|
|
| (27 intermediate revisions by 3 users not shown) |
| Line 1: |
Line 1: |
| − | 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.<ref name="ref1" /> | + | The rice '''''Os09g0457900''''' was reported as '''''OsEATB''''' in 2011 <ref name="ref1" /> by researchers from China. <ref name="ref1" /> |
| | | | |
| | ==Annotated Information== | | ==Annotated Information== |
| | + | [[File:76HF6.jpg|right|thumb|527px|'''Figure 6.''' ''Effect of transgenic OsEATB on rice yield traits.<ref name="ref1" />.'']] |
| | + | ===Gene Symbol=== |
| | + | *'''''Os09g0457900''''' '''''<=>''''' '''''OsERF#102, OsERF102, AP2/EREBP#123, AP2/EREBP123, OsEATB, EATB''''' |
| | + | |
| | ===Function=== | | ===Function=== |
| − | *'''Effect of Transgenic OsEATB on Rice Internode Elongation''' | + | * Plant height is a decisive factor in plant architecture. Rice (Oryza sativa) plants have the potential for rapid internodal elongation, which determines plant height. A large body of physiological research has shown that ethylene and gibberellin are involved in this process. |
| − | Plant height is not only a decisive factor in plant | + | * The APETALA2 (AP2)/Ethylene-Responsive Element Binding Factor (ERF) family of transcriptional factors is only present in the plant kingdom. |
| − | architecture but also an important agronomic trait that | + | * '''''OsEATB''''' reduces rice plant height and panicle length at maturity, promoting the branching potential of both tillers and spikelets. |
| − | is directly linked to yield potential <ref name="ref2" /><ref name="ref3" />. The OsEATB transgenic lines
| + | * '''''OsEATB''''' Is a Novel Rice AP2/ERF |
| − | showed dwarf phenotypes, indicating that the internodal
| + | * '''''OsEATB''''' Negatively Regulates Ethylene-Induced Enhancement of GA Responsiveness by Reducing GA Biosynthesis |
| − | elongation process was suppressed by OsEATB
| + | * '''''OsEATB''''' Regulates Rice Yield Components through the Promotion of Tillering and Panicle Branching |
| − | 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 <ref name="ref4" />. A number of genes encoding GA biosynthetic
| |
| − | or signaling pathways have been identified as being
| |
| − | associated with this rapid process <ref name="ref3" />. 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 <ref name="ref5" /><ref name="ref6" />. 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)'''<ref name="ref7" />. 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
| |
| − | <ref name="ref28" />. 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)'''.<ref name="ref1" />
| |
| − | | |
| − | *'''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 <ref name="ref8" />. 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 <ref name="ref9" /> ,
| |
| − | down-regulates the GA signaling pathway <ref name="ref10" /> . 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.<ref name="ref1" />
| |
| − | | |
| − | *'''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 <ref name="ref11" /> . 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 <ref name="ref12" />. 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).<ref name="ref1" />
| |
| − | | |
| − | *'''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 <ref name="ref13" />, and OsEREBP1 is
| |
| − | related to the regulation of defense responses <ref name="ref14" />. 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 <ref name="ref17" />. 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
| |
| − | <ref name="ref29" />. 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 <ref name="ref10" /> <ref name="ref15" /> , and many ERF genes are
| |
| − | induced by abiotic stress conditions in rice <ref name="ref16" /> <ref name="ref17" />. Thus, rice OsEATB might be a
| |
| − | negative balance regulator of the ethylene-responsive
| |
| − | pathway.<ref name="ref1" />
| |
| − | | |
| − | *'''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 <ref name="ref18" />. 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 <ref name="ref19" /> <ref name="ref10" />. 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 <ref name="ref15" />. 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
| |
| − | <ref name="ref6" />. 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
| |
| − | <ref name="ref30" />. Evidence is presented indicating
| |
| − | that OsCPS2 is involved in related secondary metabolism,
| |
| − | producing defensive phytochemicals <ref name="ref31" />.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.<ref name="ref1" />
| |
| | | | |
| − | *'''OsEATB Regulates Rice Yield Components through the Promotion of Tillering and Panicle Branching'''
| + | ===Phenotypic analysis=== |
| − | Food security for the ever-increasing world population
| + | * Overexpression of '''''OsEATB''''' Decreases the Endogenous GA Level in Rice |
| − | largely relies on the grain yield of crop plants
| |
| − | <ref name="ref32" />. 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)<ref name="ref20" />.
| |
| − | 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 <ref name="ref12" />. MOC1 positively regulates tillering by promoting
| |
| − | axillary meristem outgrowth <ref name="ref21" /><ref name="ref22" />. 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 <ref name="ref23" /><ref name="ref24" />.
| |
| − | Higher yields are typically obtained from dwarf crops
| |
| − | <ref name="ref25" />. The finding that transgenic
| |
| − | rice plants harboring the MOC1 gene are dwarf but
| |
| − | produce more tillers than wild-type plants <ref name="ref21" /> 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.<ref name="ref1" />
| |
| | | | |
| | ===Expression=== | | ===Expression=== |
| − | *'''Expression of OsEATB in Transgenic Rice Lines''' | + | * OsEATB Expression Is Negatively Regulated by Ethylene, ABA, and Abiotic Stress in Rice |
| − | To investigate the function of OsEATB, we introduced
| + | * Ectopic expression of OsEATB showed that the cross talk between ethylene and gibberellin, which is mediated by OsEATB, might underlie differences in rice internode elongation. |
| − | the plasmid 35S:OsEATB containing the
| + | * Analyses of gene expression demonstrated that OsEATB restricts ethylene-induced enhancement of gibberellin responsiveness during the internode elongation process by down-regulating the gibberellin biosynthetic gene, ent-kaurene synthase A. |
| − | OsEATB gene (Fig. 2A) into indica variety 9311. We
| |
| − | produced transgenic plants overexpressing the sense
| |
| − | strand of OsEATB. 35S:OsEATB transformants were
| |
| − | screened on antibiotic selection medium containing
| |
| − | hygromycin. The transgenic plants were checked by
| |
| − | PCR using genomic DNA as the template and specific primers (see “Materials and Methods”). Eight independent
| |
| − | transformants (T0), regenerated fromhygromycinresistant
| |
| − | calli, were shown to contain OsEATB. Eight
| |
| − | PCR-positive T1 lines (20 individual plants of each
| |
| − | line) shared the same dwarf phenotype. Two transgenic
| |
| − | lines (401003 and 401006) were selected for
| |
| − | Southern hybridization analysis. A single, specific
| |
| − | band of OsEATB transgene was observed in 35S:
| |
| − | OsEATB transgenic lines 401003 and 401006 '''(Fig. 2B)'''.
| |
| − | These results showed that OsEATB was integrated into
| |
| − | the rice genome.
| |
| − | The expression level of the OsEATB gene was examined
| |
| − | in three-leaf-stage transgenic plants and
| |
| − | control plants by real-time PCR analyses '''(Fig. 2C)'''.
| |
| − | Expression of OsEATB was 9.77-fold greater in the
| |
| − | 401006 transgenic plants and 4.00-, 2.83-, and 2.00-fold
| |
| − | greater in the 401003, 401005, and 401007 transgenic
| |
| − | plants than in 9311 control plants, respectively. These
| |
| − | results were further confirmed by RNA hybridization.<ref name="ref1" />
| |
| − | | |
| − | *'''Overexpression of OsEATB Decreases the Endogenous'''
| |
| − | GA Level in Rice CPS catalyzes the cyclization of GGDP to CDP,
| |
| − | which is then converted to ent-kaurene before going
| |
| − | through a series of processes to produce GA and various
| |
| − | GA derivatives. Since OsEATB represses the expression
| |
| − | of OsCPS2, we determined the endogenous GA
| |
| − | level in three-leaf-stage 401006 OsEATB transgenic
| |
| − | plants and 9311 control plants, six-leaf-stage 401006
| |
| − | and 401003 OsEATB transgenic plants and 9311 control
| |
| − | plants. As shown in Table II, the endogenous levels of
| |
| − | all GAs tested (i.e. GA12, GA53, GA24, GA19, GA9, GA20,
| |
| − | GA4, and GA1) were significantly lower in OsEATB
| |
| − | transgenic plants than in the wild type, while the
| |
| − | levels of abscisic acid (ABA) and indole-3-acetic acid
| |
| − | (IAA) were not significantly changed. Therefore, GA
| |
| − | levels are decreased along with the suppression of
| |
| − | OsCPS2 expression '''(Supplemental Fig. S2)'''.<ref name="ref1" />
| |
| − | | |
| − | *'''OsEATB Expression Is Negatively Regulated by Ethylene, ABA, and Abiotic Stress in Rice'''
| |
| − | OsEATB binds to the ERE, the GCC box, resulting
| |
| − | in decreases in the levels in GAs. While increased
| |
| − | responsiveness to GA is often associated with an
| |
| − | increase in ethylene <ref name="ref15" />, differential
| |
| − | expression of OsEATB was observed in 18-d-old | |
| − | (three-leaf stage) rice 9311 seedlings in response to 50
| |
| − | mM ethephon treatment '''(Fig. 5A)'''. Ethephon treatment
| |
| − | sharply decreased the expression of OsEATB, and
| |
| − | expression remained at low levels for 24 h after treatment.
| |
| − | Because the expression level of OsEATB was
| |
| − | down-regulated by ethephon treatment and OsCPS2 is
| |
| − | decreased by OsEATB, we examined the effect of
| |
| − | ethylene treatment on OsCPS2. Real-time quantitative | |
| − | PCR analysis was used to investigate the expression of
| |
| − | OsEATB and OsCPS2 at 0, 3, 6, 12, and 24 h of ethephon
| |
| − | treatment '''(Fig. 5B)'''. Expression of OsCPS2 increased
| |
| − | while that of OsEATB was sharply down-regulated by
| |
| − | the treatment. Nevertheless, after 12 h of treatment, the
| |
| − | expression of OsCPS2 was stabilized to a normal level
| |
| − | via the self-balance regulation of other pathways, for it
| |
| − | is a key enzyme in GA biosynthesis. This result indicated
| |
| − | that an increase in the amount of OsCPS2 in
| |
| − | response to ethylene is correlated with the decrease in
| |
| − | OsEATB expression responding to ethylene. In rice, most ERF genes are induced by abiotic stress | |
| − | conditions <ref name="ref17" />. To investigate the effect of
| |
| − | transgenic OsEATB on the response to abiotic stress in
| |
| − | rice, 18-d-old seedlings of 9311 control and transgenic | |
| − | 401006 plants were treated with 200 mM NaCl or
| |
| − | 100 mM ABA '''(Fig. 5, C and D)'''. The transgenic seedlings
| |
| − | treated with 200 mM NaCl showed wilting and rolling
| |
| − | of leaves, while 9311 control plants showed a significantly | |
| − | higher survival rate '''(Fig. 5C)'''. After 48 h of
| |
| − | stress followed by 48 h of watering, almost all of the
| |
| − | transgenic plants never recovered, whereas 42% of the
| |
| − | control plants survived. The transgenic seedlings
| |
| − | showed stronger chlorosis in response to the 100 mM
| |
| − | ABA treatment, while the chlorosis of the 9311 control
| |
| − | line was slower '''(Fig. 5D)'''. Thus, overexpression of
| |
| − | OsEATB made seedlings hypersensitive to NaCl and ABA. We also analyzed whether the expression of
| |
| − | OsEATB was regulated by these two conditions. Seedlings
| |
| − | (18 d old) of 9311 subjected to a salt treatment
| |
| − | (200 mM NaCl in Hoagland solution) showed rapid
| |
| − | down-regulation of OsEATB expression within 24 h
| |
| − | after the treatment compared with that in the watertreated
| |
| − | control plants (Fig. 5A). Moreover, expression
| |
| − | of OsEATB was slightly decreased in 18-d-old 9311
| |
| − | seedlings in response to ABA treatment (100 mM ABA
| |
| − | in Hoagland solution) compared with that in the
| |
| − | water-treated control plants '''(Fig. 5A)'''. OsERF3 is upregulated
| |
| − | by ethylene treatment<ref name="ref26" /> ,
| |
| − | while ABA treatment induces the expression of CPD
| |
| − | <ref name="ref276" />). Therefore, we chose OsERF3 as a
| |
| − | positive control for ethylene responsiveness and CPD
| |
| − | as a positive control for ABA responsiveness. These
| |
| − | results strongly suggest that OsEATB is responsive to
| |
| − | ethylene and environmental stresses and, therefore,
| |
| − | may be involved in the regulation of ethylene-related
| |
| − | responses.<ref name="ref1" />
| |
| − | | |
| − | ===Evolution===
| |
| − | *There are 161 known AP2/ERFs in indica rice, 103 of
| |
| − | which are potential ERFs with a single complete AP2
| |
| − | domain (http://plntfdb.bio.uni-potsdam.de). Of these
| |
| − | 103 potential ERFs, 38 cannot be classified into any of
| |
| − | the four functional subgroups <ref name="ref17" />, for
| |
| − | they lack typical group motifs. We selected OsEATB
| |
| − | from these 38 ERFs for further investigation because,
| |
| − | like the members of subgroup IV, it contains a nuclear
| |
| − | localization signal adjacent to the highly conserved
| |
| − | AP2 domain '''(Fig. 1A; Supplemental Fig. S1)'''. Multiple
| |
| − | sequence alignment of OsEATB with other known ERF
| |
| − | proteins showed that their similarity was restricted to
| |
| − | the DNA-binding domain region, and OsEATB could
| |
| − | not be classified into any of the ERF subgroups with
| |
| − | previously defined functions '''(Fig. 1B)'''. We inferred
| |
| − | that OsEATB is a rice ERF that might have a potential
| |
| − | new function.
| |
| − | Based on the data from the National Center for
| |
| − | Biotechnology Information GenBank database (http:// www.ncbi.nlm.nih.gov/), OsEATB has no intron and
| |
| − | exists as a single-copy gene. Using PCR, we isolated
| |
| − | and cloned its full-length 825-bp open reading frame
| |
| − | (ORF) from genomic DNA of indica variety 9311. Sequence
| |
| − | data for this article have been deposited at
| |
| − | GenBank under accession number EU622934. The
| |
| − | 825-bp ORF encodes a protein consisting of 274
| |
| − | amino acids.We used the yeast one-hybrid system and
| |
| − | electrophoretic mobility shift assays to examine the
| |
| − | binding activity of OsEATB to the GCC box. As shown
| |
| − | in Figure 1C, the first section of each plate shows
| |
| − | the yeast reporter strain harboring pHIS-GCC box
| |
| − | and pGAD-OsEATB, while the second section shows
| |
| − | yeast cells harboring pHIS-muGCC box and pGADOsEATB.
| |
| − | The third section of each plate shows yeast
| |
| − | cells transformed with pHIS-GCC box and the negative
| |
| − | control vector pGAD242, and the fourth section
| |
| − | shows yeast cells transformed with pGAD-OsEATB
| |
| − | and negative control vector pHIS2.1. The yeast reporter
| |
| − | strain containing pHIS-GCC box and pGADOsEATB
| |
| − | grew well on both synthetic dextrose (SD)/
| |
| − | Trp2Leu2 medium and SD/Trp2Leu2His2/50 mM
| |
| − | 3-amino-1,2,4-triazole (3-AT) medium. The other three
| |
| − | yeast reporter strains grew well on SD/Trp2Leu2
| |
| − | medium but not on SD/Trp2Leu2His2/50 mM 3-AT medium. The band corresponding to the GCC boxcontaining
| |
| − | labeled probe and purified His fusion
| |
| − | recombinant OsEATB protein complex showed a
| |
| − | marked mobility shift compared with the free probe
| |
| − | band (Fig. 1D). And the muGCC box-containing labeled
| |
| − | fragment served as a competitor. These results
| |
| − | demonstrated that OsEATB could specifically bind to
| |
| − | the GCC box.
| |
| − | If OsEATB acts as a transcription factor, then its
| |
| − | nuclear localization signal should localize it to the
| |
| − | nucleus. To confirm the subcellular localization of
| |
| − | OsEATB, we fused the coding sequence of OsEATB
| |
| − | with that of GFP under the control of the 35S promoter
| |
| − | (35S:OsEATB:GFP) and delivered the construct into
| |
| − | onion (Allium cepa) epidermal cells by particle bombardment
| |
| − | '''(Fig. 1E)'''. The OsEATB protein localized the
| |
| − | GFP signal to the nucleus '''(Fig. 1F)'''.We investigated the
| |
| − | tissue expression pattern of OsEATB by reverse transcription
| |
| − | (RT)-PCR using rice total mRNA from roots,
| |
| − | culms, leaves, and young panicles as the template. The
| |
| − | results showed that OsEATB mRNA is expressed
| |
| − | constitutively in these four tissues and is expressed
| |
| − | at higher levels in roots and leaves than in culms and
| |
| − | young panicles '''(Fig. 1G)'''.<ref name="ref1" />
| |
| | | | |
| | ==Labs working on this gene== | | ==Labs working on this gene== |
| − | *State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University,Shanghai 200433, China. | + | * State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University,Shanghai 200433, China. |
| − | *Key Laboratory of Analytical Chemistry for Biology and Medicine, Department of Chemistry, Wuhan University, Wuhan 430072, China. | + | * Key Laboratory of Analytical Chemistry for Biology and Medicine, Department of Chemistry, Wuhan University, Wuhan 430072, China. |
| | | | |
| | ==References== | | ==References== |
| Line 525: |
Line 35: |
| | | | |
| | ==Structured Information== | | ==Structured Information== |
| − | {{JaponicaGene|
| |
| − | GeneName = 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 = [[:category:Japonica Chromosome 9|Chromosome 9]]|
| |
| − | AP = Chromosome 9:17959811..17960910|
| |
| − | CDS = 17959874..17960698|
| |
| − | GCID = <gbrowseImage1>
| |
| − | name=NC_008402:17959811..17960910
| |
| − | source=RiceChromosome09
| |
| − | preset=GeneLocation
| |
| − | </gbrowseImage1>|
| |
| − | GSID = <gbrowseImage2>
| |
| − | name=NC_008402:17959811..17960910
| |
| − | source=RiceChromosome09
| |
| − | preset=GeneLocation
| |
| − | </gbrowseImage2>|
| |
| − | CDNA = <cdnaseq>atgaccaagaaggtgataccggccatggcggcggcgaggcaggattcttgcaagaccaagcttgatgagcgtgggggtagtcatcaggctccgagctccgcgcggtggatctcgtccgagcaggagcacagcatcatcgtcgcggctctgcggtacgtggtgtccgggtgcaccacgccgccgccggagatcgtcacggtggcgtgcggggaggcgtgtgctctgtgcggcatcgacggctgtctcgggtgcgacttctttggggccgaggcggcggggaacgaggaggcggtaatggcgacggattatgctgctgctgctgctgcggccgcggtggcaggaggatcaggcgggaagagggttaggcggaggaggaagaagaacgtgtaccgcggcgtgcggcatcggccgtgggggaagtgggcagcggagatacgcgacccgcgccgcgcggtgcgcaagtggctcgggacgttcgacaccgccgaggaggccgccagggcgtacgaccgcgccgccctcgagttccgcggcgcgcgcgcgaagctcaacttcccgtgctccgagcctttgcccatgcccagccaaagaaacggcaatggcggcgatgctgtcacggcggcgacgacaacggcagagcagatgactccgactctgtcgccgtgcagcgcggatgccgaggagacgacgacgccggtggattggcagatgggcgcggacgaagccggcagcaaccagctctgggatggcttgcaggacctgatgaagctggatgaagcggacacctggttcccgccattttccggtgcagcgtctagtttttga</cdnaseq>|
| |
| − | AA = <aaseq>MTKKVIPAMAAARQDSCKTKLDERGGSHQAPSSARWISSEQEHS IIVAALRYVVSGCTTPPPEIVTVACGEACALCGIDGCLGCDFFGAEAAGNEEAVMATD YAAAAAAAAVAGGSGGKRVRRRRKKNVYRGVRHRPWGKWAAEIRDPRRAVRKWLGTFD TAEEAARAYDRAALEFRGARAKLNFPCSEPLPMPSQRNGNGGDAVTAATTTAEQMTPT LSPCSADAEETTTPVDWQMGADEAGSNQLWDGLQDLMKLDEADTWFPPFSGAASSF</aaseq>|
| |
| − | DNA = <dnaseqindica>64..888#aacgacctcaagcacactactagctccggctcacttagctccctactgatcactggaacactcatgaccaagaaggtgataccggccatggcggcggcgaggcaggattcttgcaagaccaagcttgatgagcgtgggggtagtcatcaggctccgagctccgcgcggtggatctcgtccgagcaggagcacagcatcatcgtcgcggctctgcggtacgtggtgtccgggtgcaccacgccgccgccggagatcgtcacggtggcgtgcggggaggcgtgtgctctgtgcggcatcgacggctgtctcgggtgcgacttctttggggccgaggcggcggggaacgaggaggcggtaatggcgacggattatgctgctgctgctgctgcggccgcggtggcaggaggatcaggcgggaagagggttaggcggaggaggaagaagaacgtgtaccgcggcgtgcggcatcggccgtgggggaagtgggcagcggagatacgcgacccgcgccgcgcggtgcgcaagtggctcgggacgttcgacaccgccgaggaggccgccagggcgtacgaccgcgccgccctcgagttccgcggcgcgcgcgcgaagctcaacttcccgtgctccgagcctttgcccatgcccagccaaagaaacggcaatggcggcgatgctgtcacggcggcgacgacaacggcagagcagatgactccgactctgtcgccgtgcagcgcggatgccgaggagacgacgacgccggtggattggcagatgggcgcggacgaagccggcagcaaccagctctgggatggcttgcaggacctgatgaagctggatgaagcggacacctggttcccgccattttccggtgcagcgtctagtttttgagctagtgttattagatctcaaccgttggattagattactggaaggccattcatttattcgtttcattgtatagctaattagctatacttcattgtttgtgcagagcacgttttttaggagtaccgtcttatacatatttttttgtacagagtagaaagtaacaagaattttgtatattgatggaatagattatttccaattaatatttgcgc</dnaseqindica>|
| |
| − | Link = [http://www.ncbi.nlm.nih.gov/nuccore/NM_001069906.1 RefSeq:Os09g0457900]|
| |
| − | }}
| |
| | [[Category:Genes]] | | [[Category:Genes]] |
| | [[Category:Japonica mRNA]] | | [[Category:Japonica mRNA]] |
