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. 2020 Jun 17;15(8):1780405. doi: 10.1080/15592324.2020.1780405

Overexpression of a B-type cytokinin response regulator (OsORR2) reduces plant height in rice

Fachao Shi a, Min Wang b, Yuxing An a,✉
PMCID: PMC8570751  PMID: 32552330

ABSTRACT

Cytokinin plays crucial roles in regulating plant growth and development, with the signal transduction mediated by type-A and type-B response regulators (RRs).While much genetic knowledge about RRs on regulating plant height remains unclear. Here, we found that overexpressing an OsORR2 gene (a type-B RR) could reduce plant height in rice compared with the wild type (WT). Using quantitative real time (RT-qPCR) assay, OsORR2was expressed widely in most tissues such as root, culm, sheath, leaf, and panicle. Strong signals were detected in leaf mesophyll cells and anther by in situ hybridization assays (ISH). The subcellular localization of OsORR2 was in cell nucleus. In addition, we found that the transcriptional expression levels of type-A RR genes such as OsRR9, OsRR10, OsRR12, and OsRR13 were significantly up-regulated in the overexpression transgenic plants (OE) plants. Taken together, our data suggested that OsORR2was involved in the development of plant height in rice and provided a foundation for future deep molecular research of the type-B RRs.

KEYWORDS: Overexpression, cytokinin, OsORR2, plant height, rice

Introduction

Appropriate plant height is a key agronomic factor contributing to crop yield and lodging resistance in rice and other cereal crops.1,2 Semi-dwarf rice exerts superiority in lodging decrease and increasing partitioning of assimulationinto grains, finally causing yield improvement.3 Plant height was influenced by several elements such as plant hormones, cell wall biosynthesis,4 and microelement supplies.5Among plant hormones, cytokinins, as adenine derivatives, exert essential roles inthe regulation of plant growth and development such as cell division, root elongation and shoot growth, leaf senescence, and other stressresponses and pathogen resistance.6,7

It is reported that the cytokinin signal transduction was mediated by a two-component system, primarily identified in bacteria and yeast.8–10an extracellular stimulus is perceived by a plasma membrane-localized histidine kinase, which transfers the signal to a response regulator RR in theform of a phosphorylgroup.11 Furthermore, plants had a third component for cytokinin signal transduction, which involved in the transfer of a phosphoryl group from the HK to the RR.7,12These RRs, generally classified into type A and type B,are considered as the final regulating phosphorelay step. The type-A RRs, as the initial response genes, normally contain a receiverdomain with conserved D-D-K residues.13Type-B RRs, except the receiver domain, are more complex than type-A RRs, which consist of another DNA-binding motif (GARP domain).14,15Previousstudies showed that type-A RR genes in Arabidopsis such as ARR4 and ARR5are up-regulated by drought, salt, and cold.16In addition, the type-B RRs could regulate the transcription of cytokinin activated targets of type-A RRs.7

In Arabidopsis, overexpressing different type-A RRs inhibited the ability of cytokinin.8 For example, overexpression of ARR8 repressed the shoot re-generation and greening of calli tissues, while ARR15 overexpression caused decreased sensitivity to cytokinin.17ARR7 in Arabidopsis is considered as a transcriptional repressor.18In rice, thirteen type-A RR genes and six type-B RR genes in the genome were primarily identified and characterized.19Among them, the B-type RR genes (ORR1, ORR2, ORR3, ORR4 and ORR6) except ORR5, showed expression in various organs and ORR5 was expressed in callus and flower.19 A study showed that the OsRR6in rice is induced by various environmental stimuli including salinity, dehydration, and low temperature stress, suggesting its role in cross talk between abiotic stress and cytokinin signaling.20 Ehd1, a type-B RR (Pseudo-RRs), was integrated into the conserved pathway in the photoperiodic control of flowering in rice.21OsRR6 overexpressing lines displayed altered morphologies and changes in cytokinin metabolism.22 However, overexpressing OsRR3 and OsRR5 resulted in lower sensitivity to cytokinins.23

Although the gene sequence of type-B RR (OsORR2) had been reported,19its molecular function remains unclear. In the present study, we obtained overexpressing plants of OsORR2 (OE) and found that the OE lines showed decrease plant height. TheOsORR2 was expressed in different tissues with strong ISH signals in leaf mesophyll cells and pollens. And several type-A RRs genes were prominently induced in the OE lines. Our results offered a molecular basis for the future study of regulation mechanism.

Materials and methods

Plant material

Plant materials consisted of three transgenic lines (T2), namely OE-1, OE-2, OE-3, and Oryza sativa L. japonica ‘Kittake’ seedlings was as the control. Seeds were germinated for 48 h at 37°C in a growth chamber, and then seedlings were transplanted into soil-filled containers in a greenhouse at 28–30°C with 50–60% relative humidity. Plant tissues were frozen in liquid nitrogen and stored at −80°C.

Vector construction and transformation

The full-length cDNA sequence of OsORR2 was obtained from rice using the same method described in our previous study.24OsORR2 full length cDNA driven by the maize Ubipromoter was cloned into binary vector pCAMBIA1390 using the In-Fusion Advantage PCR Cloning Kits (Clontech). Resulting plasmids were transformed into Kittake plants (Control) by Agrobacterium tumefaciens-mediated transformation as described previously.25

Quantitative real-time PCR (qRT-PCR)

Three independent RNA samples per plants were used to validate gene expression levels by RT-qPCR. Total RNA was prepared from rice tissues using Trizol Reagent (Life Tech). cDNA (20 µl) was synthesized from 1 µg of RNA using a QuantiTect Reverse Transcription kit (Qiagen). RT-qPCR amplifications were performed in 20 µl reaction volumes containing 0.5 µl cDNA, 0.2 µM primer mix and SYBR Premix Ex Taq (Takara) on an ABI PRISM 7900HT sequence detection system. The UBQ gene was used as an internal control. All primers used for qRT-PCR were listed in Table S1. Data was analyzed using the relative quantification method.26

RNA in situ hybridization

According to the method described previously by Bradley,27 we carried out the RNA in situ hybridization (ISH) assay. Briefly, a 286bpOsORR2 CDS region was sub-cloned into the pGEM-T Easy vector (Tiangen). Flag leaves of control plants at heading were fixed using an RNase-free formalin/acetic acid fixative solution, followed by a series of dehydration steps and embedded in paraffin for sectioning. Digoxigenin-labeled RNA probes were prepared using a DIG Northern Starter Kit (Roche). Hybridization signals were visualized and photographed using a Leica DMR microscope.

Subcellular localization and promoter fusions

To create the integrated vector pCAMBIA1305-d35 S-OsORR2-GFP, the target gene CDS fragment was cloned into the pCAMBIA1305-GFP vector at the Bgl II site. Constructs were transiently expressed in tobacco (Nicotiana benthamiana) epidermal cells as described previously.28

Promoter binding site analysis

Eight of the type-A RR genes in rice were randomly selected in our study. B-type ORRs could bind the promoter of type-A RR genes with the sequence AGATT according to the Sakai H method.29And about 2 kb promoter sequence was analyzed and labeled in the binding sites.

Results

Overexpression of OsORR2 decreased plant height

Members of type-B RR family had the function in DNA binding signal transduction in the cytokinin pathway.30 Accordingly, we selected several type-B RR genes from different plants, such as Arabidopsis thaliana, Nicotiana attenuate, Helianthus annuus, and Apostasiashenzhenica (Figure S1).The protein structure analysis showed that all type-B RR genes possessed a conserved GARP domain in the sequence of N-terminal, indicating ORR2 might have the same function to regulate genes expression as transcriptional factor. In this study, we focused mainly on OsRR2, which belonged to the type-B RRs and its OE transgenic lines were obtained. Three positive homozygous transgenic lines (T2) with various levels of up-regulated OsRR2 transcript were identified (Figure 1a,b). OsRR2 transcript was found to be greatly increased (nearly ten times) in OE plants compared with the control (Figure 1b).OE plants apparently showed dwarfism at the heading stage in contrast to the control lines(Figure 1a,c).The plant height of control was 75 cm on average, while OE lines were decreased to about 60 cm (Figure 1c). Therefore, overexpressing OsRR2 negatively impacted plant height in transgenic lines.

Figure 1.

Figure 1.

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Overexpression OsORR2 decreased plant height. (a) Phenotype investigation of control and OE plantsat the heading stage. (b) OsORR2’sexpression in OE lines.(c) Plant height in the control and OE lines. Bar in (a) 10 cm. Data is presented as the mean ± standard deviation (n = 9) and significant difference was calculated by comparing with wild-type. **P ≤ 0.01; Student’s t test.

Expression pattern and subcellular location of OsORR2

The expression pattern of OsRR2 was analyzed by qRT-PCR in various tissues including roots, culms, leaves, sheath, and panicles at the heading stage. Results demonstrated that the OsRR2 was expressed in all tissues examined, with the highest expression in leaves (Figure 2a). To further investigate where expressed strongly, ISH assay was carried out. We finally found that strong ISH signals were detected in the leaf mesophyll cells and anther (Figure 2b–g).

Figure 2.

Figure 2.

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Expression pattern and localization of OsORR2.(a) Expression levels in various tissues revealed by qRT-PCR. Data is presented as the mean ± standard deviation (n = 9). (b-g) RNA in situ hybridization analysis of OsORR2. Flag leaves of control plant at heading were cross-sectioned and hybridized with OsRR2-specific antisense (b, c) or sense (d) probes. The anther control plant at heading stage were cross-sectioned and hybridized with OsORR2-specific antisense (e, f) or sense (g) probes. (h) Expression of OsRR2-GFP fusion protein in tobacco (Nicotianabenthamiana) epidermal cells. Scar bars: 200 μm in (b), 100 μm in (c, d), 300 μm in (e), 100 μm in (f, g), 10 μm in (h).

Subcellular localization of OsRR2 was carried out by fusing GFP, driven by the cauliflower mosaic virus 35 S promoter (35 S::OsRR2::GFP) and transiently expressed in leaf epidermal cells of N. benthamiana. Results showed that the OsRR2 GFP signal was localized in cell nucleus (Figure 2h).

DNA sequence analysis with the binding site AGATT

The type-B response regulator genes were considered as transcript factors, which played a crucial role in the early response of plants to cytokinin pathway. The conserved GARP domain could bind to the sequence 5-AGATT-3 according to the previous study and regulate the expression of type-A genes.29Thus, eight type-A response regulator genes were selected and the binding sites were highlighted and found in their promoter sequence (Figure 3). Only one binding site was labeled in the promoter sequence of OsRR8 and OsRR11, others at least had two binding sites (Figure 3).

Figure 3.

Figure 3.

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Upstream sequence analysis of promoter in several type-B RR genes. The 2 kb was selected in the promoter of type-B regulator genes from rice and analyzed the location of binding site with AGATT.

Type-A response regulator genes expression in OE lines

In order to know the expression levels of type-A genes in OE lines, eighttype-A genes were selected to detect their expression by using the qRT-PCR (Figure 4). Results showed that the relative expression of OsRR9, OsRR10, OsRR12, and OsRR13 had a significant up-regulation in OE lines compared with WT, while others were down regulated, indicating expression of type-A genes were affected in OsRR2 lines.

Figure 4.

Figure 4.

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Relative expression levels of Type-A response regulator genes in OsORR2 over-expression rice.UBQ was used as aninternal reference gene and wild type was as control. Data is presented as the mean ± standard deviation (n = 9). **P ≤ 0.01; Student’s t test.

Discussion

In this study, we found that overexpressingOsORR2 could reduce plant height and several type-A RR genes were significantly down-regulated in OE lines. Type-B RR genes are transcription factors (TFs) with a GARP-like DNA binding domain at their C-termini.31 In previous study, five Arabidopsis B-RRs were shown to act in cytokinin signaling cascades and played critical role in plant growth and development.32The B-RR TFs regulated A-RRs genes expression by binding a DNA motif (AGATT) in Arabidopsis.7Three related binding motifs were identified in OsRR2, two of these with an AGAT core sequence and the third with a palindromic sequence around the AT core.33 Significantly, a little bit different in binding site between the rice and Arobidopsis, suggesting that type-A RR genes was not merely regulated by the motif (AGATT). Therefore, we found that several A-type RRs were down-regulated in OE plants and others genes expression unchanged.

The cytokinin signaling pathway is comprised of a ‘two-component’ phosphorelay that activate the expression of type-B RRs, which modulate downstream gene expression and the biological function of these genes. For example, TOC1/APRR1, an Arabidopsis pseudo-RR, affected flowering time through the phase setting of CO expression.34 Ehd1, a special type-B RRS only existed in monocot, promoted flowering by inducing FT-like genes.21 In addition, type-B RRs genes had a function in osmotic stress. ARR1, ARR10, and ARR12 redundantly acted as negative regulators of drought responses in both ABA-dependent and independent pathways by maintenance of cell membrane stability and promotion of anthocyanin biosynthesis.35 Moreover, type-B genes were involved in cytokinin signaling and plant development. The arr1, arr10, and arr12 mutants showed insensitivity to high levels of exogenously cytokinins and exhibited shoot stature and sensitivity to light.36ARR22 overexpression transgenic lines showed dwarf phenotype with delaying primary roots development and the cytokinin-inducible genes were attenuated in ARR22-OX. In this study, OsORR2 was overexpressed under Ubi promoter in rice, more strikingly, its OE plants exhibited decreased plant height, indicating OsORR2 exerteda crucial role in plant architecture. However, type-A genes expressed differently in ARR22-OX lines by a microarray data.17Thus, we detected type-A RR genes expression in OsORR2 OE plant by qRT-PCR and found that genes such as OsRR9, OsRR10, OsRR12, and OsRR13 were up-regulated (Figure 4), indicating that although the similar phenotype existed in type-B RR transgenic lines, the molecular of function in genes regulation might be different.

In conclusion, our results showed that a conserved GARP domain existed in OsORR2 protein structure and its over-expression plants hold a dwarf phenotype. Several type-A RR genes were prominently up-regulated in OE plants. However, much knowledge of the molecular function of OsORR2 in plant remains unclear. Our study provides a basis of type-B response regulator functions in plant development for the future research.

Supplementary Material

Supplemental Material
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Supplemental Material
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Funding Statement

This work was supported by GDAS’ Project of Science and Technology Development [2019GDASYL-0103032].

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Supplementary material

Supplemental data for this article can be accessed on the publisher’s website.

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