- Open Access
A sensitive synthetic reporter for visualizing cytokinin signaling output in rice
- Jinyuan Tao†1View ORCID ID profile,
- Huwei Sun†1,
- Pengyuan Gu1,
- Zhihao Liang1,
- Xinni Chen1,
- Jiajing Lou1,
- Guohua Xu1 and
- Yali Zhang1Email author
© The Author(s) 2017
Received: 14 November 2016
Accepted: 3 October 2017
Published: 27 October 2017
Cytokinins play many essential roles in plant growth and development, mainly through signal transduction pathways. Although the cytokinin signaling pathway in rice has been clarified, no synthetic reporter for cytokinin signaling output has been reported for rice. The sensitive synthetic reporter two-component signaling sensor (TCSn) is used in the model plant Arabidopsis; however, whether the reporter reflects the cytokinin signaling output pattern in rice remains unclear.
Early-cytokinin-responsive type-A OsRR-binding element (A/G)GAT(C/T) was more clustered in the 15 type-A OsRRs than in the 13 control genes. Quantitative polymerase chain reaction analysis showed that the relative expression of seven type-A OsRRs in roots and shoots was significantly induced by exogenous cytokinin application, and that of seven OsRRs, mainly in roots, was inhibited by exogenous auxin application. We constructed a transgenic rice plant harboring a beta-glucuronidase (GUS) driven by the synthetic promoter TCSn. TCSn::GUS was expressed in the meristem of germinated rice seed and rice seedlings. Furthermore, TCSn::GUS expression in rice seedlings was induced specifically by exogenous cytokinin application and decreased by exogenous auxin application. Moreover, no obvious reduction in GUS levels was observed after three generations of selfing of transgenic plants, indicating that TCSn::GUS is not subject to transgene silencing.
We report here a robust and sensitive synthetic sensor for monitoring the transcriptional output of the cytokinin signaling network in rice.
The plant phytohormone cytokinin is a key growth regulator involved in regulating a wide range of developmental processes in diverse contexts throughout the plant life cycle [1–4]. Cytokinin regulates cell division mainly through histidyl–aspartyl phosphorelay signal transduction (also known as two-component systems), which consist of a histidine protein kinase that senses the input and a response regulator that mediates the output-control signal transduction pathways in many prokaryotes and in some eukaryotes [5–8]. The Arabidopsis cytokinin signal transduction pathway consists of four principal steps: histidine protein kinase (HK) sensing and signaling; transfer of the phosphoryl group to histidine phosphotransfer protein (HP); response regulator (RR)-dependent transcription activation; and a negative feedback loop through cytokinin-inducible RR genes [4, 6, 9–16].
Although the cytokinin multistep phosphorelay signal transduction pathway is present in Arabidopsis, the identification and functional characterization of the diverse signaling locales is challenging. The distribution of active cytokinin ligands in plants is difficult to determine, mainly due to the synthesis of the cytokinins involved in the complex enzymatic biosynthetic pathways in different cellular compartments  and the low level of precise localization (about 100-fold lower than auxin levels) with derived metabolites and different forms [18–20]. Cytokinin signaling output at the transcriptional level in Arabidopsis has been visualized using a synthetic reporter, revealing the sites of cytokinin action during wildtype development [21, 22]. Nuclear type-B RRs mediate transcriptional activation in response to phosphorelay signaling activity, whereas type-A RRs repress signaling in a negative-feedback loop. The DNA-binding domains of diverse type-B RR family members are conserved and bind a common DNA-target sequence (A/G)GAT(T/C) in vitro [23–25]. Accordingly, the concatemerized type-B ARR-binding motifs are core components of the synthetic reporter [21, 22].
Rice (Oryza sativa L.) is not only a major staple food worldwide but also an important model plant for monocot species research because of its small genome and the availability of its complete genome sequence [26, 27]. The cytokinin signaling pathway in rice has been clarified; however, no synthetic reporter for cytokinin signaling output in rice has been reported. The sensitive synthetic reporter two-component signaling sensor (TCSn) is used in the model plant Arabidopsis; however, whether the reporter reflects the cytokinin signaling output pattern in rice remains unclear. In this study, we analyzed the DNA-binding domains (5′-(A/G)GAT(T/C)-3′) in the promoters of type-A RR family members and examined the changes in the relative expression of type-A RR genes in response to phytohormones. The stronger version of TCSn::GUS was fused into rice plants through genetic transformation. We investigated GUS expression in different generations of transgenic rice seedlings, including in response to exogenous application of six cytokinin fractions and three other phytohormones. We report here a robust and sensitive synthetic sensor for monitoring the transcriptional output of the cytokinin signaling network in rice.
Results and discussion
Amino acid alignment of the Myb-like domains from type-B response regulators
We first selected and analyzed 10 rice type-B positive regulators from the phylogenetic tree described by Tsai et al. . As expected (Additional file 1), the amino acid residues of the C-terminal Myb-like domain [SH(A/L)QKY(R/F)] responsible for their DNA-binding specificity are conserved between rice and Arabidopsis, which is consistent with online registration of a conserved Myb-like motif (accession number: TIGR01557, http://www.jcvi.org/cms/home/). Also, the differences in the sequences of the Myb-like domains may result in regulation of different downstream gene sets in different species.
Analysis of the DNA-binding motif in type-A OsRRs
Cytokinins are classic plant hormones that orchestrate plant growth and physiology and affect gene expression in target cells by activating a multistep phosphorelay network. Type-B RRs, acting as transcriptional activators, mediate the final step in the signaling cascade; simultaneously, type-A RRs are immediate–early target genes of activated type-B RR proteins, which establishes a negative feedback loop to the signaling pathway [4, 15, 16, 19, 22, 29].
To identify the rice type-B RR regulator binding motif (5′-(A/G)GAT(C/T)T-3′) in rice, sharing the conserved pathway with Arabidopsis [30–32], the sequence including the promoter and part of the transcribed sequence of rice type-A RR genes (OsRR1-15) was analyzed (Additional file 2) . Additionally, 13 genes that showed stable expression and had no relationship with stress, developmental, pharmacological, or physiological treatments [34–36], were randomly selected as negative controls. The total length of the 13 control genes (69.7 kb) was similar to that of the 15 rice type-A RR genes (66.9 kb).
We analyzed the frequency of motifs based on total sequence length and gene sequence. In addition to 66.9 kb from the 15 type-A OsRRs and 69.7 kb from the 13 control genes, a 66.9-kb sequence was randomly selected from the NCBI GenBank database for use as negative control sequences (Additional file 2). The motif number, distance between neighboring motifs, and relative orientation of motifs are important for type-B regulator binding. Based on the distance, motifs were separated into two groups: one with a > 6–30-bp distance and another with a 6–30-bp distance. No difference in the total number of motifs, the motif density, or the number of motifs of distance > 6–30 bp was recorded in the sequences of the 15 type-A genes, 13 control genes, and random sequences (P > 0.05); however, a significant difference was detected in the number of motifs with a 6–30-bp distance irrespective of their direction (P < 0.05). Because motif distance is important for type-B regulator binding , our data support the notion that the motif functions in transcription factor binding in natural promoters in rice as well as Arabidopsis. Similarly, a markedly higher mean number of motifs with a 6–30-bp distance was observed in the 15 type-A OsRRs than in the 13 control genes. No apparent bias toward tandem or inverse orientation was recorded in rice (Additional file 3), as in Arabidopsis . To summarize, higher clustered motif were more in rice cytokinin signaling targets than in control genes, which supports the functionality of TCSn in rice.
Changes in the relative expression of type-A OsRRs in shoots and roots in response to cytokinin
Furthermore, OsRR6 and OsRR9/10 levels were higher both in shoots and roots in the control group (Additional file 4). Thus, OsRR6 and OsRR9/10 were selected as marker genes in subsequent experiments.
TCSn::GUS expression patterns are consistent with known cytokinin functions in rice seedlings
To generate a universal cytokinin reporter, a synthetic reporter harboring the concatemerized type-B RR-binding motifs has been tested and optimized in vivo . However, this system has the following limitations: TCS-induced expression is weak in certain developmental contexts in which cytokinin signaling is involved, such as in the shoot and in the vasculature. Furthermore, GFP expression in the root meristem of the seedling decreases as the number of generations increases. Accordingly, an improved version of the reporter, TCS new (TCSn), which, compared with TCS, is more sensitive to phosphorelay signaling in Arabidopsis and maize cellular assays while retaining its specificity, has been developed. TCSn includes variations in the number of binding sites, phasing, and identity of flanking nucleotides .
TCSn::GUS activity in response to exogenous cytokinin
Specificity of TCSn::GUS in response to exogenous cytokinin
In this study, we analyzed the DNA-binding domains 5′-(A/G)GAT(T/C)-3′ in type-A OsRR family members and examined the relative expression of type-A OsRR genes in response to treatment with phytohormones. The strong version of TCSn::GUS was fused into rice plants by genetic transformation. We evaluated GUS expression in transgenic rice seedlings, including differences between generations and responses to exogenous application of cytokinin fractions and three phytohormones. A cytokinin-responsive type-A OsRR-binding element was clustered in the upstream sequences of 15 type-A OsRR genes. TCSn::GUS was expressed in the meristem of germinated rice seeds and seedlings. Furthermore, TCSn::GUS expression was induced specifically by exogenous application of cytokinin in rice seedlings. We report here a robust and sensitive synthetic sensor for monitoring the transcriptional output of the cytokinin signaling network in rice.
Construction of reporter vectors and transformation
The TCSn promoter sequence  was commercially synthesized by the Genewiz Company (Additional file 1). To construct the TCSn::GUS vector, we inserted the synthetic TCSn promoter sequence into the SalI and BamHI sites of the pDR5::GUS vector. The pDR5::GUS construct was kindly provided by the Ping Wu laboratory at Zhejiang University, Hangzhou, China. The construct was transformed into callus initiated from rice seeds (cv. Shiokari) by Agrobacterium tumefaciens (strain EHA105)-mediated transformation. Rice embryonic calli were induced on N6 media, and transformation was performed by Agrobacterium-mediated cocultivation .
Plant growth conditions and treatments
Transgenic rice seeds were surface-sterilized with 10% (v/v) H2O2 for 30 min and rinsed thoroughly with deionized water. The sterilized seeds were germinated on a plastic support netting (mesh, 1 mm2) mounted in plastic containers for 1 week. Uniform seedlings were selected and then transferred to a tank containing 7 L of International Rice Research Institute (IRRI) nutrient solution for 2 weeks at pH 5.5. IRRI nutrient solution (1.25 mM NH4NO3, 0.3 mM KH2PO4, 0.35 mM K2SO4, 1 mM CaCl2·2H2O, 1 mM MgSO4·7H2O, 0.5 mM Na2SiO3, 20 μM NaFeEDTA, 20 μM H3BO3, 9 μM MnCl2·4H2O, 0.32 μM CuSO4·5H2O, 0.77 μM ZnSO4·7H2O and 0.39 μM Na2MoO4·2H2O; pH 5.5) was supplied and replaced every 2 days. Plants were grown in a growth chamber at 30 °C during the day and 22 °C during the night with a 16-h light/8 h dark regime. The relative humidity was controlled at ~ 70%.
The cytokinin fractions (6-benzylaminopurine, 6-BA; zeatin, Z; zeatin riboside, ZR; kinetin, KT; N6-(Δ2-isopentenyl) adenine, iP; iso-pentenyl adenosine, iPA), 1 µM indole-3-acetic acid (IAA), 10 µM gibberellic acid (GA), and 10 µM abscisic acid (ABA) were added to the hydroponic media. The seedlings were treated with 100 nM 6-benzylaminopurine (6-BA) at 0, 0.5, 1, 3, and 6 h and with 0, 1, 10, 100, and 1000 nM 6-BA for 6 h.
Quantitative reverse-transcription polymerase chain reaction analysis of gene expression
Total RNA was isolated from the roots of rice seedlings. RNA extraction, reverse transcription, and quantitative reverse-transcription polymerase chain reaction (qRT-PCR) were performed as described previously . Primer sets for type-A OsRRs are listed in Additional file 8.
Histochemical GUS staining and quantitative measurement of GUS activity
Histochemical GUS staining was performed as described previously . The tissues were treated for 1 h with 1.8 M KOH solution heated to 90 °C and then treated with 1% HCl (v/v) solution for 5 min . The stained tissues were photographed using an Olympus SZX2ILLK stereomicroscope equipped with a color charge-coupled device (CCD) camera (Olympus) .
For enzymatic GUS assays, tissues were ground in liquid nitrogen and transferred to microtubes containing 1 mL of the extraction buffer (50 mM sodium phosphate, pH 7.5; 10 mM Na2EDTA; 10 mM 2-mercaptoethanol; 0.1% Triton X-100; and 0.1% (w/v) sodium lauryl-sarco-sine) for total protein extraction. Beta-glucuronidase activity was measured fluorimetrically using 1 µg of total protein extract, as described previously .
Experimental data were pooled for calculation of means and standard deviations (SDs) and subjected to one-way analysis of variance (ANOVA), followed by least significant difference (LSD) testing to determine the significance of differences between individual treatments. Data in Additional file 3 were pooled and analyzed by nonparametric tests, followed by two independent-samples t tests to determine the significance of differences between two independent samples. All statistical procedures were performed using SPSS version 11.0 (SPSS, Chicago, IL, USA).
JY and HW conceived the study. JY and PG carried out the experiments. JY and YZ drafted the manuscript. All authors read and approved the final manuscript.
This work was funded by the National Nature Science Foundation of China (Grant Nos. 31471936 and 31672225), National key R & D projects (2017YFD0200103), Innovative Research Team Development Plan of the Ministry of Education of China (No. IRT1256), the 111 Project (No. 12009), the Priority Academic Program Development of Jiangsu Higher Education Institutions Project, China Scholarship Council (CSC) and Innovative Plan of Jiangsu Province of China (KYZZ16_0380). The Englsih in this document has been checked by at least two professional editors, both native speakers of English. For a certificate, please see: http://www.textcheck.com/certificate/vXOTa4.
The authors declare that they have no competing interests.
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