The F-Box Protein OsFBK12 Targets OsSAMS1 forDegradation and Affects Pleiotropic Phenotypes,Including Leaf Senescence, in Rice1[W][OPEN]
Yuan Chen, Yunyuan Xu, Wei Luo, Wenxuan Li, Na Chen, Dajian Zhang, and Kang Chong*
Key Laboratory of Plant Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing100093, China (Y.C., Y.X., W.Lu., W.Li., N.C., D.Z., K.C.); University of the Chinese Academy of Sciences,Beijing 100049, China (Y.C., D.Z.); and National Plant Gene Research Center, Beijing 100093, China (K.C.)
ORCID ID: 0000-0003-4364-778x (K.C.).
Leaf senescence is related to the grain-filling rate and grain weight in cereals. Many components involved in senescenceregulation at either the genetic or physiological level are known. However, less is known about molecular regulation mechanisms.Here, we report that OsFBK12 (an F-box protein containing a Kelch repeat motif) interacts with S-ADENOSYL-L-METHIONINESYNTHETASE1 (SAMS1) to regulate leaf senescence and seed size as well as grain number in rice (Oryza sativa). Yeast two-hybrid,pull-down, and bimolecular fluorescence complementation assays indicate that OsFBK12 interacts with Oryza sativa S-PHASEKINASE-ASSOCIATED PROTEIN1-LIKE PROTEIN and with OsSAMS1. Biochemical and physiological data showed that OsFBK12targets OsSAMS1 for degradation. OsFBK12-RNA interference lines and OsSAMS1 overexpression lines showed increased ethylenelevels, while OsFBK12-OX lines and OsSAMS1-RNA interference plants exhibited decreased ethylene. Phenotypically, overexpressionof OsFBK12 led to a delay in leaf senescence and germination and increased seed size, whereas knockdown lines of either OsFBK12 orOsSAMS1 promoted the senescence program. Our results suggest that OsFBK12 is involved in the 26S proteasome pathway byinteracting with Oryza sativa S-PHASE KINASE-ASSOCIATED PROTEIN1-LIKE PROTEIN and that it targets the substrateOsSAMS1 for degradation, triggering changes in ethylene levels for the regulation of leaf senescence and grain size. These datahave potential applications in the molecular breeding of rice.
F-box proteins are components of E3 ubiquitin ligaseS-PHASE KINASE-ASSOCIATED PROTEIN, CULLIN,F-BOX CONTAINING COMPLEXES (SCFs), which me-diate a wide variety of biological processes (Schulmanet al., 2000). The N terminus of F-box proteins, which in-teracts with S-Phase Kinase-Associated Protein1 (Skp1), isconserved. The C terminus generally contains one orseveral highly variable protein-protein interactiondomains, such as Leu-rich repeat, Kelch repeat, tetra-tricopeptide repeat, or WD40 repeat domains (Jainet al., 2007). Kelch motifs consist of 44 to 56 amino acidresidues, with four highly conserved residues, two ad-jacent Gly residues, and a Tyr and Trp pair separatedby about six residues. The presence of Kelch repeats is aunique characteristic of a subset of F-box proteins inplants (Prag and Adams, 2003).
F-box proteins target their substances for specificfunctions. Several key hormone signaling components,
including receptors, have been identified as F-boxproteins. The TRANSPORT INHIBITOR RESPONSE1(TIR1) F-box protein acts as an auxin receptor regu-lating the stability of auxin/indole-3-acetic acid proteinsin Arabidopsis (Arabidopsis thaliana; Gray et al., 2001;Zhang et al., 2011). CORONATINE-INSENSITIVE1 isan F-box protein that is a coreceptor with JASMONATEZIM-DOMAIN PROTEIN1 as a central regulator ofjasmonate signaling (Sheard et al., 2010). SNEEZY andSLEEPY1 regulate DELLA through interaction with theDELLA-GIBBERELLIN-INSENSITIVE DWARF1 com-plex in GA signaling (Dill et al., 2004; Strader et al.,2004). In addition, ETHYLENE-INSENSITIVE2 andETHYLENE-INSENSITIVE3 are quickly degradedby the F-box proteins ETHANOL TOLERANCEPROTEIN1/ETHANOL TOLERANCE PROTEIN2and EARLY B-CELL FACTOR1/EARLY B-CELLFACTOR2 during ethylene signaling (Guo and Ecker,2003; Potuschak et al., 2003; Qiao et al., 2009; Wang et al.,2009a). Only a few F-box proteins containing Kelchmotifs (FBKs), however, have been characterized. TheFBK proteins ZEITLUPE, FLAVIN-BINDING, KELCHREPEAT, F-BOX1, and LOV KELCH PROTEIN2 are in-volved in light signaling, flowering, and circadian controlvia a proteasome-dependent pathway in Arabidopsis(Imaizumi et al., 2005). The rice (Oryza sativa) FBK geneLARGER PANICLE/ERECT PANICLE3 was reported toregulate panicle architecture (Piao et al., 2009) and mod-ulate cytokinin levels through Oryza sativa CYTOKININOXIDASE2 expression (Li et al., 2011a). However, only a
1 This work was supported by the Major State Basic Research Pro-gram 973 (grant no. 2011CB915400), the National Natural SciencesFoundation of China (grant nos. 31070695 and 31121065).
* Address correspondence to [email protected] author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Kang Chong ([email protected]).
[W] The online version of this article contains Web-only data.[OPEN] Articles can be viewed online without a subscription.www.plantphysiol.org/cgi/doi/10.1104/pp.113.224527
Plant Physiology�, December 2013, Vol. 163, pp. 1673–1685, www.plantphysiol.org � 2013 American Society of Plant Biologists. All Rights Reserved. 1673 www.plantphysiol.orgon September 7, 2018 - Published by Downloaded from
few substances for F-box proteins with specific functionsare known in plants. In other words, it is still not clearhow F-box proteins mediate plant developmental pro-cesses such as leaf senescence and seed size.
Leaf senescence and the related ethylene regulationimpact grain filling, which is an important determi-nant of yield, especially in the last stage of maturationin rice. Delayed leaf senescence was reported to bemediated by a nucleus-localized zinc finger protein,Oryza sativaDELAYOF THEONSET OF SENESCENCE,in rice (Kong et al., 2006). The STAY GREEN RICE geneis involved in regulating pheophorbide a oxygenasethat causes alterations in chlorophyll breakdownduring senescence (Jiang et al., 2007). Physiologi-cally, the progression of leaf senescence is dependenton ethylene levels, which also regulate grain filling(Wuriyanghan et al., 2009; Agarwal et al., 2012). Inplants, it is well established that ethylene is bio-synthesized from S-adenosyl-L-methionine (SAM) via1-aminocyclopropane-1-carboxylic acid (ACC). ACCsynthase catalyzes the first step of the biosynthesis byconverting S-adenosylmethionine into ACC, and ACCoxidase catalyzes the second step by metabolizing ACCand dioxygen into ethylene. S-Adenosyl-L-methioninesynthase (SAMS) is involved in developmental regula-tion mediated by methylation alterations of DNA andhistones in rice (Li et al., 2011b). The physiologicalfunction of ethylene in rice is dependent not only on itsbiosynthesis but also on signal transduction componentssuch as the ETHYLENE-RESPONSE2 receptor (Zhu et al.,2011). However, less is known about how F-box proteinregulation is involved in the coordination of senescenceprogression.
Here, we show that a rice F-box gene, OsFBK12, thatcontains a Kelch repeat domain is involved in theregulation of ethylene-mediated senescence and seedsize. Transgenic lines with reduced or increased ex-pression of OsFBK12 showed phenotypes in germina-tion, panicle architecture, and leaf senescence as wellas in seed size. Our data suggest that OsFBK12 directlyinteracts with OsSAMS1 to induce its degradation,which affects ethylene synthesis and histone methyla-tion, leading to pleiotropic phenotypes.
RESULTS
Overexpression and Knockdown of OsFBK12 CausesPleiotropic Phenotypes, Including Leaf Senescence
To explore the network of F-box proteins, an ap-proach using transgenic rice plants as well as molecularinteraction was used. The LOC_Os03g07530 gene, locatedon chromosome 3 (DNA sequence 3,832,950–3,835,744), ispredicted to encode an F-box protein (http://rice.plantbiology.msu.edu/cgi-bin/ORF_infopage.cgi?orf=LOC_Os03g07530.1), and based on its homologs(http://rice.plantbiology.msu.edu/ca/gene_fams/1194.shtml), it is termed OsFBK12. An unrootedphylogenetic tree shows the relationship of the FBKproteins in rice and Arabidopsis (Supplemental Fig. S1).
OsFBK12 is predicted to be a protein of 431 amino acidscontaining an F-box domain (amino acid residues 94–141)at the N terminus and a Kelch repeat motif protein-proteininteraction domain (amino acid residues 159–365) at theC terminus.
To investigate the biological function of OsFBK12,transgenic lines with reduced or increased expressionwere generated. We obtained 12 transformed RNAinterference (RNAi) lines and 15 overexpression trans-genic lines. Real-time PCR analysis confirmed that thetranscription of OsFBK12 was reduced in the RNAitransgenic lines (FR) and increased in the transgenicoverexpression lines (FO; Fig. 1D). The FO transgeniclines showed delayed germination and bottle-greenleaves under normal growth conditions, whereas theFR lines developed precociously and had light greenleaves (Fig. 1, A and C). At the tillering stage, over-expression ofOsFBK12 caused a decrease in tiller numberand an increase in plant height compared with the wildtype. By contrast, the RNAi (FR) lines displayed increasedtiller numbers and decreased plant height (Fig. 1, B, E,and F). At 45 d after heading, leaves of the FR lines be-came withered and the chlorophyll a/b content showed afaster reduction than in the wild type, which is a signa-ture of earlier senescence. By contrast, the FO lines stayedgreen, with suppressed chlorophyll reduction and decel-erated senescence (Fig. 1, C and G). Overall, alterations ingermination and leaf senescence were the most dramaticdevelopmental phenotypes of the transgenic plants.
Transcription pattern analysis showed that OsFBK12was expressed in all organs and tissues but predomi-nantly in panicle and seed (Supplemental Fig. S2).Transgenic OsFBK12p::GUS rice plants showed strongGUS staining signals in panicle, root tip, young leaf, andleaf sheath but little in mature leaf and stem at theheading stage (Supplemental Fig. S3). Examination ofpanicle architecture in the FR transgenic lines revealed asignificant increase in branch numbers (both primary andsecondary) and in the total numbers of spikelets perpanicle as well as a slight decrease in panicle axis length(Fig. 2, A, B, and E). Conversely, branching and spikeletnumber per panicle were reduced in FO lines comparedwith those in the wild type, while the panicle length wasincreased (Fig. 2, A, B, and E). In addition, the number ofgrains and the grain-filling rate (the ratio of seed-filledover total florets) were altered in the transgenic lines(Fig. 2E). The grains of FR lines were a little thinner andshorter than those of the wild type (Fig. 2, C and D),causing a slight decrease in the 100-grain weight (1.71 g)compared with that of the wild type (2.25 g; Fig. 2E). Thegrains in FO lines were significantly wider and longerthan those in the wild type (Fig. 2, C and D), leading the100-grain weight to increase by 0.7 g (Fig. 2E).
Histological analysis on the spikelet hull showedthat the outer parenchyma cell layer in the FO line was
increased compared with the wild type, while the FR linewas reduced (Fig. 3, A–C). Scanning electron microscopyanalysis showed that some of the starch granules packedtogether and appeared “football like”; those football-likestarch granules in the middle zone were larger in the FOlines than in the wild type, whereas the starch granuleswere smaller in the FR lines (Fig. 3D). The cell numbersin the lemma were also different between the wild typeand the transgenic lines (Fig. 3E). The FR-4 line showedmore cells than in the wild type, whereas the FO-9 linehad fewer. There was no difference in cell size in theother zones of the endosperm. Therefore, OsFBK12 maybe involved in the regulation of cell division in the hull.
OsFBK12 Interacts with a SKP1-Like Proteinin the Nucleus
Yeast two-hybrid assays were performed to screenfor proteins that interact with OsFBK12. The entire codingregion of OsFBK12 was inserted into the pGBKT7 vector
as bait. Positive clones were identified based on bothsurvival on restrictive medium (synthetic dextrose/2His/2adenine [Ade]/2Trp/2Leu) and expressionlevels of he b-galactosidase (lacZ) reporter gene. Therewere 216 colonies that survived on restrictive medium of2His/2Ade/2Trp/2Leu, and 102 of them expressedthe b-galactosidase (lacZ) reporter gene. Among the 96positive clones that were sequenced, 16 correspondedto Oryza sativa S-PHASE KINASE-ASSOCIATEDPROTEIN1-LIKE PROTEIN (OSK1). To confirm theinteraction further, a full-length complementaryDNA (cDNA) of OSK1 was used as prey. Fragments ofOsFBK12 encoding the F-box domain (OsFBK12Dkelch),the Kelch repeat domain (OsFBK12DF-box), and the full-length cDNA were used as baits. Colonies expressinglacZ were obtained with the F-box domain and full-length OsFBK12, but not with the Kelch repeat do-main (Fig. 4A). These results suggest that OsFBK12interacts with OSK1 through the F-box domain inyeast (Saccharomyces cerevisiae) cells.
Figure 1. Phenotypes of OsFBK12 transgenic lines at different developmental stages. A, Germination. WT, The wild type; FR-4,FR-10, and FR-12, OsFBK12 RNAi lines 4, 10, and 12, respectively; FO-5 and FO-9, OsFBK12 overexpression lines 5 and 9,respectively. Bar = 1 cm. B, Vegetative growth status. Bar = 20 cm. C, Grain-filling period. Bar = 20 cm. D, Relative expressionlevel of OsFBK12 in RNAi lines and overexpression lines. ACTIN was used as a control. Quantitative reverse transcription-PCRdata represent means6 SD of three biological replicates. E, Plant height ofOsFBK12 transgenic plants at the heading stage. Dataare means6 SD of triplicate experiments. F, Tiller number ofOsFBK12 transgenic plants at the heading stage. Data are means6 SD
of triplicate experiments. G, Chlorophyll content of the upper three leaves from the main culm. Data are means 6 SD of triplicateexperiments. FW, Fresh weight; WAH, week after heading.
In bimolecular fluorescence complementation (BiFC)assays, strong GFP fluorescence was observed whenPSPYNE(R)173-OsFBK12 and SPYCE>(MR)-OSK1
were coexpressed in the nuclei of tobacco (Nicotianabenthamiana) leaf epidermal cells (Fig. 4B). Cellstransformed with single constructs alone did not show
Figure 2. Panicle morphology and seed sizephenotypes of the OsFBK12 transgenic lines.A, Panicles. OsFBK12 RNAi and overexpressionlines are shown at maturity. WT, The wild type;FR-4, FR-10, and FR-12, OsFBK12 RNAi lines 4,10, and 12, respectively; FO-5 and FO-9,OsFBK12 overexpression lines 5 and 9, respec-tively. Bar = 3 cm. B, Panicle branching. Bar = 3 cm.C, Seed width and seed length. Bar = 1 cm. Vectorindicates empty vector UN1301 transformed rice asa control. D, Tubes containing 43 seeds from theindicated lines. Bar = 1 cm. E, Statistical analysis ofseed size and panicle types. The data of FR and FOindicate averages of three individual FR lines andtwo FO lines. Data are means 6 SE. Multiple com-parisons were performed by Tukey’s honestly sig-nificant difference post hoc test. Letters indicatesignificantly different results. Tests were carried outby the R library multcomp.
Figure 3. Cell number and size of hulls and endosperm in the transgenic lines. A, Grain shape. WT, The wild type; FR-4,OsFBK12 RNAi line 4; FO-9, OsFBK12 overexpression line 9. White lines indicate the positions of the cross sections in B.Bar = 1 mm. B, Cross sections of the hulls. The boxes indicate the regions enlarged in C. Bars = 500 mm. C, Magnified view ofthe boxes in B. Bars = 50 mm. D, Scanning electron microscopy images of transections of endosperm. I, II, and III representthe outer, middle, and inner parts of the endosperm, respectively. Enlarged regions corresponding to I, II, and III are shownbelow. The wild type is shown in a, d, g, and j; FR-4 is shown in b, e, h, and k, and FO-9 is shown in c, f, i, and l. Whitearrows denote football-like starch granules. Bars = 1 mm (a–c) and 20 mm (d–l). E, Statistical analysis of the total length, cellnumber, and cell length in the outer parenchymal cell layers of the hulls. The data of FR and FO indicate averages of threeindividual FR lines and two FO lines. Data are means 6 SE. Student’s t test was performed. Asterisks represent P , 0.05compared with wild-type values.
any fluorescence. Protein localization assays showedthat the signal from an OsFBK12-GFP fusion proteinoverlapped with nuclear H33342 staining in proto-plasts (Fig. 4C; Supplemental Fig. S4A) but was notcoexpressed with the endoplasmic reticulum markermCherry-HDEL in rice protoplasts (Supplemental Fig. S5).By contrast, the OSK1-GFP fusion protein was localizedin both the nucleus and the cytoplasm (SupplementalFig. S4B).
OsFBK12 Interacts with OsSAMS1 for Degradationin Plant Cells
Kelch repeat domains in FBKs function in protein-protein interactions, which specify the protein sub-strates for degradation via the ubiquitin pathway (Sunet al., 2007). We used the Kelch repeat domain in thepGBKT7 vector as bait to screen a rice cDNA library inyeast. Of the 167 positive colonies that survived onrestrictive medium of 2His/2Ade/2Trp/2Leu, 90expressed the b-galactosidase (lacZ) reporter gene.
Among the 78 positive clones that were sequenced, oneencoded SAMS1. Assays in yeast cells using truncationconstructs showed that OsSAMS1222-316 (OsSAMS1 aminoacids 222–316) interacted with both full-length OsFBK12and the Kelch repeat domain truncation (Fig. 5A). In apull-down assay, purified glutathione S-transferase(GST)-OsSAMS1 was immobilized to glutathione-Sepharose beads, and OsFBK12 tagged with maltose-binding protein (MBP) was incubated with the beads.GST and MBP alone were used as negative controls. Aband at 92 kD was recognized by an antibody againstMBP in immunoblot assays. By contrast, the negativecontrols did not show the corresponding signal. Thissuggests that OsFBK12 can interact with OsSAMS1 invitro (Fig. 5B). To explore the possibility that OsSAMS1 isthe substrate of OsFBK12, OsSAMS1 levels were moni-tored in the transgenic lines. Immunoblot assays showedthat OsSAMS1 was increased in the knockdown trans-genic lines of OsFBK12, FR-4, FR-10, and FR-12. By con-trast, OsSAMS1 levels were decreased in the OsFBK12overexpression lines FO-5 and FO-9 (Fig. 5C). Tobaccoleaves expressing OsSAMS1-GFP were then treated with
Figure 4. Interaction of OsFBK12 with OSK1 in vivo and subcellular localization. A, Yeast two-hybrid assay for the interactionof OsFBK12 with OSK1. A schematic diagram of OsFBK12 and the truncations used is shown. The bait (BD) vector containedfull-length OsFBK12, OsFBK12△kelch, or OsFBK12 △F-box; the prey (AD) vector contained OSK1. Yeast strains were cultured onthe2Trp2Leu2His2Ade selection medium. b-Galactosidase activity of positive clones was analyzed using 5-Bromo-4-chloro-3-indolyl b-D-galactopyranoside. The proved interaction between OsGSR1 (a GA-stimulated transcript family gene in rice) andDWARF1 was used as a positive control. B, BiFC assay for interaction between OsFBK12 and OSK1 in tobacco. Shown is thecoexpression of Yellow fluorescence protein N-terminal (YN)-OsFBK12 and Yellow fluorescence protein C-terminal (YC)-OSK1(top row), YN-OsFBK12 and YC vector (middle row), and YC-OSK and YN vector as a control (bottom row). Bars = 100 mm.C, Subcellular localization of OsFBK12-GFP and OSK1-GFP fusion proteins in rice protoplasts. GFP protein alone showsfluorescent signals in nucleus, membrane, and cytoplasm. H33342 is a staining dye for the nucleus. Bars = 10 mm.
the 26S proteasome inhibitor MG132, showing thatOsSAMS1-GFP was stable in the presence of MG132for up to 8 h (Fig. 5D), whereas in the control (withoutMG132 treatment), OsSAMS1 showed a gradual de-crease. When purified OsFBK12-myc was added intothe extracts, OsSAMS1-GFP degradation was acceler-ated. Furthermore, we tested the polyubiquitination inthe transgenic leaves of OsSAMS1-GFP. The western-blot assay indicated that a series of bands with highermolecular masses were recognized by the ubiquitinantibody in the transgenic leaves of OsSAMS1-GFP
(Fig. 5F). Together, these data suggest that OsFBK12targeted OsSAMS1 for ubiquitination and subsequentdegradation by the 26S proteasome.
Similarities between OsFBK12 and OsSAMS1 TransgenicPlants in Germination and Senescence
Germination is suppressed in knockdown transgenicOsSAMS1 rice plants (Li et al., 2011b). This suppres-sion of germination was rescued by supplementation
Figure 5. Interaction of OsFBK12 with OsSAMS1 for degradation in plants. A, Yeast two-hybrid assay for interaction ofOsFBK12 with OsSAMS1222-316. The reported interacting proteins OsGSR1 (a GA-stimulated transcript family gene in rice) andDWARF1 were used as positive controls. B, Pull-down assay for the interaction of OsFBK12 with OsSAMS1222-316. Inputrepresents crude MBP-OsFBK12 and GST-OsSAMS1 protein. Lanes 1 to 3, Input sample of GST, MBP, and MBP-OsFBK12 andGST-OsSAMS1; lanes 4 to 6, elution from GST/MBP-OsFBK12, GST-OsSAMS1/MBP, and MBP-OsFBK12/GST-OsSAMS1 afterpull down. The arrow shows the band pulled down, and arrowheads show immunoblotting by the anti-MBP monoclonalantibody (Ab). C, Immunoblot analysis of the expression of OsSAMS1 protein in OsFBK12 transgenic seedlings. CoomassieBrilliant Blue staining indicates loading for total protein. WT, The wild type; FR-4, FR-10, and FR-12, OsFBK12 RNAi lines 4,10, and 12, respectively; FO-5 and FO-9, OsFBK12 overexpression lines 5 and 9, respectively. D, OsSAMS1-GFP degradationassays performed in tobacco with or without MG132 treatment. Normalized plots of the degradation of OsSAMS1-GFP areshown below. Data are means 6 SD of triplicate experiments. E, OsSAMS1-GFP degradation assays performed in tobacco withor without OsFBK12 protein. Normalized plots of the degradation of OsSAMS1-GFP are shown below. Data are means 6 SD oftriplicate experiments. F, Pull-down assay using anti-ubiquitin antibody indicated that the bands of higher molecular mass(72–170 kD) were polyubiquitinated of OsSAMS1 in tobacco. The molecular masses are shown in kD. The molecular mass forGFP is 26 kD and that for OsSAM1-GFP is 70 kD. Anti-Ub, Anti-ubiquitin monoclonal antibody.
Figure 6. Germination and senescence phenotypes of the OsFBK12 and OsSAMS1 transgenic lines. A, Effect of SAM on seedgermination in the OsFBK12 transgenic plants. The morphology of seedlings was observed after 40 h for germination. SAMconcentration was 1 mM. Bar = 1 cm. Data are means 6 SD of triplicate experiments with 30 seeds per sample. B, The timecourse of germination and the effect of SAM (1 mM). Data are means 6 SD of triplicate experiments with 30 seeds per sample.C, Effect of ethephon on seed germination in the OsFBK12 and SAMS1 transgenic plants. The morphology of seedlings wasobserved after 40 h for germination. Ethephon concentration was 50 mL L21. Bar = 1 cm. Data are means 6 SD of triplicate
with SAM (1 mM; Fig. 6A). Similarly, in OsFBK12-OX(FO) transgenic plants, there was a block of germina-tion, although the germination patterns were not dif-ferent between OsFBK12-RNAi (FR) lines and the wildtype. The germination suppression in OsFBK12-OX(FO) was also alleviated with SAM treatment (Fig. 6, Aand B). The germination suppression of OsFBK12-OX(FO) lines and OsFBK12-RNAi (FR) lines can also bealleviated with 50 mL L21 ethephon (2-chloroethylsulfonicacid; Fig. 6, C and D). These data are consistent with theidea that altered expression of SAMS caused a corre-sponding change in the production of SAM and ethyleneas well as the resulting alteration of physiological function(He et al., 2006).SAM is a precursor for ethylene biosynthesis,
raising the question of whether ethylene levels wereaffected by changes in SAM. We found that the ACCand ethylene contents were increased in OsFBK12-RNAi (FR) lines and in the OsSAMS1 overexpressiontransgenic lines (SO) relative to the wild type. Corre-spondingly, decreased ACC and ethylene levels appearedin the OsFBK12-OX (FO) plants and OsSAMS1-RNAitransgenic lines (SR) plants (Fig. 6, E and F).We further monitored the phenotypes of the
OsSAMS1 transgenic plants. In the OsSAMS1-OX (SO)transgenic plants, most leaves turned yellow and grainfilling was completed by 100 d after germination,when only a few leaves of wild-type plants were yel-low and wild-type plants remained in the grain-fillingstage. Under the same conditions, the OsSAMS1-RNAi(SR) transgenic plants were still green and not headingyet (Fig. 6G).A leaf senescence assay showed that the OsFBK12-
RNAi (FR) and OsSAMS1-OX (SO) plants displayedfaster senescence than wild-type plants, whereasOsFBK12-OX (FO) and OsSAMS1-RNAi (SR) plantsmaintained green leaves much longer than wild-typeplants (Fig. 6H). To determine whether this phenotypeis caused by ethylene content in the transgenic plants,we treated the wild-type and transgenic plants with50 mL L21 ethephon and 200 mM aminoethoxyvinylglycine(an ethylene biosynthesis inhibitor). Leaf senescencein FR and SO lines was prevented by the biosynthesisblocker, and ethylene treatment promoted the leafsenescence in the FO and SR lines (Fig. 6, H and I).These results suggest that OsSAMS1 promotes plant
developmental processes and leaf senescence and thatOsFBK12 suppresses them.
DISCUSSION
OsFBK12 Targets OsSAMS1 for 26SProteasome-Mediated Degradation
F-box proteins are subunits of E3 ubiquitin ligasecomplexes called SCFs. The F-box motif can bind toSKP1 via the N terminus to form a complex and rec-ognize the target proteins via a protein-protein inter-action domain at the C terminus (Sonnberg et al., 2009).Although F-box proteins have been identified to me-diate multiple biological processes, less is knownabout their specific degraded substrates, especially forF-box proteins containing Kelch motifs (Han et al.,2004; Imaizumi et al., 2005; Piao et al., 2009). Our datasuggest that OsFKB12, as a Kelch-type F-box protein,might form an SCF complex with SKP1 and bind toOsSAMS1, targeting it for 26S proteasome-mediateddegradation.
The well-conserved N-terminal domain of OsFBK12interacted with OSK1, and its C-terminal Kelch repeatdomain interacted with OsSAMS1 (Figs. 4 and 5). Theinteraction of the full-length proteins (Fig. 5B), alongwith three lines of biochemical and physiological evi-dence, support that OsSAMS1 is targeted by OsFBK12for degradation in the 26S proteasome. First, thedegradation of OsSAMS1 was inhibited by treatmentwith MG132 and promoted by treatment with purifiedOsFBK12 (Fig. 5, D and E). Second, OsSAMS1 was in-creased in the OsFBK12 knockdown lines. Conversely,a decrease of OsSAMS1 appeared in the OsFBK12overexpression transgenic line (Fig. 5C). Third, thegermination block in the OsSAMS1-RNAi (SR) trans-genic line could be rescued by supplementation withSAM, indicating that SAM production was likely re-duced in the OsSAMS1 knockdown lines (Li et al.,2011b). The OsFBK12-OX (FO) transgenic plantsshowed phenotypes identical to those of OsSAMS1knockdown lines, supporting that the OsFBK12 pro-motes the degradation of OsSAMS1 and causes thereduction in SAM (Fig. 6A). Moreover OsSAMS1-GFPwas detected in the nucleus and the cytoplasm(Supplemental Figs. S5 and S6), while OsFBK12 was
Figure 6. (Continued.)experiments with 30 seeds per sample. D, The time course of germination and the effect of ethephon (50 mL L21). Data aremeans 6 SD of triplicate experiments with 30 seeds per sample. E, ACC content in different transgenic plants (using 30-d-oldseedlings). Multiple comparisons were performed by Tukey’s honestly significant difference post hoc test. Letters indicatesignificantly different results. Tests were carried out by the R library multcomp. F, Ethylene content in different transgenic plants(using 30-d-old seedlings). G, Phenotypic comparison of the wild type (WT), the OsSAMS1-RNAi (SR) line, and the OsSAMS1overexpression (SO) line at 120 d after germination. Bar = 20 cm. H, Detached leaf senescence for 3 d in darkness (using30-d-old seedlings) with and without treatment of aminoethoxyvinylglycine (AVG) and ethephon. Bar = 3 cm. I, Chlorophyllcontent of the detached leaves in H. Data are means 6 SD of triplicate experiments. FW, Fresh weight. The data of FR and FOindicate averages of three individual FR lines and two FO lines; the data of SR and SO indicate averages of two individual SRlines and two SO lines.
localized in the nucleus; this result hinted that OsSAMS1may be degraded in the nucleus.
OsFBK12 Is Involved in Ethylene-MediatedLeaf Senescence
Ethylene is synthesized from Met via the interme-diates SAM and ACC (Bouvier et al., 2006). The con-version of Met to S-adenosylmethionine is catalyzedby SAMS, and the conversion from SAM to ACC iscatalyzed by ACC synthase, which are the rate-limitingenzymes in ethylene biosynthesis. Regulation of ethylenebiosynthesis occurs at both the gene expression level andthe protein activity level (Bouvier et al., 2006). Thus,OsFBK12 can be considered a negative regulator ofethylene biosynthesis by virtue of its function in pro-moting the degradation of OsSAMS1. Overexpression ofOsFBK12 and knockdown of OsSAMS1 caused a de-crease in the ethylene-responsive genes OsEATB andOsCTR1 and ethylene production, delaying leaf senes-cence, whereas overexpression of OsSAMS1 and knock-down of OsFBK12 resulted in increased ethylene levels,
promoting early leaf senescence (Fig. 6; SupplementalFig. S7). Therefore, OsFBK12 is a negative regulator ofethylene-mediated senescence through the degrada-tion of OsSAMS1.
OsFBK12 Might Regulate Seed Size
Altered OsFBK12 gene expression led to changes inseed size resulting from changes in the cell numberand the size of the football-like granules. Football-likegranules is an important part of the completion ofstarch granule packaging, which is similar to the typesof granules that associate with the grain size in wheatendosperm (Xu et al., 2010).
The cell numbers in the lemma of the OsFBK12 over-expression lines were reduced, while the cell length andthe football-like granule size were increased (Fig. 3E).This can be explained by the compensatory mechanismsin monocot species (Barrôco et al., 2006). That is, thereduced cell production can be partly compensated byan increased cell size. The cell size in the OsFBK12-OX(FO) lines was compensated through cell expansion.
Figure 7. Proposed working model for the OsFBK12regulation of leaf senescence and seed size in rice.This model proposes that OsFBK12 was involved in26S proteasome-mediated degradation by interactingwith OSK and targeted the substrate OsSAMS1.When OsSAMS1 degraded, it caused a correspond-ing change in ethylene level and regulated leafsenescence. Meanwhile, OsFBK12 might targetanother substrate or might regulate the transcriptionof some genes to affect seed size. PPi, pyrophos-phate; Pi, phosphate; MAT, 5’-methylothioadenosine.
The knockdown of OsFBK12 reduced the cell size dur-ing spikelet hull development. Cell production in theOsFBK12-RNAi (FR) lines increased while the spikelethull was smaller, and the cells size in spikelet hull andthe football-like starch granules in the endosperm wererestricted and partly compensated by decreased cellsize.OsFBK12 interacts with SAMS1 to regulate leaf se-
nescence. The mRNA levels of OsSAM1 as well asother OsSAMs in OsSAMS1-RNAi (SR) have beenreported (Li et al., 2011b). The mRNA levels of eitherOsSAMS1 orOsSAMS2were decreased in theOsFBK12-OX(FO) transgenic plants, whereas the mRNA level ofOsSAMS3 was increased in OsFBK12 RNAi (FR) trans-genic plants (Supplemental Fig. S8). This indicated thatthe transgene of OsFBK12 impacts OsSAMS1 on boththe protein interaction and RNA transcription levels,which are both upstream of ethylene biosynthesis. It isnotable that SAM is not only a precursor for ethylenebiosynthesis but also a universal methyl group donorand involved in numerous transmethylation reac-tions (Frostesjö et al., 1997; Rocha et al., 2005; Zhanget al., 2011). That SAM also functions as a precursor ofpolyamines might explain the diverse phenotypes be-tween the lines of OsFBK12-OX (FO) and OsSAMS1-RNAi (SR) except for the consensus ones (Tomosugiet al., 2006; Kusano et al., 2007; Yang et al., 2008).Based on our data, we propose a model (Fig. 7) for
how OsFBK12 regulates senescence. OsFBK12 inter-acts with OSK1 to form an SCF complex and degradeits substrate, such as OsSAMS1. The degradation ofOsSAMS1 results in a decrease in SAM content aswell as in ethylene levels, which affects germinationand leaf senescence. Additionally, it is possible thatOsFBK12 might affect cell division, resulting in changesin cell numbers in the spikelet hull. These findings maylead to a better understanding of senescence control inrice.
MATERIALS AND METHODS
Plant Germination and Transformation
For seed germination, dehulled rice (Oryza sativa) seeds of different transgeniclines and the wild type were surface sterilized and immersed in water. Seedswere placed in a growth chamber under dark conditions for the first 72 h, andthen with a 12/12-h light/dark cycle at 25°C. Germination was defined as whenthe coleoptile is 5 mm long. Every experiment was repeated three times, with30 seeds per sample.
The japonica rice cv Zhonghua 10 was used for transformation. Trans-genic plants derived from callus were defined as the T0 generation. Trans-genic plants of the T1 and T2 generations were used for phenotypicanalyses. The SAM treatment experiment was performed according to Liet al. (2011b).
Total RNA Extraction and Real-Time PCR
Total RNA was extracted by use of the TRIzol RNA extraction kit (Invi-trogen) and treated with RNase-free DNase I (MBI Fermentas). Total RNA(2 mg) was reverse transcribed into cDNA by AMV Reverse Transcriptase(Promega). Real-time PCR amplification was performed in 20-mL reactionscontaining 5 mL of 50-fold diluted cDNA, 0.2 mM of each primer, and 10 mL ofSYBR Green PCR Master Mix (Toyobo). Quantitative real-time PCR was
performed on Mx3000p (Stratagene) using SYBR Green reagent (Toyobo).Expression was normalized to that of ACTIN. Primer sequences used foramplification are listed in Supplemental Table S1.
Vector Construction and Rice Transformation
The entire open reading frame (ORF) ofOsFBK12was amplified by RT-PCRand then inserted upstream of GUS in the binary plasmid pUN1301 (Chenet al., 2011). The pTCK303-OsFBK12 construct was used to create an RNAiknockdown transgenic line. The detailed protocols for construct generationwere described previously (Wang et al., 2004). The resulting constructs wereused for transformation via Agrobacterium tumefaciens strain EHA105 as de-scribed previously (Ge et al., 2004). All primers used in this study are listed inSupplemental Table S1.
Scanning Electron Microscopy
Rice seeds were fixed in 50% (v/v) ethanol, 5% (v/v) acetic acid, and 37%(v/v) formaldehyde for more than 24 h and then dehydrated through a gradedseries of alcohol-isoamyl acetate. After being critical-point dried in carbondioxide for 1 h with a Hitachi HCP-2, the plant material was coated with goldand observed with a Hitachi S-4800 scanning electron microscope at 10.0 kV.
Histological Analysis
Rice spikelets were fixed in 50% (v/v) ethanol, 5% (v/v) acetic acid, and 37%(v/v) formaldehyde at room temperature overnight and then dehydrated in anethanol series, cleared with xylene, and embedded in Paraplast (Sigma). Tissuesections (10 mm thick) were cut and stained with 0.02% (w/v) toluidine blue for5 min at room temperature after dewaxing. Photographs were taken on anOlympus VANOX microscope.
Yeast Two-Hybrid Assay
The BD Matchmaker library construction and screening kit (ClontechLaboratories) was used for yeast two-hybrid assays. All protocols were carriedout according to the manufacturer’s user manual. The cDNA encoding the full-length OsFBK12 protein was inserted into the GAL4 DNA-binding domainvector pGBKT7. The pGADT7 clones were selected on synthetic dextrose/2Leu/2Trp/2His/2Ade/5-Bromo-4-chloro-3-indolyl b-D-galactopyranosideplates and sequenced at the Invitrogen Sequencing Facility to ensure that theprey proteins were in-frame fusions with the GAL4 pGADT7 Vector domainusing the system described previously (Wang et al., 2009a).
Pull-Down Assay
To test the interaction between OsFBK12 and OsSAMS1, the ORF of OsSAMS1was cloned into the pGEX4T-1 vector as a GST fusion protein, and OsFBK12 wassubcloned into the plasmid pMAL-c2 to allow the expression of OsFBK12 asa fusion with MBP in Escherichia coli. Expression of GST fusion proteins andin vitro binding experiments were performed as described previously (Wanget al., 2009a).
BiFC and Protein Degradation Assays in Tobacco
BiFC assays followed the described protocol (Waadt et al., 2008). For BiFCassays, the ORF of OsFBK12was cloned into the PSPYNE(R)173 vector and theORF of OSK1 was cloned into the PSPYCE(MR) vector. The plasmids wereelectroporated into A. tumefaciens (strain GV3101) and coinfiltrated into to-bacco (Nicotiana benthamiana) leaves (Liu et al., 2010). GFP fluorescence wasvisualized with a confocal scanning microscope after infiltration for 48 to 72 h.
The coinfiltrated tobacco leaves were used in the in vivo assays ofOsSAMS1 protein stability. For protein degradation assays, full-lengthOsSAMS1 (Os05g0135700) fused with GFP was transformed into tobaccoleaves. Three days after infiltration, the OsSAMS1-GFP sample, the wildtype, and the coinfiltrated PSPYNE(R)173-OsFBK12 and SPYCE(MR)-OSK1sample were separately extracted as described in NaCl-free native extractionbuffer (50 mM Tris-MES, pH 8.0, 0.5 M Suc, 1 mM MgCl2, 10 mM EDTA, 5 mM
dithiothreitol, and protease inhibitor cocktail tablets). A final concentrationof 10 mM ATP was added to preserve the function of the 26S proteasome. The
OsSAMS1-GFP extract was divided into three parts: the first part was mixedwith PSPYNE(R)173-OsFBK12 and SPYCE(MR)-OSK1, the second part wasmixed with the wild-type extract, and the third part was mixed with MG132to a final concentration of 50 mM. The mixtures were incubated at 4°C withgentle shaking. Samples were removed at different time points, and quan-tification and normalization were carried out according to the previouslydescribed protocol (Zhang et al., 2011).
ACC and Ethylene Measurements
ACCwas extracted from 1.5 g of leaves in 80% (v/v) ethanol and centrifuged,and the supernatant was evaporated to dryness. The residue was resuspended inwater, and the ACC content was determined following conversion to ethylene asdescribed previously (Comcepcion et al., 1979).
Leaves were cut into 10-cm pieces, and 10 pieces were placed in a 50-mLflask containing distilled water. After imbibition at 28°C for 60 h, a 1-mL gassample was withdrawn by syringe from the head space of each bottle and theethylene concentration was measured by gas chromatography (ShimadzuGC-2014C) on a device equipped with an activated alumina column and flameionization detectors. Separations were carried out at 50°C, using N2 as thecarrier gas, and the ethylene peak was detected with a flame ionization de-tector. The peak area was integrated and compared with an eight-pointstandard ethylene curve. Ethylene standards from 0.01 to 5 mL L21 wereused for the calibration. The quantified data, divided by fresh weight andtime, were converted to specific activities.
Chlorophyll Content
Chlorophyll was extracted from 50-mg leaf samples in 10 mL of 95% (v/v)ethanol for 16 h in the dark and measured spectrophotometrically at 660 nmusing the previously described protocol (Inskeep and Bloom, 1985).
Sequence data from this article can be found in the GenBank/EMBL datalibraries under accession numbers AC144491 (OsFBK12), AP008217 (OSK1),and AP008211 (OsSAM1).
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure S1. Unrooted phylogenetic tree of FBK protein in riceand Arabidopsis.
Supplemental Figure S2. OsFBK12 expression from the eFP browser andGene Investigator.
Supplemental Figure S3. Analysis of the expression pattern of OsFBK12 inrice.
Supplemental Figure S4. Subcellular localization of OsFBK12-GFP andOSK1-GFP fusion proteins in onion epidermal cells.
Supplemental Figure S5. OsSAMS1-GFP and OSK-GFP are co-expressedwith the endoplasmic reticulum marker mCherry-HDEL in riceprotoplasts.
Supplemental Figure S6. Subcellular localization of OsSAMS1 in onionepidermal cells.
Supplemental Figure S7. RT-PCR analysis of the expression of ethylene-induced genes in OsFBK12 and OsSAMS1 transgenic seedlings.
Supplemental Figure S8. RT-PCR analysis of the expression of OsSAMSgenes in OsFBK12 transgenic seedlings.
Supplemental Table S1. Sequences of primers used in this study.
ACKNOWLEDGMENTS
We thank Dr. Jörg Kudla (Universitat Munster) for the kind gifts of plas-mids for the BiFC assay, Dr. Jianru Zuo (Institute of Genetics and Develop-mental Biology, Chinese Academy of Sciences) for his generous gift ofantibodies against GST and GFP, Dr. Rongfeng Huang (Chinese Academy
of Agricultural Sciences) for ethylene measurements, Dr. Sheila McCormick(Plant Gene Expression Center, University of California, Berkeley) and Dr. QiXie (Institute of Genetics and Developmental Biology, Chinese Academy ofSciences) for comments on the manuscript or the project, and Dr. Hui Liu(South China Botanical Garden, Chinese Academy of Sciences) for statisticalanalyses.
Received July 8, 2013; accepted October 16, 2013; published October 21, 2013.
LITERATURE CITED
Agarwal G, Choudhary D, Singh VP, Arora A (2012) Role of ethylenereceptors during senescence and ripening in horticultural crops. PlantSignal Behav 7: 827–846
Barrôco RM, Peres A, Droual AM, De Veylder L, Nguyen SL, De Wolf J,Mironov V, Peerbolte R, Beemster GT, Inzé D, et al (2006) The cyclin-dependent kinase inhibitor Orysa;KRP1 plays an important role in seeddevelopment of rice. Plant Physiol 142: 1053–1064
Bouvier F, Linka N, Isner JC, Mutterer J, Weber AP, Camara B (2006)Arabidopsis SAMT1 defines a plastid transporter regulating plastid bio-genesis and plant development. Plant Cell 18: 3088–3105
Chen N, Xu Y, Wang X, Du C, Du J, Yuan M, Xu Z, Chong K (2011) Os-RAN2, essential for mitosis, enhances cold tolerance in rice by pro-moting export of intranuclear tubulin and maintaining cell divisionunder cold stress. Plant Cell Environ 34: 52–64
Comcepcion M, Lizada C, Yang SF (1979) A simple and sensitive assay for1-aminocyclopropane-1-carboxylic acid. Anal Biochem 100: 140–145
Dill A, Thomas SG, Hu J, Steber CM, Sun TP (2004) The Arabidopsis F-boxprotein SLEEPY1 targets gibberellin signaling repressors for gibberellin-induced degradation. Plant Cell 16: 1392–1405
Frostesjö L, Holm I, Grahn B, Page AW, Bestor TH, Heby O (1997) In-terference with DNA methyltransferase activity and genome methyla-tion during F9 teratocarcinoma stem cell differentiation induced bypolyamine depletion. J Biol Chem 272: 4359–4366
Ge L, Chen H, Jiang JF, Zhao Y, Xu ML, Xu YY, Tan KH, Xu ZH, Chong K(2004) Overexpression of OsRAA1 causes pleiotropic phenotypes intransgenic rice plants, including altered leaf, flower, and root develop-ment and root response to gravity. Plant Physiol 135: 1502–1513
Gray WM, Kepinski S, Rouse D, Leyser O, Estelle M (2001) Auxin reg-ulates SCF(TIR1)-dependent degradation of AUX/IAA proteins. Nature414: 271–276
Guo H, Ecker JR (2003) Plant responses to ethylene gas are mediated bySCF(EBF1/EBF2)-dependent proteolysis of EIN3 transcription factor.Cell 115: 667–677
Han L, Mason M, Risseeuw EP, Crosby WL, Somers DE (2004) Formationof an SCF(ZTL) complex is required for proper regulation of circadiantiming. Plant J 40: 291–301
He J, Deng J, Zheng Y, Gu J (2006) A synergistic effect on the productionof S-adenosyl-L-methionine in Pichia pastoris by knocking in ofS-adenosyl-L-methionine synthase and knocking out of cystathionine-betasynthase. J Biotechnol 126: 519–527
Imaizumi T, Schultz TF, Harmon FG, Ho LA, Kay SA (2005) FKF1 F-boxprotein mediates cyclic degradation of a repressor of CONSTANS inArabidopsis. Science 309: 293–297
Inskeep WP, Bloom PR (1985) Extinction coefficients of chlorophyll a and bin N,N-dimethylformamide and 80% acetone. Plant Physiol 77: 483–485
Jain M, Nijhawan A, Arora R, Agarwal P, Ray S, Sharma P, Kapoor S,Tyagi AK, Khurana JP (2007) F-box proteins in rice: genome-wideanalysis, classification, temporal and spatial gene expression duringpanicle and seed development, and regulation by light and abioticstress. Plant Physiol 143: 1467–1483
Jiang H, Li M, Liang N, Yan H, Wei Y, Xu X, Liu J, Xu Z, Chen F, Wu G(2007) Molecular cloning and function analysis of the stay green gene inrice. Plant J 52: 197–209
Kong Z, Li M, Yang W, Xu W, Xue Y (2006) A novel nuclear-localizedCCCH-type zinc finger protein, OsDOS, is involved in delaying leafsenescence in rice. Plant Physiol 141: 1376–1388
Kusano T, Yamaguchi K, Berberich T, Takahashi Y (2007) Advances inpolyamine research in 2007. J Plant Res 120: 345–350
Li M, Tang D, Wang K, Wu X, Lu L, Yu H, Gu M, Yan C, Cheng Z (2011a)Mutations in the F-box gene LARGER PANICLE improve the paniclearchitecture and enhance the grain yield in rice. Plant Biotechnol J 9:1002–1013
Li W, Han Y, Tao F, Chong K (2011b) Knockdown of SAMS genes encodingS-adenosyl-L-methionine synthetases causes methylation alterations ofDNAs and histones and leads to late flowering in rice. J Plant Physiol168: 1837–1843
Liu L, Zhang Y, Tang S, Zhao Q, Zhang Z, Zhang H, Dong L, Guo H, XieQ (2010) An efficient system to detect protein ubiquitination by agro-infiltration in Nicotiana benthamiana. Plant J 61: 893–903
Piao R, Jiang W, Ham TH, Choi MS, Qiao Y, Chu SH, Park JH, Woo MO,Jin Z, An G, et al (2009) Map-based cloning of the ERECT PANICLE 3gene in rice. Theor Appl Genet 119: 1497–1506
Potuschak T, Lechner E, Parmentier Y, Yanagisawa S, Grava S, Koncz C,Genschik P (2003) EIN3-dependent regulation of plant ethylene hor-mone signaling by two Arabidopsis F box proteins: EBF1 and EBF2. Cell115: 679–689
Prag S, Adams JC (2003) Molecular phylogeny of the Kelch-repeat super-family reveals an expansion of BTB/Kelch proteins in animals. BMCBioinformatics 4: 42
Qiao H, Chang KN, Yazaki J, Ecker JR (2009) Interplay between ethylene,ETP1/ETP2 F-box proteins, and degradation of EIN2 triggers ethyleneresponses in Arabidopsis. Genes Dev 23: 512–521
Rocha PS, Sheikh M, Melchiorre R, Fagard M, Boutet S, Loach R, MoffattB, Wagner C, Vaucheret H, Furner I (2005) The Arabidopsis HOMOLOGY-DEPENDENT GENE SILENCING1 gene codes for an S-adenosyl-L-homocysteine hydrolase required for DNA methylation-dependentgene silencing. Plant Cell 17: 404–417
Schulman BA, Carrano AC, Jeffrey PD, Bowen Z, Kinnucan ER, FinninMS, Elledge SJ, Harper JW, Pagano M, Pavletich NP (2000) Insightsinto SCF ubiquitin ligases from the structure of the Skp1-Skp2 complex.Nature 408: 381–386
Sheard LB, Tan X, Mao H, Withers J, Ben-Nissan G, Hinds TR, KobayashiY, Hsu FF, Sharon M, Browse J, et al (2010) Jasmonate perception byinositol-phosphate-potentiated COI1-JAZ co-receptor. Nature 468: 400–405
Sonnberg S, Fleming SB, Mercer AA (2009) A truncated two-alpha-helixF-box present in poxvirus ankyrin-repeat proteins is sufficient for binding theSCF1 ubiquitin ligase complex. J Gen Virol 90: 1224–1228
Strader LC, Ritchie S, Soule JD, McGinnis KM, Steber CM (2004)Recessive-interfering mutations in the gibberellin signaling gene SLEEPY1 are
rescued by overexpression of its homologue, SNEEZY. Proc Natl Acad Sci USA101: 12771–12776
Sun YJ, Zhou XF, Ma H (2007) Genome-wide analysis of Kelch repeat-containing F-box family. J Integr Plant Biol 49: 940–952
Tomosugi M, Ichihara K, Saito K (2006) Polyamines are essential for thesynthesis of 2-ricinoleoyl phosphatidic acid in developing seeds of cas-tor. Planta 223: 349–358
Waadt R, Schmidt LK, Lohse M, Hashimoto K, Bock R, Kudla J (2008)Multicolor bimolecular fluorescence complementation reveals simulta-neous formation of alternative CBL/CIPK complexes in planta. Plant J56: 505–516
Wang L, Wang Z, Xu Y, Joo SH, Kim SK, Xue Z, Xu Z, Wang Z, Chong K(2009a) OsGSR1 is involved in crosstalk between gibberellins andbrassinosteroids in rice. Plant J 57: 498–510
Wang X, Kong H, Ma H (2009b) F-box proteins regulate ethylene signalingand more. Genes Dev 23: 391–396
Wang Z, Chen C, Xu YY, Jiang RX, Han Y, Xu ZH, Chong K (2004) Apractical vector for efficient knockdown of gene expression in rice (Oryzasativa L.). Plant Mol Biol Rep 22: 409–417
Wuriyanghan H, Zhang B, Cao WH, Ma B, Lei G, Liu YF, Wei W, Wu HJ,Chen LJ, Chen HW, et al (2009) The ethylene receptor ETR2 delaysfloral transition and affects starch accumulation in rice. Plant Cell 21:1473–1494
Xu SB, Yu HT, Yan LF, Wang T (2010) Integrated proteomic and cyto-logical study of rice endosperms at the storage phase. J Proteome Res 9:4906–4918
Yang J, Yunying C, Zhang H, Liu L, Zhang J (2008) Involvement of polyaminesin the post-anthesis development of inferior and superior spikelets in rice.Planta 228: 137–149
Zhang Z, Chen H, Huang X, Xia R, Zhao Q, Lai J, Teng K, Li Y, Liang L,Du Q, et al (2011) BSCTV C2 attenuates the degradation of SAMDC1 tosuppress DNAmethylation-mediated gene silencing in Arabidopsis. Plant Cell23: 273–288
Zhu G, Ye N, Yang J, Peng X, Zhang J (2011) Regulation of expressionof starch synthesis genes by ethylene and ABA in relation to thedevelopment of rice inferior and superior spikelets. J Exp Bot 62:3907–3916
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