WRKY72 Negatively Regulates Seed Germination Through Interfering Gibberellin Pat

Letter

WRKY72 Negatively Regulates Seed Germination Through Interfering Gibberellin Pathway in Rice

Seed germination is associated with grain yield and quality in crop production. Gibberellic acid (GA) serves as a major phytohormone in the promotion of seed germination. It is synthesized in the embryos and transmitted to the aleurone layers, where GA triggers the synthesis and secretion of a set of hydrolases, especially α-amylase. Subsequently, the storage nutrients such as starch in the endosperm are digested by these hydrolases and absorbed by the embryo to sustain seed germination and early seedling establishment (Kaneko et al, 2002). The detailed GA biosynthesis process has been well studied and thoroughly reviewed in several literatures (Sakamoto et al, 2004; Reinecke et al, 2013). Briefly, geranylgeranyl diphosphate (GGDP) is turned into-kaurene by two terpene synthases,-copalyl diphosphate synthase (CPS) and-kaurene synthase (KS). Subsequently, the conversion of GA precursor-kaurene to-kaurenoic acid is catalyzed by-kaurene oxidase (KO), and that from-kaurenoic acid to GA12is catalyzed by-kaurenoic acid oxidase (KAO). Ultimately, GA12is converted to various GA intermediates and bioactive GAs by GA20-oxidase (GA20ox) and GA3-oxidase (GA3ox), respectively.

WRKY transcription factors (TFs), one of the largest TF families in higher plants, usually bind to the W-box motif (T)(T)TGAC(C/T) in the promoter of the downstream target genes (Eulgem et al, 2000; Ulker and Somssich, 2004). Several WRKY family members have been reported to participate in GA-mediated seed germination in the past decade. In, AtWRKY27, which is directly regulated by GA signaling component RGA, is involved in GA-mediated seed germination (Zentella et al, 2007). Rushton et al (1995) reported that AfWRKY1 and AfWRKY2 inhibit the expression of α-amylase, therefore delay seed germination in. OsWRKY51 and OsWRKY71, which are homologous of AfWRKY1 and AfWRKY2, function as heterologous dimers and interact with GA signal positive regulator GAMYB to inhibit the expression of α-amylase in rice (Zhang et al, 2004; Xie et al, 2006). Here we report that WRKY72 acts as a negative regulator in rice seed germination by restricting GA accumulation through modulating ‘’ pathway, which would provide novel insights into the finely regulated mechanism of WRKY72-mediated seed germination in rice.

Previous studies have shown thatis predominantly expressed in rice developing seeds, especially in aleurone layers, indicating it can participate in the regulation of seed maturation or germination (Xie et al, 2005; Hou et al, 2019). In this study, we mainly focused on the role ofin rice seed germination process. Firstly,over-expression lines () andmutants () were generated. Two independentover-expression lines (and) showed about 90-fold higher transcript level compared with the wild type (WT) (Fig. S1-A). Twomutants (and) harbored a G insertion and a T insertion in the 1st exon ofrespectively, which shifted the open reading frame, though the transcript level ofremained unchanged (Fig. S1-B to -D). Seeds of T2generation from bothandlines were subjected to seed germination assay. The germination rates oflines were significantly lower than that of the WT (Fig. 1-A). In consistent with the retarded seed germination, the seedling heights oflines were also lower than that of the WT (Fig. 1-B and -C). However, the germination rates and seedling growths oflines were similar to the WT, possibly due to its functional redundancy with other WRKY family members (Fig. 1-A to -C). As GA is a major activating phytohormone in seed germination, the retarded seed germination ofseeds intrigued us to measure the endogenous GA level inas well as the WT. The results showed that the GA3content was significantly reduced in thegerminating embryos, indicating thatdefected in GA accumulation, rather than GA signaling (Fig. 1-D). As expected, the retarded germination rates and seedling growths oflines were restored to the same level as the WT when 1.5 µmol/L exogenous GA3was applied (Fig. 1-E to -G). Hence, the suggestion is that WRKY72 inhibits seed germination at least partly by blocking GA accumulation.

Fig. 1. Seed germination characteristics of overexpression linesand mutant lines.

A,Germination time courses of the wild type (WT), overexpression linesand mutant lines, respectively. B, Germination phenotypes of the WT,andgrown on 1/2 Murashige and Skoog (MS) medium for 4 d. Scale bars, 1 cm.C, Seedling heights of the WT,andin accordance to B. D,GA3content in the germinating embryos of the WT and. E, Germination time courses of the WT andunder mock or 1.5 µmol/L GA3treatment. F, Germination phenotypes of the WT andunder mock or GA3treatment for 4 d. Scale bars, 2 cm. G, Seedling heights of the WT andlines in accordance to F. Error bars indicate SD with triple biological replicates (each replicate containing 50 seeds) in A andE, 50 biological replicates in C andG, and triple biological replicates in D. Asterisks indicate the significant differences between the WT and transgenic lines as determined by the Student’stest analysis. **,< 0.01.

Germinating embryos ofand the WT grown on half-strength Murashige and Skoog(MS) medium for 2 d were collected for RNA-sequencing (RNA-seq)assay to clarify the regulatory mechanism underlying the WRKY72-governed seed germination. As a result, we totally identified 2457 differentiallyexpressed genes (DEGs), including 727 down-regulated and 1730 up-regulated genes in(|log2 ratio|≥1; False discovery rate <0.01) (Table S1). To validate the transcriptome analysis, 13 DEGs, which are functionally relevant to GA biosynthesis or seed germination, were selected for gene transcript abundance verification (Table S2). As shown in Fig. 2-A, the transcript levels of most of the selected genes were consistent with the RNA-seq results, suggesting the high-reliability of the RNA-seq data. Interestingly, among these detected DEGs, several have been reported to be functionally involved in GA biosynthesis or metabolism. For example,(gibberellin 20 oxidase 2, a major GA biosynthesis enzyme) () was down-regulated in, and mutation ofreduces GA biosynthesis and thereby delays seed germination (Ye et al, 2015).(a C2C2-type zinc finger protein) () was also reduced in, and it can interact with OsbZIP58 to promote seed germination through activating the gibberellin biosynthesis gene(Wu et al, 2014).(a leucine-rich repeat receptor-like kinase, LRR-RLKs)() was significantly elevated in, and it represses GA biosynthesis through inhibiting the activity of the GA biosynthesis enzyme OsKO2 (Itoh et al, 2004; Yang et al, 2013). We further analyzed the-element distribution in the promoter region of these selected DEGs using the online tool PlantCARE(http://bioinformatics.psb.ugent.be/webtools/plantcare/html/), and found that onlyandcontain W-box (TTGAC[C/T]) or W-box like (TGAC[C/T])-elements (Fig. S2). Theincreased transcript levelofinindicated thatmight be involved in WRKY72-mediated GA biosynthesis repression(Fig. 2-A). Therefore, we mainly focused on whethercan be the direct target of WRKY72. To test this hypothesis, the EMSA(electrophoresis mobility shift assay) was firstly performed to detect the DNA binding ability of WRKY72 with. As shown in Fig. 2-B and -C, GST-WRKY72 protein can bind to the probe 3 (P3), which contains a conserved W-box motif close to the transcription starting site, andthe shift band signal was gradually weakened by the addition of unlabeled, competitive P3 probe in a dosage-dependentmanner, suggesting that this binding is highly specific (Fig. 2-B and -C). Subsequently, ChIP-qPCR (chromatin immunoprecipitation-quantitative PCR) assay was performed to validate the binding pattern of WRKY72 onpromoter. In consistent with the results of EMSA, WRKY72 was significantly enriched in the P3 region ofpromoter, while there was no significant enrichment in the other fragments, except that P1 region located inpromoter exhibited slightly WRKY72enrichment,strongly suggesting that the W-box in the P3 region acts as a core binding site for WRKY72 (Fig. 2-B and -D).Finally, a dual-luciferase (LUC) transient transcriptional activity assay was performed to determine the regulatory effect of WRKY72 ontranscription (Fig. 2-E and -F). In comparison with the empty effector,drastically elevated the transcript level ofreporter, but such induction was significantly reduced when the W-box in the P3 promoter region ofwas mutated, which was in accordancewith the transcription pattern ofintransgenic lines (Fig. 2-A, -E and -F). Taken together, these experiments clearly demonstrated that WRKY72 specifically binds to thepromoter containing a W-box-element and induces the latter’s transcription.

Fig. 2. WRKY72 mediates seed germination by WRKY72--pathway.

A, Real-time PCR (qRT-PCR) validation of the differentially expressed genes (DEGs) revealed by RNA-sequencing (RNA-seq) experiments. cDNA of germinating embryos grown on 1/2 Murashige and Skoog (MS) medium for 2 d was used as templates. B, Probe positions onpromoter and genome. Grey, black and yellow boxes represent untranslational regions, coding sequence and promoter regions, respectively. Transcription starting site (TSS) was set as 0. Numbers indicate the distances (bps) to the TSS. C, Electrophoretic mobility shift assay (EMSA) to show GST-WRKY72 specifically binds with the probe 3 (P3) region on the promoter ofin B. Purified GST, GST-bZIP72 was detected with anti-GST antibody. The 5-, 10- and 100-fold excess non-labeled probes were applied for competition. D, Chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) assay to show WRKY72 binding to the promoter regions of. P1‒P7 represent the regions shown in B detected by ChIP-qPCR, respectively. The enrichment values were normalized to the Input. IgG immunoprecipitated DNA was used as a control. E and F, Luciferase (LUC) transient transcriptional activity assay in rice protoplast., The promoter ofwith G-box mutated. G, qRT-PCR analysis for the transcript accumulation ofingerminating embryos grown on half-strength MS medium for 2 d. H, Germination time courses of the wild type (WT) andunder mock, 3 µmol/L-kaurenoic acid or 10 µmol/L paclobutrazol (PAC) treatments, respectively. I, Germination phenotypes of the WT andunder mock,-kaurenoic acid or PAC treatments for 4 d. Scale bars, 2 cm. J,Seedling heights of the WT andin accordance to I. Data represent Mean ± SD (= 3) in A, D, F and G,= 3 (each replicate containing 50 seeds) in H, and= 50 in J. Asterisks indicate the significant differences as determined by the Student’stest analysis (*,< 0.05; **,< 0.01).

It is reported thatrestricts rice internode elongation through suppressingand thereby results in reduced endogenous GA level (Yang et al, 2013). OsKO2, a key-kaurene oxidase, promotes GA biosynthesis by catalyzing GA precursor-kaurene intokaurenoic acid, and the mutation ofcauses severe GA deficiency and dwarf phenotype (Itoh et al, 2004). These evidences intrigued us to speculate that the function of OsKO2 could be interrupted by WRKY72. In consistent with the up-regulation of, thetranscription was significantly reduced in(Fig. 2-G). Moreover, the effects of-kaurenoic acid and paclobutrazol (PAC, a KO inhibitor) (Swain et al, 2005) were further determined on the seed germination of. Interestingly,-kaurenoic acid, the product of OsKO2 catalyzed reaction, fully restored the delayed seed germination and seedling growth of(Fig. 2-H to -J). On the contrary, PAC significantly inhibited the seed germination and seedling growth of all the tested seeds (Fig. 2-H to -J). These results strongly suggested that WRKY72 negatively regulates seed germination and GA accumulation via the ‘WRKY72-’ pathway.

Up to date, over 100 WRKY gene family members have been identified in rice (Ramamoorthy et al, 2008). Rice WRKY proteins have been shown to regulate the cross-talk between multiple hormone-mediated signaling pathways in various biological processes, but most notably in biotic stress responses (Qiu et al, 2007; Peng et al, 2012; Wang et al, 2015). Previous studies have shown thatis induced by polyethylene glycol, NaCl, naphthalene acetic acid, abscisic acid (ABA) and heat stress in rice, indicating the versatile roles of WRKY72 in multiple physiological processes (Song et al, 2010). Very recently, our group revealed that WRKY72 acts negatively in rice resistance to bacterial blast disease through repressing jasmonic acid (JA) accumulation (Hou et al, 2019). WRKY72 can directly bind to the promoter of a key JA biosynthesis gene, and repress thetranscription possibly via a RNA-directed DNA methylation mechanism. Meanwhile, the WRKY72 transrepression activity depends on its phosphory-lation status mediated by SAPK10, which is a core component in ABA signaling (Hou et al, 2019). Hence, WRKY72 likely serves as an important node in the ABA-JA interaction. Due to its predominant expression pattern in rice developing seeds, especially in aleurone layers, WRKY72 might also participate in the regulation of seed maturation or germination (Xie et al, 2005; Hou et al, 2019). Indeed, when WRKY72 is ectopically expressed in, seed germination of the transgenic lines is drastically retarded (Song et al, 2010). Nevertheless, how WRKY72 functions in rice remains unclear. In this study, we revealed that over-expression ofinhibited seed germination and seedling growth (Fig. 1-A to -C). Several cases have demonstrated that WRKYs involve in seed germination by interfering GA biosynthesis or signaling. For example, heterologous dimmers of OsWRKY51 and OsWRKY71 are found to negatively regulate GA signaling through direct interacting with GAMYB, a GA signal positive regulator, and ultimately inhibit the expression of α-amylase (Zhang et al, 2004; Xie et al, 2006). In our case, it is clear that GA-deficiency resulted in the retarded germination and seedling growth of, becauseexhibited reduced endogenous GA level, and the addition of GA completely restored the phenotype (Fig. 1-D to -G). Therefore, WRKY72 can be a key player in the interaction of phythormones including ABA, JA and GA.

Since WRKY72is annotated as a transcription factor, identifying its direct target gene is crucial to clarify the regulatory mechanism underlying the WRKY72-governed seed germination. Our RNA-seq and qRT-PCR analyses identified a long list of DEGs which are functionally related to GA biosynthesis and metabolism. Among the DEGs, a leucine-rich repeat receptor-like kinase (LRR-RLKs), which is up-regulated in, is of particular interest (Fig. 2-A). EMSA experiment, ChIP-qPCR and rice protoplasts transient transcriptional activity assaydemonstrated that WRKY72 canspecifically bind to theW-box-element ofpromoter and activate its transcription, suggesting thatis a direct target of WRKY72 (Fig. 2-B to -D). It is reported thatrestricts rice internode elongation through suppressing the-kaurene oxidaseand thereby results in reduced endogenous GA level (Yang et al, 2013). In agreement with the up-regulation of,was significantly reduced in(Fig. 2-G). OsKO2 has been known as a key enzyme catalyzing the conversion of-kaurene tokaurenoic acid, and mutation ofcauses severe GA deficiency and dwarf phenotype (Itoh et al, 2004). This hypothesis is further supported by the fact that addition of-kaurenoic acid, the product of OsKO2 catalyzed reaction, fully rescued the retarded germination of(Fig. 2-H to -J). Thus, WRKY72 inhibits seed germination and GA accumulation via the‘WRKY72-’pathway.

ACKNOWLEDGEMENTS

This study was supported by the National Natural Science Foundation of China (Grant No. 31701395), the special research funds for the Central Public Research Institute of the China National Rice Research Institute (Grant No. 2017RG002-5) and the special research funds of State Key Laboratory of Rice Biology (Grant No. 2017ZZKT10105).

SUPPLEMENTAL DATA

The following materials are available in the online version of this article at http://www.sciencedirect.com/science/journal/ 16726308; http://www.ricescience.org.

File S1. Methods.

Fig. S1. Molecular characterization ofandmutants.

Fig. S2. Occurrence of-regulatory elements in promoters ofand.

Table S1. Differentially expressed genes between wild type and.

Table S2. Selected differentially expressed genes used for RNA-seq verification.

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Wang Huimei1, Hou Yuxuan1, Wang Shuang1, 2, Tong Xiaohong1, Tang Liqun1, Abolore Adijat Ajadi1, Zhang Jian1, Wang Yifeng1

(State Key Lab of Rice Biology, China National Rice Research Institute, Hangzhou 311400, China; College of Life Science, Yangtze University, Jingzhou 434025, China)

Copyright © 2021, China National Rice Research Institute. Hosting by Elsevier B V

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)

Peer review under responsibility of China National Rice Research Institute

http://dx.doi.org/10.1016/j.rsci.2020.11.001

s:Wang Yifeng (wangyifeng@caas.cn); Zhang Jian (zhangjian@caas.cn)

20 December 2019;

30 May 2020