Os04g0169100

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Annotated Information

Function

• In rice, ETR2 reduces ethylene sensitivity, delays the transition fromthe vegetative stage to the floral stage, and affects starch accumulation.

• ETR2 is a Ser/Thr kinase that can phosphorylate its own receiver domain and an in vitro substrate MBP. Complete abolishment of ETR2 kinase activity by N box mutation suggests that the N box is the most important motif for ETR2 function. Overexpression of ETR2 leads to reduced ethylene sensitivity and late flowering, whereas T-DNA insertion mutant etr2 showed enhanced ethylene sensitivity and early flowering. Other yield-related traits, starch accumulation, and gene expressions were also affected by ETR2 overexpression or reduction. This study reveals conserved and diverged aspects in ethylene receptor signaling between the monocotyledous rice plant and dicotyledonous plant.

Mutation

• To investigate ETR2 function, the T-DNA insertion mutant, etr2, for the ETR2 gene was requested from the Rice Mutant Database (http://rmd.ncpgr.cn) and analyzed. For comparison, the T-DNA insertion mutant etr3 and ers2 for rice ETR3 and ERS2, respectively, were also studied. These mutants were in the background of Zhong Hua 11 (ZH), a japonica variety. In etr2, the T-DNA was inserted at 0.6 kb upstream of the ATG start codon of ETR2 gene (Figure 1A). The three T-DNA insertion mutants were treated with ethylene, and their coleoptile growth response was examined. The etr2 mutant exhibited the longest coleoptile at all ethylene concentrations tested compared with controls (Figure 1C). The etr3 and ers2 mutants showed moderate length of coleoptiles with these treatments compared with controls (Figure 1C). These results indicate that the three mutants have an enhanced ethylene response, suggesting that reduction of ethylene receptor gene expression results in increased ethylene sensitivity.

Figure 1. Identification of T-DNA Insertion Mutants and Their Ethylene Sensitivity. (A) Schematic representation of T-DNA insertions in ETR2, ETR3, and ERS2 genomic sequences and PCR identification of mutants. The etr2, etr3, and ers2 are mutant plants for ETR2, ETR3, and ERS2, respectively. ZH (Zhong Hua 11) is the parental control plant. ZH/etr3 and ZH/ers2 are heterozygous plants. p1 and p2 are primers from the T-DNA regions. The 59- and 39-primers are from the genes examined. ATG indicates the start codon. (B) Examination of ETR2, ETR3, and ERS2 expression in their corresponding mutants by RT-PCR. Actin was amplified as a control. Three biological replicates for RT-PCR were performed, and the results were consistent. One set of the results is shown. (C) Coleoptile growth of etr2, etr3, and ers2 in response to ethylene treatment. Left panel: phenotype of the etiolated seedlings after ethylene treatment. Right panel: coleoptile growth of various mutants after ethylene treatments. Each data point represents the average of 20 to 30 seedlings. Bars indicate SD. .

• The mutants were grown under field conditions to observe the effect of reducing expression of the receptor. We found that etr2 and etr3 developed panicles earlier than ZH control plants (Figure 2A). During the heading period, etr2 and etr3 plants showed early heading panicles compared with ZH controls (Figure 2B). The heading time distribution of these mutants was also analyzed. The etr2 mutant showed a 3-d early heading, whereas the etr3 had a 7-d early heading compared with ZH control plants (Figure 2C). These results indicate that reduction of ethylene receptor gene expression leads to early flowering. Since overexpression of ETR2 causes starch accumulation in stems, the researchers examined whether reduction of ETR2 and ETR3 gene expression would affect starch accumulation. In both the etr2 and etr3 mutants, starch granules were almost absent in stems (Figure 2D). However, in the ZH control, starch granules were still abundant (Figure 2D). These results indicate that reduction of ethylene receptor gene expression causes disappearance of starch granules. The seed phenotypes were compared, and the etr2 and etr3 mutant seeds appeared slightly larger than those of the ZH controls and ers2 (Figure 2E). The thousand-seed weight was also examined, and etr2 seeds had significantly higher thousandseed weight than ZH control (Figure 2E). The researcers examined whether other genes were altered in the SAM of the three mutant rice plants. Both OsGI and RCN1 expression were reduced in the three mutants compared with the ZH controls (Figure 2F). By contrast, RAmy3D and monosaccharide transporter gene expression were upregulated in the three mutants compared with the ZH control (Figure 2F). These results indicate that suppression of ethylene receptor gene expression in T-DNA insertion mutants leads to inhibition of OsGI and RCN1 but upregulation of RAmy3D and the monosaccharide transporter gene.

Figure 2. Phenotype of etr2 and etr3 Mutants in Comparison with the Control ZH (Zhong Hua 11). .

Expression

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Evolution

• The rice ETR2 kinase domain was compared with those from other ethylene receptors or homologs (Figure 3). Alignment of the amino acid sequences of the ethylene receptor kinase domains of various ethylene recpetors. The positions of the H, N, G1, F, and G2 box are indicated on top of the sequence based on the corresponding boxes from Arabidopsis ETR1. The amino acids shaded in black are identical to each other. Five mutated amino acids (G to A, E to Q, R to Q, F to A, and G to A) upstream of and within the N box are also indicated, and the mutated protein N was used for kinase analysis. At ETR1, At ETR2, and At EIN4 are from Arabidopsis. NTHK1 and NTHK2 are from tobacco. Zm ETR2 is from maize. The Os ETR2/Os PK1, Os ETR3/Os PK2, and Os ETR4/Os PK3 receptors are from rice.

Figure 3. Alignment of the Amino Acid Sequence of the Kinase Domain with That of Other Ethylene Receptors. .

Labs working on this gene

• Plant Gene Research Center, National Key Lab of Plant Genomics, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China