Os09g0457900

The rice gene Os09g0457900,namely OsEATB,is a Ethylene-Response AP2/ERF Factor and restricts ethylene-induced enhancement of gibberellin responsiveness during the internode elongation process by down-regulating the gibberellin biosynthetic gene, ent-kaurene synthase A.

Annotated Information

Function

• Effect of Transgenic OsEATB on Rice Internode Elongation

Plant height is not only a decisive factor in plant architecture but also an important agronomic trait that is directly linked to yield potential (Huang et al., 1996; Yang and Hwa, 2008). The OsEATB transgenic lines showed dwarf phenotypes, indicating that the internodal elongation process was suppressed by OsEATB overexpression. For experiments, we selected two transgenic lines: 401003 and 401006 (20 individual plants of each line). The transgenic plants and 9311 control plants were cultivated in the same conditions, and plant height was determined at maturity. The average plant heights of the control, 401003, and 401006 lines were 121, 113, and 106 cm, respectively. Thus, 401003 and 401006 plants were 6.61% and 12.40% shorter than the control, respectively (Fig. 3A; Supplemental Table S1). Furthermore, every elongated internode was shortened, especially the fourth internode, which was shortened by an average of 56.30% (Fig. 3A). OsEATB negatively regulated plant height at every growth stage of rice plants. The transgenic plants showed a dwarf phenotype from the three-leaf stage to the mature stage (Fig. 3B; see Fig. 6, A and B, below). The panicles also showed reduced elongation, demonstrating that panicle internodes were shortened as well (see Fig. 6C below). The plant hormone GA is involved in deepwaterresponsive internode elongation (Raskin and Kende, 1984). A number of genes encoding GA biosynthetic or signaling pathways have been identified as being associated with this rapid process (Yang and Hwa, 2008). To examine the regulation of GA sensitivity in OsEATB transgenic lines, we treated wild-type and transgenic seedlings with 10, 50, and 100 mM GA3 at the four-leaf stage. Under these conditions, both seedlings showed rapid elongation after the 24-h treatments, and OsEATB transgenic seedlings did not show negative sensitivity to GA treatments compared with the control seedlings during this process (Fig. 3, B and C). Together, these results suggested that OsEATB negatively regulates the elongation process of every internode, in every organ, at every growth stage and that the responsiveness of transgenic seedlings to GA is not impaired. Many GAresponsive dwarf plants that are deficient in the biosynthesis of active GAs have been characterized in various plant species (Ross et al., 1997; Magome et al., 2004). These findings, together with our results, led us to conclude that there is negative regulation of GA biosynthesis in the transgenic plants. To test this hypothesis, we used microarray analysis to compare the expression of GA biosynthetic genes between OsEATB transgenic 401006 seedlings and control seedlings at the four-leaf stage. ent-Kaurene is an early intermediate in the GA biosynthesis pathway: ent-kaurene synthase A (CPS) catalyzes the cyclization of geranylgeranyl diphosphate (GGDP) to ent-copalyl diphosphate (CDP), which is then converted to ent-kaurene by ent-kaurene synthase B (KS; Supplemental Fig. S2; Yamaguchi et al., 1998). The microarray analysis results showed that the expression of rice CPS OsCPS2 in OsEATB transgenic seedlings was sharply down-regulated (10.32-fold less than the expression level in 9311 wild-type seedlings). In contrast, the expression of GIBBERELLIN 20-OXIDASE (GA20ox2) was 2.77-fold greater in OsEATB transgenic seedlings than in 9311 wild-type seedlings (Table I). GA20ox is one of the major GA biosynthetic genes, and the levels of GAs are homeostatically modulated through negative feedback regulation of GA20ox expression (Xu et al., 1995). In addition, no significant differences were found between the OsEATB transgenic seedlings and the wild type in the expression of OsCPS1, OsKS1, OsKO2, and OsKAO. To confirm these results, we used real-time PCR analysis to examine the expression of OsCPS2 and GA20ox2 genes in four-leafstage transgenic line 401006 and 401003 plants and control plants (Fig. 3D). Expression of these two GA biosynthesis-related genes was similarly affected by the overexpression of OsEATB to the microarray results. We also analyzed the expression of OsCPS1, OsKS1, OsKO2, and OsKAO, and in agreement with the microarray data, no significant differences in expression of these genes were found (Fig. 3E).

• Effect of Rice OsEATB on GA Biosynthesis during

Internode Elongation To examine the negative regulatory relationship between OsEATB and OsCPS2 during internode elongation, we examined the expression of these two genes in elongating internodes of 64- to 73-d-old 9311 plants (Fig. 4A). In elongating internodes of 64-dold plants, OsEATB was expressed at a high level while OsCPS2 was expressed at a much lower level. The expression of OsEATB sharply decreased 3 d later, while that of OsCPS2 increased approximately 10-fold. Expression of OsEATB was remarkably decreased from 67 to 73 d, while expression of OsCPS2 was slightly increased and then remained almost stable. We compared gene expression in elongating internodes of 67-d-old 401006 transgenic and control plants using real-time quantitative PCR analysis. The genes examined were involved in the GA biosynthesis and GA signaling pathways (Fig. 4B). First, we reexamined the expression of OsCPS2 and GA20ox2 at this growth stage. In elongating internodes of transgenic plants, OsCPS2 was down-regulated by 11.21-fold while GA20ox2 was up-regulated by 2.03- fold, compared with their respective expression in wild-type plants. In rice, the expression levels of XET (for xyloglucan endotransglycosylase) and UROD (for uroporphyrinogen decarboxylase) are up-regulated by GA treatment, while that of GOX (for glycolate oxidase) is down-regulated (Yang et al., 2004). We investigated the effect of OsEATB on the expression of these three genes. Transgenic lines overexpressing OsEATB showed decreased expression of XET and UROD but slightly increased expression of GOX compared with their respective expression in the wild type. These findings suggested that the GA response was suppressed in the transformants, possibly due to the lack of bioactive GAs. SLENDER RICE1 (SLR1), the only DELLA protein in rice (Achard and Genschik, 2009), down-regulates the GA signaling pathway (Fukao and Bailey-Serres, 2008). We evaluated the effect of OsEATB overexpression on this gene and found that expression of SLR1 was down-regulated in transgenic plants. Together, all of these results confirmed that OsEATB negatively regulates the internode elongation process, not via down-regulating GA sensitivity but through restricting GA biosynthesis.

• Effect of Transgenic OsEATB on Rice Tillering and

Panicle Branching To investigate the function of rice OsEATB on yield traits, we analyzed traits in two transgenic lines (401003 and 401006; 20 individual plants of each line). The transgenic lines and 9311 control plants were cultivated in the same conditions, and we evaluated four yield components: panicles per plant, panicle length, spikelets per panicle, and grain number per panicle. The transgenic plants produced more tillers than the control 9311 lines at the six-leaf stage (Fig. 6B). In rice, the tiller is a specialized grain-bearing branch that forms on the unelongated basal internode. The tiller grows independently of the mother stem (culm) by means of its own adventitious roots (Li, 1979). At maturity, transgenic plants had produced 16.95% more panicles than wild-type plants (Fig. 6, A and D; Supplemental Table S1). Spikelets are grass-specific, flower-bearing branches that form on each panicle branch. They consist of primary, secondary, and sometimes higher order panicle branches (Furutani et al., 2006). We counted the spikelets per panicle in transgenic plants and wildtype lines. The 35S:OsEATB transformants produced more spikelets than control plants (Fig. 6, C and E).We also counted the numbers of primary and secondary branches per panicle. Transgenic lines showed 8.31% more primary spikelets per panicle and 34.41% more secondary branches per panicle compared with the wild type (Fig. 6D; Supplemental Table S1). The finding that 35S:OsEATB transformants producedmore secondary braches, more spikelets per panicle, and more panicles per plant suggested that OsEATB activity is responsible for branching in rice. To investigate the effects of OsEATB on rice grain weight, we examined the 1,000-grain weight at random and observed a slight change. The 1,000-grain weight of the control and transgenic lines is about 30.0 and 25.5g, respectively. Average numbers of grains per plant of the control, 401003, and 401006 lines were 776, 1,096, and 1,254, respectively. The calculated grain weights per plant were about 23.28, 27.95, and 31.98 g, respectively. These results indicated that 401003 and 401006 possess increases in grain yield per plant over the control of 20.06% and 37.37% (Fig. 6E; Supplemental Table S1).

• OsEATB Is a Novel Rice AP2/ERF

ERFs are an important subfamily of AP2/ERF transcription factors, and they have a variety of functions. Several rice ERF genes have been isolated and identified; for example, OsEBP-89 regulates transcription of the rice Wx gene (Zhu et al., 2003), and OsEREBP1 is related to the regulation of defense responses (Cheong et al., 2003). In this study, we investigated and confirmed the function of a rice AP2/ERF gene, OsEATB. ERFs can be classified into different subgroups based on their function and group motif. Subgroup I, which includes Arabidopsis (Arabidopsis thaliana) AtERF1 and AtERF2 and tomato (Solanum lycopersicum) Pti4, functions as transcriptional activators. Subgroup II, which includes tobacco (Nicotiana tabacum) NtERF3 and Arabidopsis AtERF3 and AtERF4, functions as transcriptional repressors. However, the functions of ERF subgroups III and IV remain unclear (Cao et al., 2006). OsEATB cannot be classified into any of the function-known ERF subgroups (Fig. 1). Our data show that OsEATB encodes a transcriptional factor that localizes to the nucleus and is constitutively expressed in various tissues (Fig. 1). Experimental data from transgenic plants showed that OsEATB plays a crucial role in regulating the rice internode elongation process (Figs. 2–4). Ethylene is perceived by a family of His kinase-like receptors and, downstream, by EIN2, a novel protein containing an integral membrane domain. In the nucleus, the EIN3 family of DNA-binding proteins regulates ethylene-responsive transcription, and an immediate target of EIN3 is the AP2/EREBP family (Chang and Shockey, 1999). As a result, most ERFs are regulated by ethylene. Many ERF proteins have been shown to bind to the specific ERE, the GCC box. In our study, we examined the binding activity of OsEATB to the GCC box (Fig. 1). At the same time, we found that the expression level of OsEATB was sharply decreased by ethylene (Fig. 5). Consequently, our results show that this gene is involved in the regulation of ethylene-related. responses. OsEATB overexpression dramatically suppressed the internode elongation process (Fig. 3) and made seedlings hypersensitive to NaCl and ABA. In addition, the expression of this gene was down-regulated by both NaCl and ABA (Fig. 5). Ethylene is mostly reported to trigger the internodal elongation process via GA (Fukao and Bailey-Serres, 2008; Hattori et al., 2009), and many ERF genes are induced by abiotic stress conditions in rice (Thara et al., 1999; Cao et al., 2006). Thus, rice OsEATB might be a negative balance regulator of the ethylene-responsive pathway.

• OsEATB Negatively Regulates Ethylene-Induced

Enhancement of GA Responsiveness by Reducing GA Biosynthesis Plant height is a decisive factor in plant architecture. In rice, there is the potential for rapid internodal elongation, and the degree of elongation determines plant height. This rapid growth response is best demonstrated in deepwater rice, especially in the process of submergence tolerance (Kende et al., 1998). Sub1A is an ERF that confers submergence tolerance to rice. This tolerance is mediated by SLR1, which restricts the response to GA. Sub1A augments SLR1 and SLRL1 gene expression, which counteract the elevated responsiveness to GA promoted by the increase in ethylene (Xu et al., 2006; Fukao and Bailey-Serres, 2008). The ERF genes SK1 and SK2 allow rice to adapt to deep water. Under deepwater conditions, ethylene accumulates in the plant and induces the expression of these two genes. These SK genes encoding ERFs trigger internode elongation in deepwater rice via GA. In contrast to Sub1A, SK1 and SK2 may stimulate GA responses (Hattori et al., 2009). Both SK genes and SUB1A encode ERFs and are related toGA, but they have opposing functions in regulating plant height in response to flooding. It is interesting that gene family members in the same subgroup confer different functions related to internode elongation. In conclusion, there is a self-balance of ethylene-induced enhancement of the GA response during the internodal elongation process. It is possible that cross talk mediated by OsEATB between ethylene and GA underlies the differences in rice internode elongation. The OsEATB transgenic seedlings did not show negative sensitivity to GA treatments (Fig. 3), and the expression-level analysis of the genes involved in GA biosynthesis and GA signaling pathways showed that the GA response is suppressed via down-regulating a key enzyme involved in GA biosynthesis and not through up-regulating the important GA sensitivity repressor (Fig. 4). The Arabidopsis GA-deficient mutant dwarf and delayed flowering (ddf1) phenotypes are caused by increased or ectopic expression of a putative AP2 transcription factor, DDF1. DDF1 is involved in the down-regulation of GA biosynthesis (Magome et al., 2004). As a potential negative balance regulator of the ethylene-responsive pathway, OsEATB suppresses the internode elongation process through the restriction of GA biosynthesis, specifically downregulating the expression of OsCPS2 (Table I; Fig. 3). The mutation line of OsCPS1 showed a dwarf phenotype without flower or seed development, which is a typical phenotype of GA-deficient rice dwarf mutants (Sakamoto et al., 2004). Evidence is presented indicating that OsCPS2 is involved in related secondary metabolism, producing defensive phytochemicals (Prisic et al., 2004).The expression of OsCPS2 is sharply negatively related to OsEATB expression, and the endogenous GA level is decreased in OsEATB transgenic plants alongside suppressed expression of OsCPS2 (Table II). No significant differences between the OsEATB transgenic seedlings and the wild type in the expression of OsCPS1, OsKS1, OsKO2, and OsKAO were found, and the expression of GA20ox2 was slightly up-regulated for the feedback of GA deficiency. All these experimental results strongly demonstrated that OsCPS2 is also associated with GA biosynthesis. Promoter regions (1,300 bp upstream of the translation site) of OsCPS2 do not contain the GCC box motif, suggesting that this gene may not be a direct target of OsEATB. We propose a model for OsEATB-dependent hormonal regulation of internode elongation in rice (Fig. 7). It was suggested that the important function of OsEATB is to negatively regulate the ethylene-induced enhancement of GA responsiveness during the internode elongation process by decreasing GA biosynthesis.

• OsEATB Regulates Rice Yield Components through the

Promotion of Tillering and Panicle Branching Food security for the ever-increasing world population largely relies on the grain yield of crop plants (Xue et al., 2008). The critical components to determine rice yield include grain number and grain weight. Grain number is contingent on the number of spikelets per panicle and the number of panicles per plant. The number of panicles is mainly determined by the plant architecture and the spikelets per panicle by panicle morphology (i.e. the number of primary/secondary/ tertiary branches on each panicle; Zha et al., 2009). Overexpression of OsEATB decreased plant height (Fig. 3) and increased the numbers of panicles per plant and spikelets per panicle (Fig. 6). More panicles and spikelets resulted in a 37.37% increase in grain yield, notwithstanding that the 1,000-grain weight was 15.00% lower in transgenic lines than in the control (Fig. 6). The results of this study show that OsEATB, as a member of the AP2/ERF family, positively regulates rice yield components through the promotion of rice tillering and panicle branching. The basic structure of a rice panicle is determined by the pattern of branch formation. In rice, the MONO CULM1 (MOC1) and LAX PANICLE genes are necessary for branch meristem formation (Furutani et al., 2006). MOC1 positively regulates tillering by promoting axillary meristem outgrowth (Li et al., 2003; Leyser, 2005). Although the molecular mechanisms that underlie the cross talk between plant height and branching are poorly understood, it is well known that rice plant height is strongly negatively correlated with tiller number (Hong et al., 2003; Booker et al., 2004). Higher yields are typically obtained from dwarf crops (Spielmeyer et al., 2002). The finding that transgenic rice plants harboring the MOC1 gene are dwarf but produce more tillers than wild-type plants (Li et al., 2003) provides a good opportunity to investigate the genetic control network. Our results indicate that overexpression of the OsEATB gene (Fig. 2) reduces rice plant height and panicle length at maturity, promoting rice branching potential in both tillers and spikelets, possibly via the regulation of both shoot elongation and axillary outgrowth. The short stature reflects the decreased growth of the mother stem, which allows the growth of more tillers, leaves, and panicles and enhances the energy utilization ratio and biomass production. The functions of OsEATB in regulating rice plant architecture include its effects on plant height (decreased internode elongation) and panicle morphology (increased tiller formation). Studies on the rice AP2/ERF gene OsEATB provide an opportunity to identify agriculturally important functions that can be used to improve rice yield components. In addition, this gene provides a model for investigating cross talk between ethylene and GA in the internodal elongation process.

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Structured Information

Gene Name	Os09g0457900
Description	Similar to AP2 domain containing protein RAP2.6 (Fragment)
Version	NM_001069906.1 GI:115479554 GeneID:4347266
Length	1100 bp
Definition	Oryza sativa Japonica Group Os09g0457900, complete gene.
Source	Oryza sativa Japonica Group ORGANISM Oryza sativa Japonica Group Eukaryota; Viridiplantae; Streptophyta; Embryophyta; Tracheophyta; Spermatophyta; Magnoliophyta; Liliopsida; Poales; Poaceae; BEP clade; Ehrhartoideae; Oryzeae; Oryza.
Chromosome	Chromosome 9
Location	Chromosome 9:17959811..17960910
Sequence Coding Region	17959874..17960698
Expression	GEO Profiles:Os09g0457900
Genome Context	<gbrowseImage1> name=NC_008402:17959811..17960910 source=RiceChromosome09 preset=GeneLocation </gbrowseImage1>
Gene Structure	<gbrowseImage2> name=NC_008402:17959811..17960910 source=RiceChromosome09 preset=GeneLocation </gbrowseImage2>
Coding Sequence	<cdnaseq>atgaccaagaaggtgataccggccatggcggcggcgaggcaggattcttgcaagaccaagcttgatgagcgtgggggtagtcatcaggctccgagctccgcgcggtggatctcgtccgagcaggagcacagcatcatcgtcgcggctctgcggtacgtggtgtccgggtgcaccacgccgccgccggagatcgtcacggtggcgtgcggggaggcgtgtgctctgtgcggcatcgacggctgtctcgggtgcgacttctttggggccgaggcggcggggaacgaggaggcggtaatggcgacggattatgctgctgctgctgctgcggccgcggtggcaggaggatcaggcgggaagagggttaggcggaggaggaagaagaacgtgtaccgcggcgtgcggcatcggccgtgggggaagtgggcagcggagatacgcgacccgcgccgcgcggtgcgcaagtggctcgggacgttcgacaccgccgaggaggccgccagggcgtacgaccgcgccgccctcgagttccgcggcgcgcgcgcgaagctcaacttcccgtgctccgagcctttgcccatgcccagccaaagaaacggcaatggcggcgatgctgtcacggcggcgacgacaacggcagagcagatgactccgactctgtcgccgtgcagcgcggatgccgaggagacgacgacgccggtggattggcagatgggcgcggacgaagccggcagcaaccagctctgggatggcttgcaggacctgatgaagctggatgaagcggacacctggttcccgccattttccggtgcagcgtctagtttttga</cdnaseq>
Protein Sequence	<aaseq>MTKKVIPAMAAARQDSCKTKLDERGGSHQAPSSARWISSEQEHS IIVAALRYVVSGCTTPPPEIVTVACGEACALCGIDGCLGCDFFGAEAAGNEEAVMATD YAAAAAAAAVAGGSGGKRVRRRRKKNVYRGVRHRPWGKWAAEIRDPRRAVRKWLGTFD TAEEAARAYDRAALEFRGARAKLNFPCSEPLPMPSQRNGNGGDAVTAATTTAEQMTPT LSPCSADAEETTTPVDWQMGADEAGSNQLWDGLQDLMKLDEADTWFPPFSGAASSF</aaseq>
Gene Sequence	<dnaseqindica>64..888#aacgacctcaagcacactactagctccggctcacttagctccctactgatcactggaacactcatgaccaagaaggtgataccggccatggcggcggcgaggcaggattcttgcaagaccaagcttgatgagcgtgggggtagtcatcaggctccgagctccgcgcggtggatctcgtccgagcaggagcacagcatcatcgtcgcggctctgcggtacgtggtgtccgggtgcaccacgccgccgccggagatcgtcacggtggcgtgcggggaggcgtgtgctctgtgcggcatcgacggctgtctcgggtgcgacttctttggggccgaggcggcggggaacgaggaggcggtaatggcgacggattatgctgctgctgctgctgcggccgcggtggcaggaggatcaggcgggaagagggttaggcggaggaggaagaagaacgtgtaccgcggcgtgcggcatcggccgtgggggaagtgggcagcggagatacgcgacccgcgccgcgcggtgcgcaagtggctcgggacgttcgacaccgccgaggaggccgccagggcgtacgaccgcgccgccctcgagttccgcggcgcgcgcgcgaagctcaacttcccgtgctccgagcctttgcccatgcccagccaaagaaacggcaatggcggcgatgctgtcacggcggcgacgacaacggcagagcagatgactccgactctgtcgccgtgcagcgcggatgccgaggagacgacgacgccggtggattggcagatgggcgcggacgaagccggcagcaaccagctctgggatggcttgcaggacctgatgaagctggatgaagcggacacctggttcccgccattttccggtgcagcgtctagtttttgagctagtgttattagatctcaaccgttggattagattactggaaggccattcatttattcgtttcattgtatagctaattagctatacttcattgtttgtgcagagcacgttttttaggagtaccgtcttatacatatttttttgtacagagtagaaagtaacaagaattttgtatattgatggaatagattatttccaattaatatttgcgc</dnaseqindica>
External Link(s)	NCBI Gene:Os09g0457900, RefSeq:Os09g0457900