1

The F-box protein OsFBK12 targets OsSAMS1 for degradation and

affects pleiotropic phenotypes including leaf senescence in rice

Yuan Chen,a, b

Yunyuan Xu,a Wei Luo,

a Wenxuan Li,

a Na Chen,

a Dajian Zhang,

a, b

Kang Chong a, c, 1

a Key Laboratory of Plant Molecular Physiology, Institute of Botany, Chinese Academy

of Sciences, Beijing 100093, China

b University of the Chinese Academy of Sciences, Beijing 100049, China

c National Plant Gene Research Center, Beijing 100093, China

1 Corresponding author. E-mail: chongk@ibcas.ac.cn; Fax: +86-10-6283-6517

Abstract

Leaf senescence is related to the grain filling rate and grain weight in cereals. Many

components involved in senescence regulation at either the genetic or physiological level

are known. However, less is known about molecular regulation mechanisms. Here we

report that OsFBK12 (F-box protein containing a kelch repeat motif) interacts with

S-adenosyl-l-methionine synthetase1 (SAMS1) to regulate leaf senescence and seed size,

as well as grain number in rice. Yeast two-hybrid, pull-down and bimolecular

fluorescence complementation assays indicate that OsFBK12 interacts with OSK1 (Oryza

sativa SKP1-like protein) and with OsSAMS1. Biochemical and physiological data

showed that OsFBK12 targets OsSAMS1 for degradation. OsFBK12-RNAi (FR) lines

and OsSAMS1 overexpression (SO) lines showed increased ethylene levels, while

OsFBK12-OX (FO) lines and OsSAMS1-RNAi (SR) plants exhibited decreased ethylene.

Phenotypically, overexpression of OsFBK12 led to a delay in leaf senescence and

germination, and increased seed size, whereas knockdown lines of either OsFBK12 or

OsSAMS1 promoted the senescence program. Our results suggest that OsFBK12 is

involved in the 26S proteasome pathway by interacting with OSK1 and that it targets the

substrate OsSAMS1 for degradation, triggering changes in ethylene levels for regulation

of leaf senescence and grain size. These data have potential applications in molecular

breeding of rice.

Plant Physiology Preview. Published on October 21, 2013, as DOI:10.1104/pp.113.224527

Copyright 2013 by the American Society of Plant Biologists

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Introduction

F-box proteins are components of E3 ubiquitin ligase SCF complexes, which mediate

a wide variety of biological processes (Schulman et al., 2000). The N-terminus of F-box

proteins, which interacts with Skp1, is conserved. The C-terminus generally contains one

or several highly variable protein-protein interaction domains, such as Leu-rich repeat

(LRR), kelch repeat, tetratricopeptide repeat (TPR), or WD40 repeat domains (Jain et al.,

2007). Kelch motifs consist of 44–56 amino acid residues, with four highly conserved

residues, two adjacent glycines (G) and a tyrosine (Y) and trytophan (W) pair separated

by about six residues. The presence of kelch repeats is a unique characteristic of a subset

of F-box proteins in plants (Prag and Adams, 2003).

F-box proteins target their substances for their specifical functions. Several key

hormone signaling components, including receptors, have been identified as F-box

proteins. The TIR1 F-box protein acts as an auxin receptor regulating the stability of Aux

⁄ IAA proteins in Arabidopsis thaliana (Gray et al., 2001; Zhang et al., 2011). COI1 is an

F-box protein that is a co-receptor with JAZ1 as a central regulator of jasmonate

signaling (Sheard et al., 2010). SNEEZY (SNE) and SLY1 regulate DELLA through

interaction with the DELLA-GID1 complex in gibberellin signaling (Dill et al., 2004;

Strader et al., 2004). In addition, EIN2 and EIN3 are quickly degraded by the F-box

proteins ETP1/ETP2 and EBF1/EBF2 during ethylene signaling (Guo and Ecker, 2003;

Potuschak et al., 2003; Qiao et al., 2009; Wang et al., 2009). Only a few F-box proteins

containing kelch motifs (FBK), however, have been characterized. The FBK proteins

(ZTL, FKF, LKP2) are involved in light signaling, flowering and circadian control via a

proteasome-dependent pathway in Arabidopsis (Imaizumi et al., 2005). The rice FBK

gene LARGER PANICLE (LP) /ERECT PANICLE 3 (EP3) was reported to regulate

panicle architecture (Piao et al., 2009) and modulate cytokinin levels through OsCKX2

expression (Li et al., 2011). However, a few substances for F-box proteins for the specific

functions are known in plants. On the other words, it is still not clear how F-box proteins

mediate plant developmental processes such as leaf senescence and seed size.

Leaf senescence and the related ethylene regulation impact grain filling, which is an

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important determinant of yield, especially in the last stage of maturation in rice. Delayed

leaf senescence was reported to be mediated by a nuclear-localized zinc finger protein

OsDOS in rice (Kong et al., 2006). The SGR (Stay Green Rice) gene is involved in

regulating pheophorbide a oxygenase that causes alterations in chlorophyll breakdown

during senescence (Jiang et al., 2007). Physiologically, the progression of leaf senescence

is dependent on ethylene levels, which also regulate grain filling (Wuriyanghan et al.,

2009; Agarwal et al., 2012). In plants, it is well established that ethylene is

biosynthesized from S-adenosyl-L-methionine (SAM) via 1-aminocyclopropane

-1-carboxylic acid (ACC). ACC synthase (ACS) catalyzes the first step of the

biosynthesis by converting AdoMet into ACC, and ACC Oxidase (ACO) catalyzes the

second step by metabolizing ACC and dioxygen into ethylene. SAM synthase (SAMS) is

involved in developmental regulation mediated by methylation alterations of DNA and

histones in rice (Li et al., 2011). The physiological function of ethylene in rice is

dependent not only on its biosynthesis but also on signal transduction components such

as the ETR2 receptor (Zhu et al., 2011). However, less is known about the about the

F-box proteins regulation involved in coordination of senescence progression.

Here we show that a rice F-box gene OsFBK12 that contains a kelch repeat domain is

involved in regulation of ethylene-mediated senescence and seed size. Transgenic lines

with reduced or increased expression of OsFBK12 showed phenotypes in germination,

panicle architecture and leaf senescence, as well as in seed size. Our data suggest that

OsFBK12 directly interacts with OsSAMS1 to induce its degradation, which affects

ethylene synthesis and histone methylation, leading to pleiotropic phenotypes.

Results

Overexpression and knockdown of OsFBK12 causes pleiotropic phenotypes

including leaf senescence

To explore the network of F-box proteins, an approach of transgenic rice plants as well

as that of molecular interaction were used. The LOC_Os03g07530 gene, located on

Chromosome 3 (genome sequence 3,832,950–3,835,744) is predicted to encode an F-box

protein, and based on its homologues (http://rice.plantbiology.msu.edu/ca/gene_fams/

1194.shtml), it is termed as OsFBK12. An unrooted phylogenetic tree shows the

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relationship of the FBK (F-box proteins containing kelch motifs) proteins in rice and

Arabidopsis (Supplemental Fig. S1). OsFBK12 is predicted to be a protein of 431

amino acids containing an F-box domain (94th

– 141th

amino acid residues) at the

N-terminus and a kelch repeat motif protein-protein interaction domain (159th

– 365th

) at

the C terminus.

To investigate the biological function of OsFBK12, transgenic lines with reduced

expression or increased OsFBK12 expression were generated. We obtained 12 lines of

transformed RNAi and 15 overexpression transgenic lines. Real-time PCR analysis

confirmed that the transcription of OsFBK12 was reduced in the RNAi (FR) transgenic

lines and increased in the transgenic overexpression (FO) lines (Fig. 1). The FO

transgenic lines showed delayed germination and bottle-green leaves under normal

growth conditions whereas the FR lines developed precociously and had light green

leaves (Fig. 1A). At the tillering stage, overexpression of OsFBK12 caused a decrease in

tiller number and an increase in plant height compared with WT. By contrast, the Ri

transgenic lines displayed increased tiller numbers and decreased plant height (Fig. 1, B,

E and F). At 45 days after heading, leaves of the FR lines became withered and the

chlorophyll a/b content showed a faster reduction than in WT, which is a signature of

earlier senescence. By contrast, the FO lines stayed green with suppressed chlorophyll

reduction and decelerated senescence (Fig. 1, C and G). Overall, alterations in

germination and leaf senescence were the most dramatic developmental phenotypes of

the transgenic plants.

OsFBK12 expression levels impact panicle architecture and grain size

Transcription pattern analysis showed that OsFBK12 was expressed in all organs

and tissues but predominantly in panicle and seed (Supplemental Fig. S2).

Transgenic OsFBK12p::GUS rice plants showed strong GUS staining signals in panicle,

root tip, young leaf and leaf sheath, but little in mature leaf and stem at the heading stage

(Supplemental Fig. S3). Examination of panicle architecture in the FR transgenic lines

revealed a significant increase in branch numbers (both primary and secondary) and in

the total numbers of spikelets per panicle as well as a slight decrease in panicle axis

length (Fig. 2, A, B and E). Conversely, branching and spikelet number per panicle were

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reduced in FO lines compared with those in WT, while the panicle length was increased

(Fig. 2, A, B and E). In addition, the number of grains and the grain filling rate (the ratio

of seed-filled over total florets) were altered in the transgenic lines. (Fig. 2, C and E). The

grains of FR lines were a little thinner and shorter than those of wild type (Fig. 2, C and

D), causing a slight decrease in the 100-grain weight (1.71 g) compared with that of WT

(2.25 g) (Fig. 2E). The grains in FO lines were significantly wider and longer than those

in WT (Fig. 2, C and D), leading the 100-grain weight to increase by 0.7 g (Fig. 2E).

Histological analysis on the spikelet hull showed that the outer parenchyma cell layer

in the FO line was increased compared to WT, while the FR line was reduced (Fig. 3, A,

B and C). Scanning electron microscopy analysis showed that some of the starch granules

packed together and appeared “football-like”, those football-like starch granules in the

middle zone were larger in the FO lines than in WT, whereas the starch granules were

smaller in the FR lines (Fig. 3D). The cell numbers in the lemma were also different

between WT and the transgenic lines (Fig. 3E). The FR-4 line showed more cells than in

WT, whereas the FO-9 line had fewer. There was no difference in cell size in the other

zones of the endosperm. Therefore, OsFBK12 may be involved in regulation of cell

division in the hull.

OsFBK12 interacts with a SKP1-like protein in the nucleus

Yeast two-hybrid assays were performed to screen for proteins that interact with

OsFBK12. The entire coding region of OsFBK12 was inserted into the pGBKT7 vector as

bait. Positive clones were identified based on both survival on restrictive medium

(SD/-His/-Ade/-Trp/-Leu) and on expression levels of the β-galactosidase (lacZ) reporter

gene. There were 216 colonies that survived on restrictive medium of

-His/-Ade/-Trp/-Leu, and 102 of them expressed the β-galactosidase (lacZ) reporter gene.

Among the 96 positive clones that were sequenced, 16 corresponded to OSK1 (Oryza

sativa SKP1-like) (Supplemental Table 2). To confirm the interaction further, a full-length

cDNA of OSK1 was used as prey. Fragments of OsFBK12 encoding the F-box domain

(OsFBK12Δkelch), the kelch repeat domain (OsFBK12ΔF-box) and the full-length cDNA

were used as baits. Colonies expressing lacZ were obtained with the F-box domain and

full-length OsFBK12, but not with the kelch repeat domain did not (Fig. 4A). These

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results suggest that OsFBK12 interacts with OSK1 through the F-box domain in yeast

cells.

In bimolecular fluorescence complementation (BiFC) assays, strong GFP

fluorescence was observed when PSPYNE(R)173-OsFBK12 and SPYCE(MR)-OSK1 were

co-expressed in the nuclei of tobacco leaf epidermal cells (Fig. 4B). Cells transformed

with single constructs alone did not show any fluorescence. Protein localization assays

showed that the signal from an OsFBK12-GFP fusion protein overlapped with nuclear

H33342 staining in protoplasts (Fig. 4C; Supplemental Fig. S4A), but not co-expressed

with ER marker mCherry-HDEL in rice protoplasts (Supplemental Fig. S5). By contrast,

the OSK1-GFP fusion protein was localized in both the nucleus and the cytoplasm

(Supplemental Fig. S4B).

OsFBK12 interacts with OsSAMS1 for degradation in plant cells

Kelch repeat domains in FBKs function in protein-protein interactions, which specify

the protein substrates for degradation via the ubiquitin pathway (Sun et al., 2007). We

used the kelch repeat domain in the pGBKT7 vector as bait to screen a rice cDNA library

in yeast. Of the 167 positive colonies that survived on restrictivve medium of

-His/-Ade/-Trp/-Leu, 90 expressed β-galactosidase (lacZ) reporter gene. Among the 78

positive clones that were sequenced, one encoded S-adenosyl-l-methionine synthetase1

(SAMS1). Assays in yeast cells using truncation constructs showed that OsSAMS1222-316

(OsSAMS1 amino acids 222–316) interacted with both full-length OsFBK12 and the

kelch repeat domain truncation (Fig. 5A). In a pull-down assay, purified GST-OsSAMS1

was immobilized to glutathione-sepharose beads, and OsFBK12 tagged with

maltose-binding protein (MBP-OsFBK12) was incubated with the beads. GST and MBP

alone were used as negative controls. A band at 92 kDa was recognized by an antibody

against MBP in immunoblot assays. By contrast, the negative controls did not show the

corresponding signal. This suggests that OsFBK12 can interact with OsSAMS1 in vitro

(Fig. 5B). To explore the possibility that OsSAMS1 is the substrate of OsFBK12,

OsSAMS1 levels were monitored in the transgenic lines. Immunoblot assays showed that

OsSAMS1 was increased in the knockdown transgenic lines of OsFBK12, FR-4, FR-10

and FR-12. By contrast, OsSAMS1 levels were decreased in the OsFBK12

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overexpression lines FO-5 and FO-9 (Fig. 5C). Tobacco leaves expressing

OsSAMS1-GFP were then treated with the 26S proteasome inhibitor MG132, showing

that OsSAMS1-GFP was stable in the presence of MG132 for up to 8 h (Fig. 5D),

whereas in the control (without treatment of MG132), OsSAMS1 showed a gradual

decrease. When purified OsFBK12-myc was added into the extracts, OsSAMS1-GFP

degradation was accelerated. Furthermore, we tested the poly-ubiquitination in the

transgenic leaves of OsSAMS1-GFP. The Western blot assay indicated that a series of

bands with higher molecular weights were recognized by the ubiquitin antibody in the

transgenic leaves of OsSAMS1-GFP (Fig. 5F). Together, these data suggest that

OsFBK12 targeted OsSAMS1 for ubiquitination and subsequent degradation by the 26S

proteasome.

Similarities between OsFBK12 and OsSAMS1 transgenic plants in germination and

senescence

Germination is suppressed in knockdown transgenic OsSAMS1 rice plants (Li et al.,

2011). This suppression of germination was rescued by supplementation with SAM (1

mM) (Fig. 6A). Similarly, in OsFBK12-OX (FO) transgenic plants, there was a block of

germination, although the germination patterns were not different between

OsFBK12-RNAi (FR) lines and WT. The germination suppression in OsFBK12-OX (FO)

was also alleviated with treatment of SAM (Fig. 6, A and B).The germination suppression

of OsFBK12-OX (FO) lines and OsFBK12-RNAi (FR) lines can also alleviated with 50

ppm ethephon (2- chloroethylsulfonic acid) (Fig. 6, C and D).These data are consistent

with the idea that altered expression of SAMS caused a corresponding change in the

production of SAM and ethylene, as 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 were affected by changes in SAM. We found that the ACC and ethylene

content were increased in OsFBK12-RNAi (FR) lines and in the OsSAMS1

overexpression (SO) lines, relative to WT. Correspondingly, decreased ACC and

ethylene levels appeared in the OsFBK12-OX (FO) plants and OsSAMS1-RNAi (SR)

plants (Fig. 6, E and F).

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We further monitored the phenotypes of the OsSAMS1 transgenic plants. In the

OsSAMS1-OX (SO) transgenic plants, most leaves turned yellow and grain filling was

completed by 100 days after germination, when only a few leaves of WT plants were

yellow and WT plants remained in the grain-filling stage. Under the same conditions, the

OsSAMS1-RNAi (SR) transgenic plants were still green and not heading yet (Fig. 6E).

A leaf senescence assay showed that the OsFBK12-RNAi (FR) and OsSAMS1-OX (SO)

plants displayed faster senescence than wild type, whereas OsFBK12-OX (FO) plants and

OsSAMS1-RNAi (SR) maintained green leaves much longer than wild type (Fig. 6, F

and G). To determine whether this phenotype is caused by ethylene content in the

transgenic plants, we treated the wild-type and transgenic plants in with 50 ppm

ethylephon and 200mM Aminoethoxyvinylglycine (AVG) (an ethylene biosynthesis

inhibitor). Leaf senescence in FR and SO lines was prevented by the biosynthesis blocker,

and ethylene treatment promotes the leaf senescence in the FO and SR lines. (Fig. 6, H

and I).These results suggest that OsSAMS1 promotes plant developmental processes and

leaf senescence and that OsFBK12 suppresses them.

Discussion

OsFBK12 targets OsSAMS1 for 26S proteasome-mediated degradation

F-box proteins are subunits of E3 ubiquitin ligase complexes called SCFs (containing

SKP1, Cullin1, an F-box protein, and Rbx1). The F-box motif can bind to SKP1 via the

N-terminus to form a complex and recognize the target proteins via a protein-protein

interaction domain at the C-terminus (Sonnberg et al., 2009). Although F-box proteins

have been identified to mediate multiple biological processes, less is known about their

specific degraded substrates, especially for F-box proteins containing kelch motifs (Han

et al., 2004; Imaizumi et al., 2005; Piao et al., 2009). Our data suggest that OsFKB12, as

a kelch-type F-box protein, might form a SCF complex with SKP1 and bind to

OsSAMS1, targeting it for 26S proteasome-mediated degradation.

The well-conserved N-terminal domain of OsFBK12 interacted with OSK1, and its

C-terminal kelch repeat domain interacted OsSAMS1 (Fig. 4 and Fig. 5). The interaction

of the full-length proteins (Fig. 5B), along with three lines of biochemical and

physiological evidence, support that OsSAMS1 is targeted by OsFBK12 for degradation

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in the 26S proteasome. First, the degradation of OsSAMS1 was inhibited by treatment

with MG132 and promoted by treatment with purified OsFBK12 (Fig. 5, D and E).

Second, OsSAMS1 was increased in the OsFBK12 knockdown lines. Conversely, a

decrease of OsSAMS1 appeared in the OsFBK12 overexpression transgenic line (Fig.

5C). Third, the germination block in the OsSAMS1-RNAi (SR) transgenic could be

rescued by supplementation with SAM, indicating that SAM production was likely

reduced in the OsSAMS1 knockdown lines (Li et al., 2011). The OsFBK12-OX (FO)

transgenic plants showed phenotypes identical to those of OsSAMS1 knockdown lines,

supporting that the OsFBK12 promotes degradation of OsSAMS1 and causes the

reduction in SAM (Fig. 6A). Moreover OsSAMS1-GFP was detected in the nucleus and

the cytoplasm (Supplemental Fig.S5 and Fig.S6), while OsFBK12 was localized in the

nucleus; this result hinted that OsSAMS1 may be degraded in the nucleus.

OsFBK12 is involved in ethylene-mediated leaf senescence

Ethylene is synthesized from methionine via the intermediates SAM and ACC (Bouvier

et al., 2006). The conversion of methionine to AdoMet is catalysed by SAMS and the

conversion from SAM to ACC is catalyzed by ACS, which are the rate-limiting enzymes

in ethylene biosynthesis. Regulation of ethylene biosynthesis occurs at both the gene

expression level and the protein activity level (Bouvier et al., 2006). Thus, OsFBK12 can

be considered a negative regulator of ethylene biosynthesis by virtue of its function in

promoting degradation of OsSAMS1. Overexpression of OsFBK12 and knockdown of

OsSAMS1 caused a decrease in ethylene responsive genes OsEATB and OsCTR1 and

ethylene production, delaying leaf senescence, whereas overexpression of OsSAMS1 and

knockdown of OsFBK12 resulted in increased ethylene levels, promoting early leaf

senescence (Fig. 6, Supplemental Fig. S7). Therefore, OsFBK12 is a negative regulator

of the ethylene-mediated senescence through degradation of OsSAMS1.

OsFBK12 might regulate seed size

Altered OsFBK12 gene expression led to changes in seed size resulting from changes

in the cell number and the size of the “football-like” granules. “football-like” granules is

an important part of completion of starch granule packaging, which is similar with the

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one types of granules which associate with the grain size in wheat endosperm (Xu et

al.,2010).

The cell numbers in the lemma of the OsFBK12 overexpression lines were reduced

while the cell length and the “football-like” granules size were increased (Fig. 3E). This

can be explained by the compensatory mechanisms in monocot species (Barroco et al.,

2006). That is the reduced cell production can be partly compensated by an increased cell

size. The cell size in the OsFBK12-OX (FO) lines was compensated through cell

expansion. The knockdown of OsFBK12 reduced the cell size during spikelet hull

development. The cell production in the OsFBK12-RNAi (FR) lines increased while the

spikelet hull is smaller, the cells size in spikelet hull and the football-like starch granules

in the endosperm are restricted and partly compensated by decreased cell size.

OsFBK12 interacts with SAMS1 to regulate leaf senescence. The mRNA levels of

OsSAM1 as well as other OsSAMs in the OsSAMS1-RNAi (SR) had been reported (Li et

al.,2011). The mRNA levels of either OsSAMS1 or OsSAMS2 were decreased in the

OsFBK12-OX (FO) transgenic plants, whereas the mRNA level of OsSAMS3 was

increased in OsFBK12 Ri (FR) transgenic plants (Supplemental Fig. S8). This indicated

that the transgene of OsFBK12 impacts OsSAMS1 on both the protein interaction and

RNA transcription levels, which are both upstream of ethylene biosynthesis. It is notable

that SAM is not only a precursor for ethylene biosynthesis, but also is a universal methyl

group donor and involved in numerous transmethylation reactions (Frostesjo et al., 1997;

Rocha et al., 2005; Zhang et al., 2011). That SAM also functions as a precursor of

polyamines, might explain the diverse phenotypes between the lines of OsFBK12-OX

(FO) and OsSAMS1-RNAi (SR) except for the consensus ones (Tomosugi et 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 interacts with OSK1 to form a SCF-complex and degrade its

substrate, such as OsSAMS1. The degradation of OsSAMS1 results in a decrease in SAM

content, as well as in ethylene levels, which affects germination and leaf senescence.

Additionally, it is possible that OsFBK12 might affect cell division, resulting in changes

in cell numbers in the spikelet hull. The findings may help a better understanding of the

senescence control in rice.

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Materials and Methods

Plant germination and transformation

For seed germination, dehulled rice seeds (Oryza sativa L) of different transgenic

lines and wild type (WT) were surface-sterilized and immersed in water. Seeds were

placed in a growth chamber under dark conditions for the first 72 h, and then with 12/12

light/dark cycle at 25°C. Germination is defined as when the coleoptile is 5mm long.

Every experiment was repeated three times, 30 seeds per sample.

Oryza sativa L. ssp. japonica cv Zhonghua 10 (ZH10) was used for transformation.

Transgenic plants derived from callus were defined as the T0 generation. Transgenic

plants of the T1 and T2 generations were used for phenotypic analyses. The SAM

treatment experiment was performed according to (Li et al., 2011).

Total RNA extraction and real-time PCR

Total RNA was extracted by use of the TRIzol RNA extraction kit (Invitrogen) and

treated with RNase-free DNase I (MBI Fermentas, Lithuania). Total RNA (2 μg) was

reverse transcribed into cDNA by AMV Reverse Transcriptase (Promega). Real-time

PCR amplification was performed in 20 μL reactions containing 5 μL 50-fold diluted

cDNA, 0.2 μM of each primer, and 10 μL SYBR Green PCR Master Mix (TaYOBO).

Quantitative real-time PCR was performed on Mx3000p (STRATAGENE) using SYBR

Green reagent (Toyobo). Expression was normalized to that of ACTIN. Primer sequences

used for amplification are listed in Supplemental Table 1.

Vector construction and rice transformation

The entire open reading frame (ORF) of OsFBK12 was amplified by RT-PCR, and

then inserted upstream of GUS in the binary plasmid pUN1301 (Chen et al., 2011). The

pTCK303-OsFBK12 construct was used for creating an RNAi knockdown transgenic line.

The detailed protocols for construct generation were previously described (Wang et al.,

2004). The resulting constructs were used for transformation via Agrobacterium

tumefaciens strain EHA105 as described previously (Ge et al., 2004). All primers used in

this study are listed in Supplemental Table 1.

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Scanning electron microscopy

Rice seeds were fixed in FAA (50% ethanol, 5% acetic acid and 37% formaldehyde)

for more than 24 h and then dehydrated through a graded series of alcohol–isoamyl

acetate. After being critical-point dried in carbon dioxide for 1 hr with a Hitachi HCP-2,

the plant material was coated with gold and observed with a Hitachi S-4800 SEM at 10.0

kV.

Histological analysis

Rice spikelets were fixed in FAA (50% ethanol, 5% acetic acid and 37%

formaldehyde) at room temperature overnight and then dehydrated in an ethanol series,

cleared with xylene and embedded in Paraplast (Sigma). Tissue sections (10 m thick)

were cut and stained with 0.02% toluidine blue for 5 min at room temperature after

dewaxing. Photographs were taken on an Olympus VANOX microscope.

Yeast two-hybrid assay

The BD Matchmaker library construction and screening kit (Clontech Laboratories,

Inc.) was used for yeast two-hybrid assays. All protocols were carried out according to

the manufacturer’s user manual. The cDNA encoding the full-length OsFBK12 protein

was inserted into the GAL4 DNA binding-domain vector pGBKT7. The pGADT7 clones

were selected on SD/-Leu/-Trp/-His/-Ade/X-gal plates and sequenced at the Invitrogen

Sequencing Facility to ensure that the prey proteins were in-frame fusions with the GAL4

AD domain using the system described previously (Wang et al., 2009).

Pull-down assay

To test the interaction between OsFBK12 and OsSAMS1, the ORF of OsSAMS1 was

cloned into the pGEX4T-1 vector as a glutathione S-transferase (GST) fusion protein, and

OsFBK12 was subcloned into the plasmid pMAL-c2 to allow expression of OsFBK12 as

a fusion with maltose-binding protein (MBP) in Escherichia coli. Expression of GST

fusion proteins and in vitro binding experiments were performed as described previously

(Wang et al., 2009).

Bimolecular fluorescence complementation (BiFC) and protein degradation assays

in tobacco

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BiFC assays followed the described protocol (Waadt et al., 2008). For BiFC assays,

the ORF of OsFBK12 was cloned into the PSPYNE(R)173 vector, and the ORF of Oryza

sativa SKP1-like (OSK1) was cloned into the PSPYCE(MR) vector. The plasmids were

electroporated into Agrobacterium (strain GV3101) and co-infiltrated into tobacco leaves

(Liu et al., 2010). GFP fluorescence was visualized with a confocal scanning microscope

after infiltration for 48–72 h.

The co-infiltrated Nicotiana benthamiana leaves were used in the in vivo assays of

OsSAMS1 protein stability. For protein degradation assays, full-length OsSAMS1

(Os05g0135700) fused with GFP was transformed into tobacco leaves. Three days after

infiltration, the OsSAMS1-GFP sample, WT and the co-infiltrated

PSPYNE(R)173-OsFBK12 and SPYCE(MR)-OSK1 sample were separately extracted as

described in NaCl-free native extraction buffer (NB1, 50 mM Tris-MES pH 8.0, 0.5 M

sucrose, 1 mM MgCl2, 10 mM EDTA, 5 mM DTT, protease inhibitor cocktail tablets). A

final concentration of 10 μM ATP was added to preserve the function of the 26S

proteasome. The OsSAMS1-GFP extract was divided into three parts: the first part was

mixed with PSPYNE(R)173-OsFBK12 and SPYCE(MR)-OSK1, the second part was

mixed with WT extract, and the third part was mixed with MG132 to a final

concentration of 50 μM. The mixtures were incubated at 4°C with gentle shaking.

Samples were removed at different time points, and quantification and normalization was

carried out according to the previously described protocol (Zhang et al., 2011).

ACC (1-aminocyclopropane-1-carboxylic acid) and Ethylene measurements

ACC was extracted from 1.5g leaves in 80% ethanol, centrifuged and the supernatant

was evaporated to dryness. The residue was resuspended in water and the ACC content

was determined following conversion to ethylene as previously described (Lizada and

Yang 1979).

Leaves were cut into 10 cm pieces, and ten pieces were placed in a 50 mL flask

containing distilled water. After imbibition at 28°C for 60 h, a 1 mL gas sample was

withdrawn by syringe from the headspace of each bottle and the ethylene concentration

was measured by gas chromatography (SHIMADZU GC-2014C, Japan) equipped with

an activated alumina column and flame ionization detectors. Separations were carried out

at 50°C, using N2 as the carrier gas, and the ethylene peak was detected with a flame

14

ionization detector. The peak area was integrated and compared to an 8-point standard

ethylene curve. Ethylene standards from 0.01–5 ppm were used for the calibration. The

quantified data, divided by fresh weight and time, were converted to specific activities.

Chlorophyll Content

Chlorophyll was extracted from 50 mg leaf samples in 10 mL 95% ethanol for 16 h in

the dark and measured spectrophotometrically at 660 nm using the previously described

protocol (Inskeep and Bloom, 1985).

Acknowledgment

We thank Dr. Weihua Wu (China Agriculture University) and Dr. J. Kudla (Universitat

Munster) for the kind gifts of plasmids for BiFC assay. We thank Dr. Jianru Zuo

(Institute of Genetics and Developmental Biology, Chinese Academy of Science) for his

generous gift of antibodies against GST and GFP. We thank Dr. Rongfeng Huang

(Chinese Academy of Agricultural Sciences) for Ethylene measurements. We are grateful

Dr. J. Kudla (Universitat Munster) for the kind gifts of plasmids for BiFC assay. We

thank Dr. Sheila McCormick (Plant Gene Expression Center, UC-Berkeley) for

comments on the manuscript. We thank Dr. Hui Liu (South China Botanical Garden,

Chinese Academy of Sciences) for statistical analyses. This work was supported by the

Major state basic research program (973) (2011CB915400) and the National Natural

Sciences Foundation (31070695) and NSFC for the innovation team (31121065).

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Legends

Figure 1. Phenotypes of OsFBK12 transgenic lines at different developmental stages. A，

Germination. WT, 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 = 20cm. D,

Relative expression level of OsFBK12 in RNAi lines and overexpression lines. ACTIN

was used as a control. Quantitative RT-PCR data represent mean SD of 3 biological

replicates. E, Plant height of OsFBK12 transgenic plants at the heading stage. Data are

mean SD of triplicate experiments. F, Tiller number of OsFBK12 transgenic plants at

the heading stage. Data are mean SD of triplicate experiments. G, The chlorophyll

content of the upper three leaves from the main culm. Data are mean SD of triplicate

experiments. FW, fresh weight. WAH, week after heading.

Figure 2. Panicle morphology and seed size phenotypes of the OsFBK12 transgenic lines.

A, Panicles. OsFBK12 RNAi and overexpression line at maturity. WT, wild type; FR-4,

FR-10，FR-12， OsFBK12 RNAi line 4, line 10, line 12; FO-5, FO-9，OsFBK12

overexpression line 5, line 9. Bar = 3 cm. B, Panicle branching. Bar = 3 cm. C, Seed

width and seed length. Bar =1 cm. Vector: empty vector UN1301 transformed rice as

control. D, Tubes containing 43 seeds from the indicated lines. Bar = 1 cm. E, Statistical

analysis of seed size and panicle types. The data of FR and FO indicate the average of

three individual FR lines and two FO lines. The data represent mean ± SE. Multiple

comparisons were performed by Tukey HSD post-hoc tests. Letters indicated

significantly different results. Tests were carried out by R library multcomp.

Figure 3. Cell number and size of hulls and endosperm in the transgenic lines. A, Grain

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shape. WT, wild type; FR-4, OsFBK12 RNAi line 4; FO-9, OsFBK12 overexpression line

9. White lines indicate the position 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 m.

C, Magnified view of the boxes in B. Black arrow shows the outer parenchymal cell layer.

Bars = 50 m. D, SEM images of transections of endosperm. I, II and III represent the

outer, middle, and inner parts of endosperm, respectively. Enlarged regions

corresponding to I, II and III are shown below. WT is shown in a, d, g and j. FR-4 is in b,

e, h and k. FO-9 is in c, f, i and l. White arrows denote “football-like” starch granules.

Bars = 1 mm (a-c), 20 m (d-l). E, Statistical analysis of the total length, cell number and

cell length in the outer parenchymal cell layers of the hulls. The data of FR and FO

indicate the average of three individual FR lines and two FO lines. The data represent

mean ± SE. Student’s t-test was performed. Single asterisks represent P < 0.05, compared

to wild type values.

Figure 4. Interaction of OsFBK12 with OSK1 in vivo and subcellular localization. A,

Yeast two-hybrid assay for interaction of OsFBK12 with OSK1. A schematic diagram of

OsFBK12 and the truncations used is shown. The bait (BD) vector contained full length

OsFBK12, OsFBK12△kelch or OsFBK12 △F-box; the prey (AD) vector contained OSK1.

Yeast strains were cultured on the -Trp-Leu-His-Ade selection medium. β-Galactosidase

activity of positive clones was analyzed using X-Gal. The proved interaction between

OsGSR1 and DWF1 was used as a positive control. B, BiFC assay for interaction

between the OsFBK12 and OSK1 in Nicotiana benthamiana. Shown is co-expression of

YN-OsFBK12 and YC-OSK1 (top row), YN- OsFBK12 and YC vector (middle), and

YC-OSK and YN vector as a control (bottom). Bars = 100 µm. C, Subcellular

localization of OsFBK12-GFP and OSK1-GFP fusion proteins in rice protoplasts. GFP

protein alone shows fluorescent signals in nucleus, membrane and cytoplasm. H33342 is

a staining dye for the nucleus. Bar = 10 µm.

Figure 5. Interaction of OsFBK12 with OsSAMS1 for degradation in plant. A, Yeast

two-hybrid assay for interaction of OsFBK12 with OsSAMS1222-316. The reported inter

19

-acted proteins OsGSR1 and DWF1 were used as positive control. B, Pull-down assay for

interaction of OsFBK12 with OsSAMS1222-316. Input, crude MBP-OsFBK12 and

GST-OsSAMS1 protein. Lane1-3: Input sample of GST, MBP, MBP-OsFBK12 and

GST-OsSAMS1; Lane4-6: Elution from GST/MBP-OsFBK12, GST-OsSAMS1/MBP and

MBP-OsFBK12/GST-OsSAMS1 after pull down. Black arrows show the band pulled

down and immunobloting by the anti-MBP monoclonal antibody. IB: immunobloting. C,

Immunoblot analysis of the expression of OsSAMS1 protein in OsFBK12 transgenic

seedlings. CBB staining as loading for total protein. D, OsSAMS1-GFP degradation

assays performed in Nicotiana benthamiana with or without MG132 treatment.

Normalized plots of the degradation of OsSAMS1-GFP are shown below Data are mean

SD of triplicate experiments. E, OsSAMS1-GFP degradation assays performed in N.

benthamiana with or without OsFBK12 protein. Normalized plots of the degradation of

OsSAMS1-GFP are shown below. Data are mean SD of triplicate experiments. F,

Pull-down assay using anti-ubiquitin indicated that the bands of higher molecular

weights(72 kDa-170 kDa) were poly-ubiquitinated of OsSAMS1 in N. benthamiana. The

molecular weights are shown in kDa，The molecular weights for GFP is 26kDa and

OsSAM1-GFP is 70kDa. Anti-Ub stands for anti-ubiquitin monoclonal antibody.

Figure 6. Germination and senescence phenotypes of the OsFBK12 and OsSAMS1

transgenic lines. A, Effect of SAM on seed germination in the OsFBK12 transgenic

plants. Morphology of seedlings was observed after 40 hrs for germination. SAM

concentration was 1 mM. Bar = 1 cm. Data are mean SD of triplicate experiments and

30 seeds per sample. B, The time course of germination rate and effect of SAM (1 mM).

Data are mean SD of triplicate experiments and 30 seeds per sample. C Effect of

Ethylephon on seed germination in the OsFBK12 and SAMS1 transgenic plants.

Morphology of seedlings was observed after 40 hrs for germination. Ethylephon

concentration was 50 ppm. Bar = 1 cm. Data are mean SD of triplicate experiments and

30 seeds per sample. D, The time course of germination rate and effect of Ethylephon (50

ppm). Data are mean SD of triplicate experiments and 30 seeds per sample. E, ACC

content in different transgenic plants (using 30-day-old seedlings). Multiple comparisons

were performed by Tukey HSD post-hoc tests. Letters indicated significantly different

20

results. Tests were carried out by R library multcomp. F, Ethylene content in different

transgenic plants (using 30-day-old leaves seedlings). G, Phenotypic comparison of WT

and the OsSAMS1-RNAi (SR) line and OsSAMS1 overexpression (SO) line at 120 days

after germination. Bars = 20 cm. H, Detached leaf senescence for 3 days in the darkness

(using 30-day-old leaves seedlings) with and without treatment of AVG and Ethylephon.

Bar = 3 cm I, The chlorophyll content of detached leaf in H. Data are mean SD of

triplicate experiments. FW, fresh weight. The data of FR and FO indicate the average of

three individual FR lines and two FO lines. The data of SR and SO indicate the average

of two individual SR lines and two SO lines.

Figure 7. Proposed working model for OsFBK12 regulation of leaf senescence and seed

size in rice. This model proposes that OsFBK12 was involved in 26S proteasome

mediated degradation by interacting with OSK and targeted the substrate OsSAMS1.

When the OsSAMS1 degraded, it caused a corresponding change on ethylene level and

regulated the leaf senescence. Meanwhile, OsFBK12 might target other substrate or

might regulate the transcription of some genes to affect seed size.

21

Figures

Figure 1

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Figure 2

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Figure 3

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Figure 4

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Figure5

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Figure 6

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Figure 7