Os04g0673300

Please input one-sentence summary here. OsRR6 is a kind of CK-inducible type-A response regulator in rice[2].

Annotated Information

Function

OsRR6 is a kind of CK-inducible type-A response regulator[2].The type-A response regulators are relatively small, containing a receiver domain along with short N- and C-terminal extensions.

The expression of a majority of OsRR genes was not significantly altered under stress, with the notable exception of OsRR6. The expression of OsRR6 gene was induced to significant levels by salt, dehydration and low temperature treatments (Fig. 1), and results were reproducible. This indicates that OsRR6 may play an important role in abiotic stress signaling in rice, besides acting as a component in cytokinin signaling[1].

Fig.1

The induction of OsRR6 by different abiotic stress stimuli provides a molecular link between stress and cytokinin signaling as well[1].

Overexpression of OsRR6 also affected the expression of CK-responsive genes[2].

OsRR6-ox plants displayed altered morphologies and changes in CK metabolism, probably due to changes in the gene regulatory network[2].

Fig.1: Changes in transcript levels of the OsRR6 gene in response to different stress treatments. The transcript levels of OsRR6 gene in 6-day-old light-grown seedlings treated with ABA, NaCl, mannitol and cold for 6 h, were plotted as the relative expression (fold) of the seedlings mock-treated for the same duration.

Generated transgenic rice plants that overexpress OsRR6 (OsRR6-ox) by fusing its coding sequence to the rice actin1 (Act1) promoter, because this promoter generally produces much higher levels of constitutive expression in rice than the cauliflower mosaic virus(CaMV) 35S promoter. Callus transformed with Act1::OsRR6 showed severe retardation of shoot regeneration compared with callus transformed with a control vector (Fig. 2)[2].

Fig.2

Fig.2: OsRR6 represses shoot regeneration in rice callus. Callus was transformed with Agrobacterium carrying a binary vector pActnos/Hmz (Vec.), Act1::OsRR6 (OsRR6) or Act1::OsRR6D103E (OsRR6D103E). Calli were selected with hygromycin B and grown on regeneration agar plates for 6 weeks.

Moreover, each of 20 OsRR6D103E-ox independent lines was indistinguishable from plants transformed with a control vector (control plants; Fig. 3A, B). These results support the hypothesis that growth defects associated with OsRR6 overexpression are due to a requirement for phosphorylation of OsRR6[2].

Fig.3

Fig.3: Morphologies of transgenic plants overexpressing OsRR6. OsRR6-ox transgenic plants (T0 generation) were grown on MS-agar plates containing hygromycin B for 7 d, and then hydroponically grown for 75 d. Transgenic plants, transformed with vector pActnos/Hmz (Vec.) or Act1::OsRR6D103E (OsRR6D103E-ox), were also grown under the same conditions. The typical phenotype of each transgenic line and their panicles are shown in (A) and (B). Total RNA samples were prepared from the shoots of each transgenic plant. (C) Semiquantitative RT–PCR analysis of the OsRR6 gene in the transgenic lines. OsAct1 is an extraction and loading control.

Future analyses of knockout or RNA interference mutants of OsRR6 will enable us to define further its possible participation in stress responses.

Expression

OsRR6 is found as repeats on the top arm of chromosome 4. This area of chromosome 4 is included in a segmental duplication with a region on the upper arm of chromosome 2 (Fig. 4)[4].

Fig.4

Fig.4:Locations and duplications of putative cytokinin two-component regulators in the rice chromosomes (cv. Nipponbare). Ovals on the chromosomes represent centromeres.

The OsRR genes express differentially in various organs examined, and also in response to light[1]. Most of the OsRR genes were expressed at relatively higher level in mature tissues (leaves and flowers).The majority of the type-A OsRR genes (OsRR2–9 and OsRR11) were expressed at various levels in roots, stems, leaves, and spikelets (Fig.5)([4]. OsRR6 was expressed mostly in roots and leaves.

The transcript levels of OsRR2, 3, 4, 6, 7,and 9 were significantly higher in etiolated seedlings as compared to green seedlings (Fig. 6)[4].

Fig.5

Fig.5:The analysis of the response of the system genes family to cytokinin by RT-PCR. RNA was isolated from roots and leaves from Nipponbare rice growing in liquid nutrient medium either with 1 μM 6-benzylaminopurine (6-BA) for 4 h or with no 6-BA. OsACTIN primers were used as a template control.

Fig.6

Fig.6:Real-time PCR analysis showing the organ-specific expression profiles of individual OsRR genes. The relative mRNA levels of individual OsRR genes normalized with respect to housekeeping gene, UBQ5, in different tissues (GS, green seedlings; ES, etiolated seedlings; S, green shoots

Evolution

The type-A RRs are mainly composed of a receiver domain with short N- and C-terminal extensions [2], essentially similar to the E. coli response regulator (RR) CheY involved in chemotaxis, and lack a typical output domain(3). All the OsRR proteins also contain the highly conserved Lys and two Asp residues (D-D-K) in the receiver domain (Fig. 7B, C).However, OsRR6 and OsRR7 have N-terminal extensions rich in gly and asp residues (Fig. 7C). These N- and C-terminal variable regions may play a role in their localization to different cellular compartments.No homolog of OsRR6 was found within the duplicated region, suggesting the involvement of gene loss or more localized duplications[4].

Fig.7

Fig.7:type-A response regulators in rice. (A) Exon-intron organization of OsRR genes. Exons and introns are represented by black boxes and lines, respectively. (B) Schematic representation of OsRR proteins (aligned with respect to the second conserved Asp (D) residue). The conserved receiver domain is represented as gray box with D-D-K residues. The black vertical bars represent intron position. The numbers 0, 1 and 2 above the vertical bars represent the phase 0, 1 and 2 introns, respectively. (C) Multiple alignments of the OsRR proteins obtained with ClustalX. Fully and partially conserved (present in more than 50% of aligned sequences) residues are highlighted in black and gray boxes, respectively. Gaps (marked with dashes) have been introduced to maximize the alignments. The conserved receiver domain has been underlined. Conserved Asp and Lys residues are marked with asterisks.

OsRR6 were found as repeats on the top arm of chromosome 4. This area of chromosome 4 is included in a segmental duplication with a region on the upper arm of chromosome 2 that contains the OsRR11 gene [4](Fig. 4).

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Labs working on this gene

1. Mukesh Jain, Akhilesh K Tyagi and Jitendra P Khurana

2. Liming Du, Fangchan Jiao, Jun Chu, Ming Chen, Ping Wu

3. X. Cheng, H. Jiang, J. Zhang, Y. Qian, S. Zhu and B. Cheng

References

1. Mukesh Jain, Akhilesh K Tyagi: Molecular characterization and differential expression of cytokinin-responsive type-A response regulators in rice (Oryza sativa)BMC Plant Biology 2006, 6:1

2.Hirose N, Makita N, Kojima M, Kamada-Nobusada T, et al. Overexpression of a type-A response regulator alters rice morphology and cytokinin metabolism. Plant Cell Physiol. 2007,48: 523-539.

3.Imamura A, Hanaki N, Umeda H, Nakamura A, Suzuki T, Ueguchi C, Mizuno T: Response regulators implicated in His-to-Asp phosphotransfer signaling in Arabidopsis. Proc Natl Acad Sci USA 1998,95:2691-2696

4.Liming Du, Fangchan Jiao, Jun Chu:The two-component signal system in rice (Oryza sativa L.): A genome-wide study of cytokinin signal perception and transduction.Genomics 2007,89: 697–707