulation. Furthermore, we demonstrated that the grains the OsmiR408 knockout plants were smaller than normal, providing further evidence of the positive effects of OsmiR408 on rice grain size.

Hormone signaling pathways along with other factors are important for controlling seed size. Among these pathways, BR signaling is the most extensively studied. BR is generally a positive regulator of grain size, and GSK2 is a negative regulator of BR signaling (Tong et al., 2012). Additionally, GSK2 is involved in several pathways influencing grain size, including those related to GS2 (OsGRF4; Che et al., 2015), GS9 (Zhao et al., 2018), GW5 (Liu et al., 2017). Moreover, Big Grain1 (BG1) regulates grain size via the auxin signaling pathway (Liu et al., 2017). Previously, we revealed that OsmiR396 regulates panicle branching by modulating the auxin biosynthesis pathway (Gao et al., 2015), and Tang et al. found that miR396d regulates rice architecture through both the BR and GA signaling pathway (Tang et al., 2018). OsmiR159 and its target genes regulate grain size and affect both BR and GA signaling (Gao et al., 2018), indicating GA signaling influences grain size. In this study, we revealed that BR, NAA, and GA treatments induced extensive expression-level changes to almost all OsmiR396 members and at least some of their target GRF genes (Supplemental Figure S8). Thus, OsmiR396 and hormone signaling pathways might coordinately mediate grain size regulation.

Therefore, OsmiR396 is likely a pivotal regulator of rice grain size. Elucidating the network consisting of OsmiR396 and other miRNAs and hormone signaling pathways might reveal new regulatory mechanisms underlying grain development. We propose that the OsmiR396/OsGRF module may be potentially useful for breeding new rice varieties with increased grain size and yield.

Materials and methods

Plant material and trait measurements

The WT rice varieties used in this study were ZH11 (Oryza sativa L. subsp. japonica), XS134 (Oryza sativa L. subsp. japonica) and R8015 (Oryza sativa L. subsp. japonica), and YuetaiB (Oryza sativa L. subsp. indica). Rice plants were grown in a paddy field or in pots in a greenhouse under standard growth conditions.

Grain length, width and the KGW were measured using fully filled grains after maturation. Panicle length and plant height were measured on the main culm, tiller number counting was carried out before harvest when there is no more effective tillers producing.

At least 30 grains with three biological repeats were calculated for grain length and width analysis. The KGW was calculated by measuring 100 grains in three biological repeats. Panicle length and plant height was measured on the main culm with three biological repeats, 30 plants were used in each biological repeat. Tiller number was determined by counting the effective tillers of 30 plants with three biological repeats. Datum was shown as mean ± sd.

SEM analysis

The corresponding places in the lemma of MIM396, GRF8OE, and ZH11 were used for scanning using ZEISS Merlin Compact, and the average length of the epidermis cells were calculated by measuring 10 cells each time at 10 repeats. The grains were tentatively cut and handily broken in halves, and the starch granules in cross surfaces were scanned.

Isolation of young embryos

Flowers were marked when the palea and lemma are opening for pollination. And the developmental status of the embryos was indicated by the DAF. Before 3 DAF, the young seed composed of the embryo and the endosperm is <3 mm in length, and it is difficult to separate the embryo and endosperm apart, so that we used the whole young seed to represent the embryo. At other stages, the embryo and endosperm were separated manually, and collected in liquid Nitrogen as quickly as possible.

Vector construction and transformation of rice and Arabidopsis

Vector over-expressing OsmiR396 target mimicry (MIM396) and OsGRF8 (pHBGRF8-GFPOE) was constructed previously (Gao et al., 2015; Dai et al., 2019).

For OsGRF1 and OsGRF9 over-expression, full-length respective cDNA was amplified by primers GRF1OEF and GRF1OER, and GRF9OEF and GRF9OER and cloned into pHBGFP vector under 2 × 35S promoter. For over expression of OsMIR408 and OsMIR396b, pre-miR408 and pre-miR396b were amplified and cloned into the p1301-35SNos vector, respectively. For construction of p408::GUS, the 1.5-kb promoter of OsMIR408 was amplified and cloned into the p1301proGUSnos vector. For knocking out of OsmiR408 and OsGRF8, guider DNA was synthesized through annealing oligo 408casF and 408casR, and GRF8cas9F and GRF8cas9R respectively, and cloned in the pOs-sgRNA vector, and then transferred to the pH-Ubi-cas9-7 vector through LR reaction (Miao et al., 2013).

miR396dOE grains were kindly provided by Professor Kang Chong from Institute of Genetics and Developmental Biology, Chinese Academy of Sciences. The GS2ZH11 grains were kindly provided by Professor Qianqian from Zhejiang Academy of Agricultural Sciences.

Rice transformation was carried out by agrobacterium-mediated method (Hiei et al., 1994).

Histochemical GUS staining

Histochemical GUS staining was carried out as previously described (Dai et al., 2018).

RNA isolation and RT–qPCR analysis

Total RNAs were extracted using TRIzol (Life technologies) and reverse transcribed using the First Strand cDNA Synthesis Kit (Toyobo). RT-qPCR was performed with the SYBR Green Real-time PCR Master Mix Kit (Toyobo), and actin was used as an internal reference. Each sample was performed in triplicate and the mean value of technical replicates was recorded for each biological replicate. Data from three biological samples were collected, and the mean value with standard error (se) was plotted.

For BR, NAA and GA treatment, 3-week-old rice seedlings were sprayed with 1 µm BR, 100 µM NAA and 10 µM GA, respectively, and leaves were collected after 0, 0.5, 1, 2, 4, 6, 9, 12 h for RNA extraction and RT-qPCR analysis.

Yeast-one-hybrid assays

The full-length cDNAs of OsGRF8 were amplified and fused with the activation-domain (AD) of pPC86 vector. Fragments each containing two putative GRF binding motifs in OsMIR408 promoter were amplified and fused into vector p178 at the XhoI site to get p178:408P1 and p178:408P2. The fragments with mutant GRF-binding motifs were constructed based on the p178:408P plasmids.

The respective p178-derived and pPC86-derived constructs were transformed into the yeast (Saccharomyces cerevisiae) strain EGY48 and grew on SD selective medium (SD-His-Leu) and observed on Chromogenic medium. Void plasmid pPC86 and p178 constructs were used as negative controls.

miRNA northern blot analysis and RT-qPCR

miRNA northern blot was carried out as previously described (Dai et al., 2019). RT-qPCR of miRNA was carried out as described (Chen et al., 2005).

In situ hybridization

In situ Hybridization was carried out as described (Dai et al., 2016).

ChIP analysis

ChIP analysis was carried out as described (Dai et al., 2019).

EMSA

For protein expression and purification, the DNA binding domain of OsGRF8 (WRC) was cloned into the pET44b vector and transformed into Escherichia coli strain BL21 to produce His-tagged fusion protein. His-WRC fusion protein was induced by adding 0.5 mM isopropyl-d-1-thiogalactopyranoside to the culture medium and incubating the cells for 14 h at 20°C and purified using Ni-NTA (nitrilotriacetic acid) agarose (GenScript) according to the manufacturer’s instructions. The EMSAP1, EMSAP2, EMSAP1t, and EMSAP2t DNA probes from the OsMIR408 promoter were synthesized and cy5 labeled. The DNA probes and proteins were co-incubated in the reaction buffer, purified and incubated with the Cy5-labeled probe at 25°C for 20 min in EMSA buffer (25 mM HEPES (pH 7.5), 40 mM potassium chloride, 3 mM dithiothreitol, 10% (v/v) glycerol, 0.1 mM EDTA, 0.5 mg/ml bovine serum albumin, 0.5 mg/ml poly-glutamate). After incubation, the reaction mixture was electrophoresed on a 6% native polyacrylamide gel, and then labeled DNA was detected using a Starion FLA-9000 instrument (Fujifilm, Tokyo, Japan).

miRNA microarray and data analysis

miRNA microarray was carried out under the help of Oebiotech company. Briefly, Total RNAs were extracted from 6-cm young panicles of the MIM396 and WT plants, and quantified by the NanoDrop ND-2100 (Thermo Scientific) and the RNA integrity was assessed using Agilent 2100 (Agilent Technologies). The sample labeling, microarray hybridization and washing were performed based on the manufacturer’s standard protocols. Briefly, total RNA were tailed with PolyA and then labeled with Biotin. Then, the labeled RNAs were hybridized onto the microarray. After washing and staining the slides, the arrays were scanned by the Affymetrix Scanner 3000 (Affymetrix).

Affymetrix GeneChip Command Console software (version 4.0, Affymetrix) was used to analyze array images to get raw data and then offered RNA normalization. Next, Genespring software (version 14.9; Agilent Technologies) was used to proceed the following data analysis. Differentially expressed miRNAs were then identified through fold change as well as P value calculated using t test. The threshold set for up- and downregulated genes was a fold change ≥2.0 and a P ≤0.05. Target genes of differentially expressed miRNAs were the intersection predicted with databases (Targetscan, PITA, microRNAorg). GO analysis and KEGG analysis were applied to determine the roles of these target genes. Hierarchical Clustering was performed to show the distinguishable miRNAs expression pattern among samples.

Primer sequences

All primer sequences used in this study are listed in Supplemental Table S2.

Accession numbers

The ID of protein coding Genes referenced in this article can be found in the Rice Genome Annotation Project under the following accession numbers: OsGRF1 (LOC_Os02g53690), OsGRF2 (LOC_Os06g10310), OsGRF3 (LOC_Os04g51190), OsGRF4 (LOC_Os02g47280), OsGRF5 (LOC_Os06g02560), OsGRF6 (LOC_Os03g51970), OsGRF7 (LOC_Os12g29980), OsGRF8 (LOC_Os11g35030), OsGRF9 (LOC_Os03g47140), OsGRF10 (LOC_Os02g45570), OsGRF11 (LOC_Os07g28430), OsGRF12 (LOC_Os04g48510). The MIRNA genes could be searched in the miRbase using their respective name (http://www.mirbase.org/).

Supplemental data

The following materials are available in the online version of this article.

Supplemental Figure S1. Grain characteristics and KGW of the YM396 and WT YuetaiB plants.

Supplemental Figure S2. Northern blot analysis of the expression of each mature OsmiR396 in the MIM396 and WT plants, and SEM analysis of the husk of MIM396, GRF8OE,408KO-1, 408KO-2 and ZH11 plants, and SEM analysis of the starch granules of the MIM396, GRF8OE and ZH11 plants.

Supplemental Figure S3. Grain morphology of the transgenic plants overexpressing OsmiR396b or OsmiR396d.

Supplemental Figure S4. Phenotypic analysis of the plants derived from hybridization between MIM396 and A989 plants, and between MIM396 and cd mutant plants.

Supplemental Figure S5. Statistical analysis of some characteristics of the hybrids derived from crosses between the MIM396 and A989 plants as well as between the MIM396 and cd mutant plants.

Supplemental Figure S6. Analysis of the GRF8KO lines.

Supplemental Figure S7. Grain shape of the ZH11, MIM396, GRF1OE, GRF8OE, and GS2 (GRF4OE) lines.

Supplemental Figure S8. Responses of the OsmiR396s and OsGRF1, OsGRF8, and OsGRF9 to BR, NAA, and GA treatments.

Supplemental Table S1. The filtered differentially expressed miRNAs in the miRNA microarray analysis of the MIM396 and WT plants.

Supplemental Table S2. The oligo sequences used in this study.

Supplementary Material

kiab084_Supplementary_Data
Click here for additional data file. (4.7MB, zip)

Acknowledgments

We are very grateful to Prof. Kang Chong from Institute of Genetics and Developmental Biology, Chinese Academy of Sciences for providing us the OsmiR396dOE grains, Prof. Qianqian from Chinese National Rice Research Institute for providing us with the GS2ZH11 grains, and Prof. Shaoqing Li from Wuhan University for providing us the YM396 grains. We thank Liwen Bianji, Edanz Editing China (https://www.liwenbianji.cn/ac) for editing the English text of a draft of this manuscript.

Funding

This work was supported by the National Key R&D Program of China (2016YFD0100603), the National Natural Science Foundation of China (32072029, 31870232, 31371949), the National Transgenic Great Subject from the Ministry of Agriculture of China (2016ZX08009-003-001), and the grant from State Key Laboratory of Hybrid Rice (KF201805).

Conflict of interest statement. The authors declares no conflict of interest.

 

X. Y. and X. Z. carried out the function analysis of the miR396, Z. D. carried out the expression and function analysis of the miR408-2, X. Z. carried out the genetic analysis of the MIM396 plant and the miR408KO, X. Y. and F. M. carried out the biochemical experiments, X. M. and Z. S. designed the project and wrote the MS.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (https://academic.oup.com/plphys/pages/general-instructions) is: Zhenying Shi (zyshi@cemps.ac.cn).

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