Overexpression of OsSPL9 enhances accumulation of Cu in rice grain and improves its digestibility and metabolism

Accepted Manuscript Overexpression of OsSPL9 enhances accumulation of Cu in rice grain and improves itsdigestibility and metabolism Mingfeng Tang, Chuanshe Zhou, Lu Meng, Donghai Mao, Chengbing Liu, Can Peng, Yuxing Zhu, Dechun Zhang, Daoyou Huang, Zhiliang Tan, Caiyan Chen PII:

S1673-8527(16)30140-0

DOI:

10.1016/j.jgg.2016.09.004

Reference:

JGG 483

To appear in:

Journal of Genetics and Genomics

Received Date: 2 June 2016 Revised Date:

19 September 2016

Accepted Date: 23 September 2016

Please cite this article as: Tang, M., Zhou, C., Meng, L., Mao, D., Liu, C., Peng, C., Zhu, Y., Zhang, D., Huang, D., Tan, Z., Chen, C., Overexpression of OsSPL9 enhances accumulation of Cu in rice grain and improves itsdigestibility and metabolism, Journal of Genetics and Genomics (2016), doi: 10.1016/ j.jgg.2016.09.004. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Overexpression of OsSPL9 enhances accumulation of Cu in rice grain and

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improves itsdigestibility and metabolism

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Copper (Cu) is an essential trace mineral element for all forms of life, and is an

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important structural component and co-factor for a variety of metalloenzymes (Peña

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et al., 1999; Bertinato and L’Abbé,2004). In humans, Cu deficiency is not common

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because of the ubiquitous occurrence of Cu and ease of gastrointestinal absorption

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(Zidar et al., 1977; Uauy et al., 1998). However, because of the low Cu content in

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most sources of feed, the use of Cu as a growth promoter to maximize animal

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production has been well documented in animal feeding (Braude, 1945; Lu et al.,

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2010). Previous studies have shown that dietary supplementation of Cu can improve

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reproductive performance in beef cattle and affect steer lipid metabolism including

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decreasing the backfat thickness while increasing the unsaturated fatty acid

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composition of the longissimus muscle (Engle and Spears, 2000). Generally, up to

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250 mg/kg actual inorganic Cu is added to the diet of starter pigs to improve growth

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and feed efficiency (Dove, 1995). In addition, Cu supplementation improves the

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piglets’ immunity and reduces mortality (Højberg et al., 2005). However, high levels

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of inorganic Cu usually lead to Cu poisoning in animals, and can be detrimental to the

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environment (Armstrong et al., 2004; Johnston et al., 2014).CuSO4 has been shown to

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decrease the bacterial degradation of manure in lagoons(Hatfield et al., 1998).

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Different sources or forms of Cu have different levels of bioavailability and therefore,

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different effects on animal production. Previous studies have shown that compared

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with inorganic Cu, Cu in the form of chelates, complexes, or proteinates can reduce

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the Cu dose in supplements and decrease excretion to the environment due to

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improved bioavailability and digestibility (Creech et al., 2004; Gonzales-Eguia et al.,

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2009). More than 98% of the Cu in plants is present in complexed forms bound to

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cysteine-rich proteins and carboxylic and phenolic groups (Broadley et al., 2012). As

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a result, decreasing the inorganic Cu dose and enhancing the amount of organic Cu

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derived from crops are highly desirable. Therefore, investigating Cu accumulation in

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crops and biofortification of feed with Cu is of great interest to enhance feed

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ACCEPTED MANUSCRIPT efficiency and for environmental sustainability.In the present study, we used rice as a

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model system to evaluate the potential to increase organic Cu levels in the edible parts

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of the plant to enhance feed efficiency without destroying Cu homeostasis and

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impairing plant growth.

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Using a comparative genetics approach, we searched for the rice ortholog of

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SQUAMOSA PROMOTER BINDING PROTEIN-LIKE7 (AtSPL7), which precisely

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controls Cu uptake and relocation in Arabidopsis. Through BLASTp searches using

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the conserved domain sequence of AtSPL7, we identified 16 SPL homologs in the

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Arabidopsis genome and seven homologous genes in the rice genome (Table S1).

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Using the protein sequences of these 23 genes, we constructed a phylogenetic tree of

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the rice and Arabidopsis proteins using the neighbor-joining method (Fig. S1A).

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Based on this phylogenetic tree, we identified a total of six SPL subgroup clusters;

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OsSPL9 (Os05g33810) and AtSPL7 (At5g18830) were closest to each other in one

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cluster compared to other rice or Arabidopsis homologs. Also, the OsSPL9 genomic

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region exhibits microsynteny with the regions surrounding AtSPL7 in Arabidopsis

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(Fig. S1B). Taken together, this is evidence thatOsSPL9 is the rice ortholog of AtSPL7

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in Arabidposis.

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Aligning the protein sequences of OsSPL9 and AtSPL7 showed that they share only

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37.17% identity. Based on a previous prediction (Xie et al., 2006; Yang et al., 2008),

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six protein motifs are present in OsSPL9：AHA, Zn1, JP, ZN2-NLS, and two

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unknown motifs (Fig. S2). To confirm the transcriptional activity of OsSPL9 and to

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identify which motifs are responsible for this activity, the full length coding region

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and a series of truncated fragments of OsSPL9 were inserted into the pGBKT7 vector

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and transformed into yeast strain Y2HGold. As shown in Figure 1A, all transformants

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grew well on SD/Trp- medium. However, only transformants containing pGBKT7-a,

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pGBKT7-b, pGBKT7-c, pGBKT7-d, and pGBKT7-e were able to grow on

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SD/Trp-/His-/Ade- medium and showed β-galactosidase activity, while those

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containing pGBKT7, pGBKT7-f, and pGBKT7-g could not. These results provided

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evidence that OsSPL9 functions as a transcriptional activator, and the N-terminal 200

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ACCEPTED MANUSCRIPT amino acid resides containing AHA motif, which has a high density of aromatic (A),

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bulky hydrophobic (H) as well as acidic (A) amino acid residues(Yang et al., 2008),

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and the first zinc finger 1 (Zn1) domain accounted for this transactivation activity.

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Subcellular localization analysis was carried out by constructing a fusion of OsSPL9

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and GFP, driven by the 35S promotor (35S:OsSPL9-GFP), which was then

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co-agroinfiltrated into N. benthamianaleaves with a nuclear marker HY5-DsRed

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(35S:HY5-DsRed). The GFP signal was co-localized in the nucleus with DsRed,

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indicating that OsSPL9 is a nuclear protein (Fig. 1B).The tissue- and stage-specific

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expression profile of OsSPL9 was investigated using qRT-PCR in different tissues at

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the tilling stage, booting stage, flowering stage, and grain filling stage. We can detect

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the transcription of OsSPL9 in all assayed tissues; its expression was relatively high in

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the leaf blade, and the highest level of expression occurred at flowering stage (Fig.

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S3). We found that the expression level was not affected by the availability of Cu or

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other heavy metals from the culture solution (Fig. S4), which is consistent with

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previous studies showing that AtSPL7 transcript levels remain constant regardless of

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Cu availability and maybe regulated post-transcriptionally (Yamasaki et al., 2009).

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We generated transgenic rice plants overexpressingOsSPL9 (Fig. S5). A total of 32

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independent lines were generated, and five `lines with increased levels of

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OsSPL9-specificmRNAwere chosen for further analysis except for hydrophilic culture,

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in which only three independent lines were tested (Fig. S6).To investigate the

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differences in Cu accumulation in the roots and shoots at the vegetative stage, we

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determined the mineral contents of 4-week-old seedlings that were grown in 0.5X MS

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liquid medium containing 50 nmol/L Cu2+. The results showed that there was no

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difference in the concentration of Fe, Mn, and Zn in both the roots and shoots

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between the overexpression lines and the wild type (WT) seedlings (Fig. S6); however,

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the Cu concentration in the shoots was higher in the overexpression lines than in the

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WT except OE2 (P=0.073), while it was comparable in the roots (Fig. S7A and B). In

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the shoots, the transgenic lines accumulated13.14-16.97 mg/kg Cu, an increase of up

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to 32% compared to the WT at 12.86 mg/kg Cu. These results indicate that

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ACCEPTED MANUSCRIPT overexpression of OsSPL9 affects Cu accumulation in the shoot but not in the root. As

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shown in Fig. S7C, the root-shoot translocation was higher in the overexpression lines

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than in the WT. Subsequently, the total amount of Cu in transgenic plants is slightly

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higher than in WT (Fig S7D), suggesting that overexpression of OsSPL9 also

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increased Cu uptake in rice seedlings.

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A field experiment was performed in two consecutive years at different sites with

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various soil Cu levels to study Cu accumulation at the reproductive stage. Cu

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accumulation data from two years at four sites is presented in Table S2, and Cu levels

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in different tissues at one site are shown as an example in Figs.1C and S8. When Cu

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accumulation in the grain was measured, the transgenic lines contained 3.22-4.47

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mg/kg Cu in the brown rice, an increase of 39%-94% over the WT (Fig.1C). However,

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the levels of other metals such as the Zn, Fe, and Mn were not affected in the

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transgenic plants compared to the WT plants (Fig. S9). We also measured Cu

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accumulation in different tissues including node III, internode II, node II, the lower

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leaf sheath, the lower leaf blade, node I, the flag leaf sheath, the flag leaf blade,

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internode I, the rachis, and the husk, and there were no statistical differences between

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the transgenic and WT plants (Fig. S8). These results indicate that increased Cu

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accumulation in the grain had little, if any, effect on the accumulation of Cu in other

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tissues or the accumulation of other mineral elements in the grain.

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Because

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OsSPL9-overexpressing and WT plants, we investigated different growth parameters

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such as plant height, panicle number, grain number/panicle, and grain yield per plant

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before and after harvesting to define the growth vigor and yield potential of the

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transgenic plants. In most cases, the transgenic plants behaved similarly to the WT.

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No reliable differences between the WT and the transgenic plants were observed (data

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not shown). We compared the growth vigor at different Cu concentrations to

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determine whether over-accumulation of Cu results in disruption of Cu homeostasis

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and negatively affects Cu tolerance. Plant root length, shoot length, and dry weight

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(Fig. S10) were compared between the WT plants and OsSPL9-overexpressing plants

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relative

differences

in

Cu

accumulation

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ACCEPTED MANUSCRIPT after they were grown for two weeks in liquid 0.5X MS medium and then for another

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two weeks where the medium was supplemented with different concentrations of Cu.

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The results showed that the roots of overexpression and WT plants had similar growth

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patterns, suggesting that overexpression of OsSPL9 did not change the ability of rice

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seedlings to maintain Cu homeostasis.

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To investigate the mechanism(s) underlying Cu accumulation in the transgenic plants,

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we first performed qRT-PCR to evaluate the expression of Cu transporter genes under

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normal conditions between WT and the OsSPL9-overexpressing plants. The results

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showed that mRNA accumulation of the copper transporter1 (COPT1) and COPT5

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genes, which are involved in Cu absorption and translocation (Yuan et al., 2010), and

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alsoCOPT6, were higher in OsSPL9 transgenic plants than in the WT plants, while the

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expression level of genes for other transporters such as COPT3, COPT4, and COPT7

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was unchanged in the transgenic plants (Figs. 1D and S11). We then used a

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reporter-effector transient expression assay (Li et al., 2016) to verify that

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OsSPL9upregulates the downstream transporters (Fig. 1E). The promoters of

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COPT1,COPT5,COPT6, and COPT7 were fused individually with the GFP reporter

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gene containing anuclear localization sequence (NLS) for the construction of the

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reporter expression cassettes (Fig. S12). The effector OsSPL9 was driven by the

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CaMV35S promoter. When the reporter was expressed separately or was co-expressed

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with an empty vector, an extremely weak GFP signal was observed. Consistent with

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the qRT-PCR results, dramatically enhanced GFP signals were detected when OsSPL9

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was

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COPT6promoters, while no GFP signals were detected with the reporter driven by the

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COPT7 promoter (Fig. 1E). Because SPL7 in Arabidopsis also regulates several

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microRNAs, the targets of which are Cu-containing proteins, we also examined the

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expression levels of miR528 and miR408 and found that they were upregulated in the

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transgenic plants (Fig. S13A). However, none of the plants overexpressing the two

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miRNAs either independently or together in the ‘Nipponbare’ background showed

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altered levels of Cu in the grain (Fig. S13B-D). All of these results indicate that

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co-expressed

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driven

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COPT1,COPT5

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ACCEPTED MANUSCRIPT overexpression OsSPL9 affects Cu accumulation mainly though by regulatingthe

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expression transporter genes rather than through microRNAs.

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The nutrient digestibility was further compared between grain from OsSPL9

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-overexpressing and WT plants. Both grains showed similar chemical composition

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including dry matter (DM), ash, crude protein (CP), crude fat (CF) and acid detergent

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fiber (ADF) (Table S3). We measured the total gas production, rumen pH value,

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volatile fatty acid (VFA) contents, and in vitro dry matter disappearance (IVDMD)

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using an in vitro ruminal fermentation technique. The total volume of gas produced

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and the fermentation parameters were used as indicators of the relative utilization

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efficiency of the fermentation substrates. Grain from plants overexpressing OsSPL9

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produced more gas and had an increased rate of degradation during early incubation

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(FRD0) compared with WT grain (Fig. 1F and G). Also, the IVDMD was significantly

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higher for OsSPL9-overexpression grain than that for WT grain (Fig.1H).

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Furthermore, a lower rumen fluid pH value and a lower ratio of acetic acid to

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propionic acid were observed for grain from the OsSPL9-overexpressing plants

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compared to the grain from WT plants (Fig.1I and Table S4). These results indicate

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that using grain from the OsSPL9-overexpressing plants with elevated levels of Cu as

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fermentation substrates has better performance in ruminal fermentation and higher

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nutrient digestibility.

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In summary, overexpression of OsSPL9 in rice resulted in increased Cu accumulation

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in shoots at the seedling stage and in the grain after maturation. Our data showed that

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this is due mainly to the upregulation of Cu transporter genes including COPT1,

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COPT5, and COPT6 that are involved in Cu uptake and distribution. The transgenic

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plants showed similar growth and vigor, Cu tolerance, and other mineral nutrient

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values in the grain compared to WT plants, suggesting that Cu homeostasis is

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maintained. The transgenic rice grain with enhanced Cu contents was of greater

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nutritional value and was more digestible compared to WT grain in an in vitro ruminal

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fermentation assay. Our study suggests that OsSPL9 and its orthologs in other crops

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could be valuable genes for improving organic Cu accumulation, and could pave the

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ACCEPTED MANUSCRIPT 1

way toward Cu biofortification of crops for feed efficiency and environmental

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sustainability.

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Acknowledgements

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We thank Dr. Diqiu Yu for kindly providing NLS-GFP vector, Dr. Dayong Li for his

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critical comments and Dr. David Zaitlin for language editing. This work was jointly

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supported by the National Key Technology Support Program (No. 2015BAD05B02)

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and by the National Natural Science Foundation of China (Nos. 31270426,

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31470443and 31371596).

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Mingfeng Tang1, Chuanshe Zhou1, Lu Meng1, Donghai Mao1, Chengbing Liu2, Can

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Peng1, Yuxing Zhu1, Dechun Zhang2, Daoyou Huang1, Zhiliang Tan1 and Caiyan

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Chen1,*

1 Key Laboratory of Agro-Ecological Processes in Subtropical Region, Institute of

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Subtropical Agriculture, Chinese Academy of Sciences, Changsha, 410125, China

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2 Biotechnology Research Center, Three Gorges University, Yichang, 443002, China

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* To whom correspondence should be addressed.

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E-mail: [email protected]

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REFERENCES

20

Armstrong, T., Cook, D., Ward, M., Williams, C. and Spears, J., 2004. Effect of

21

dietary copper source (cupric citrate and cupric sulfate) and concentration on

22

growth performance and fecal copper excretion in weanling pigs. J. Anim. Sci.

23

82, 1234-1240.

24

Bertinato, J., L’Abbé,MR., 2004. Maintaining copper homeostasis: regulation of

25

copper-trafficking proteins in response to copper deficiency or overload. J. Nutr. Biochem. 15, 316-322.

26 7

ACCEPTED MANUSCRIPT 1

Braude, R., 1945. Some observations on the need for copper in the diet of fattening pigs. J. Agric. Sci. 35, 163-167.

2

Broadley, M., Brown, P., Cakmak, I., Rengel, Z. and Zhao, F. , 2012. Chapter 7 -

4

Function of Nutrients: Micronutrients. In: Marschner's Mineral Nutrition of

5

Higher Plants (Third Edition) pp. 191-248. San Diego: Academic Press.

6

Creech, B., Spears, J., Flowers, W., Hill, G., Lloyd, K., Armstrong, T. and Engle, T.,

7

2004. Effect of dietary trace mineral concentration and source (inorganic vs.

8

chelated) on performance, mineral status, and fecal mineral excretion in pigs

9

from weaning through finishing. J. Anim. Sci.82, 2140-2147.

SC

Dove, C., 1995. The effect of copper level on nutrient utilization of weanling pigs. J. Anim. Sci.73, 166-171.

11

M AN U

10

RI PT

3

Engle, T. and Spears, J., 2000. Dietary copper effects on lipid metabolism,

13

performance, and ruminal fermentation in finishing steers. J. Anim. Sci.78,

14

2452-2458.

TE D

12

Gonzales-Eguia, A., Fu, C.-M., Lu, F.-Y. and Lien, T.-F., 2009. Effects of nanocopper

16

on copper availability and nutrients digestibility, growth performance and

17

serum traits of piglets. Livest. Sci. 126, 122-129.

EP

15

Højberg, O., Canibe, N., Poulsen, H.D., Hedemann, M.S. and Jensen, B.B. , 2005.

19

Influence of dietary zinc oxide and copper sulfate on the gastrointestinal

20

ecosystem in newly weaned piglets. Appl. Environ. Microb. 71, 2267-2277.

AC C

18

21

Hatfield, J., Brumm, M. and Melvin, S., 1998.In: Wright R. J., Kemper, W.D., Millner,

22

P.D., Power J. F. and Korcak R. F.(Eds.), Agricultural Uses of Municipal,

23

Animal and Industrial by Products. USDA-ARS Conservation. Research

24

Report Number 44, 78-90.

25

Johnston, H., Beasley, L. and MacPherson, N., 2014. Copper toxicity in a New Zealand dairy herd. Irish Vet. J. 67, 1.

26 27

Li, X., Zhang H., Ai, Q., Liang, G. and Yu, D., 2016. Two bHLH transcription factors, 8

ACCEPTED MANUSCRIPT 1

bHLH34 and bHLH104, regulate iron homeostasis in Arabidopsis thaliana.

2

Plant Physiol. 170, 2478-2493.

3

Lu, L., Wang, R.L., Zhang, Z.J., Steward, F.A., Luo, X. and Liu, B., 2010. Effect of

5

dietary supplementation with copper sulfate or tribasic copper chloride on the

6

growth performance, liver copper concentrations of broilers fed in floor pens,

7

and stabilities of vitamin E and phytase in feeds. Biol. Trace Elem. Res. 138,

8

181-189.

SC

9

Peña, M.M., Lee, J. and Thiele, D.J., 1999. A delicate balance: homeostatic control of copper uptake and distribution. J. Nutr. 129, 1251-1260.

M AN U

10 11

RI PT

4

Uauy, R., Olivares, M. and Gonzalez, M., 1998. Essentiality of copper in humans. Am. J. Clin. Nutr. 67, 952S-959S.

12

Wang, Z., He, Z., Beauchemin, K.A., Tang, S., Zhou, C., Han, X., Wang, M., Kang, J.,

14

Odongo, N.E. and Tan, Z., 2016. Evaluation of different yeast species for

15

improving in vitrofermentation of cereal straws. Asian Austral. J. Anim. 29,

16

230-240.

TE D

13

Xie, K., Wu, C. and Xiong, L., 2006. Genomic organization, differential expression,

18

and interaction of SQUAMOSA promoter-binding-like transcription factors

19

and microRNA156 in rice. Plant Physiol.142, 280-293.

AC C

20

EP

17

Yang, Z., Wang X., Gu S., Hu Z., Xu H., Xu C., 2008. Comparative study of SBP-box gene family in Arabidopsis and rice. Gene 407,1-11.

21 22

Yamasaki, H., Hayashi, M., Fukazawa, M., Kobayashi, Y. and Shikanai, T., 2009.

23

SQUAMOSA promoter binding protein–like7 is a central regulator for copper

24

homeostasis in Arabidopsis. Plant Cell 21, 347-361.

25

Zidar, B.L., Shadduck, R.K., Zeigler, Z. and Winkelstein, A., 1977. Observations on

26

the anemia and neutropenia of human copper deficiency. Am. J. Hematol. 3, 177-185.

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ACCEPTED MANUSCRIPT Figure legends

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Fig. 1 OsSPL9regulates grain Cu accumulation in rice.

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A: Transactivation assay of OsSPL9. Fusion proteins consisting of the GAL4

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DNA-binding domain fused to different regions of OsSPL9 were expressed in yeast

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strain Y2HGold. a is the full-length OsSPL9 coding region; b,c,d,e,f and g are

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truncated forms of OsSPL9 (amino acid positions are labeled in the diagrams). p

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represents the yeast transformant carrying the empty pGBKT7 vector that was used as

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the negative control. The transformants (a through g in the diagram) were incubated

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on SD/Trp- and SD/Trp-/His-/Ade- media with or without X-α-gal to examine their

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growth and to test for β-galactosidase activity.B: OsSPL9 localizes to the nucleus.

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Confocal laser scanning microscopy was used to take images of N. benthamiana leaf

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epidermal cells transiently co-expressing the OsSPL9-GFP fusionor the GFP

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controlwith the nuclear marker HY5-DsRedunder the control of the CaMV35S

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promoter. Merged images of whole-cell viewsare shown. The GFP and DsRed signals

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are shown in green and red, respectively. Scale bars = 20 µm. C: Cu concentrations in

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brown riceharvested from the WT and OsSPL9-overexpression lines. The WT rice

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line ‘Nipponbare’ and the OsSPL9-overexpression lines were grown in a paddy field

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until ripening, and grains of WT and OsSPL9-overexpression lines were subjected to

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Cu concentration analysis by ICP-OES.OE represents overexpression.D: The Cu

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transporter genes COPT1, COPT5, and COPT6 are up-regulated in rice plants

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overexpressing OsSPL9. RNA was extracted from ~0.2 g of mixed tissues from

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7-day-old seedlings for each sample. Gene expression was quantified by qRT-PCR

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using OsUBCas the internal control.E: OsSPL9 activates the promoters of COPT1,

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COPT5, COPT6, but not COPT7 in transient gene expression assays. The results

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shown are single representatives of at least three biological repeats. Scale bars =

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100µm. F-I: Gas production parameters, IVDMD and butyrate for the two

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fermentation substrates. Vf, The maximum gas production; IVDMD, in vitro dry

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matter disappearance; FDR0, rate of degradation during early incubation.All data are

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means ± SD of at least three biological replicates.

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ACCEPTED MANUSCRIPT Fig. S1 Phylogeny and microsynteny of Arabidopsis and rice SPL proteins.A: The

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phylogenetic analysis was performed using the neighbor-joining method in ClustalW2,

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and the tree was edited and viewed with TreeView software. OsSPL9 is indicated with

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a black circle. Bootstrap values from 1,000 replicates are indicated at each node. Scale

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bar represents 0.1 amino acid substitution per site. B:Microsynteny of SPL

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orthologous regions between Arabidopsis (top) and rice (bottom).The homologs of

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AtSPL7

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AT5G18890,were found in a region of ~200 kb containing OsSPL9 in the rice

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genome.

its

adjacent

duplicate

genes,

AT5G18860,

AT5G18870,

and

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Fig. S2 Putative sequence motifspresent in OsSPL9. The organization of putative

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sequence motifs in OsSPL9 predicted by MEME according to Xie (2006) and Yang

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(2008) with some modifications is shown. Numbered colored boxes represent

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different putative motifs and the annotations for each motif are given in the box below

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the figure.

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Fig. S3 Relative expression levels of OsSPL9 in various rice tissues at different

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growth stages. Plants were grown in a paddy field until ripening, and the various

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tissues were sampled at 6, 9, 11, and 13 weeks. The expression levels of OsSPL9 were

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determined by quantitative real-time RT-PCR. OsUBC was used as the internal

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control. Data are means ±SD of at least three biological replicates.

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Fig. S4 Response of OsSPL9 expression to metal deficiency in roots and shoots. Rice

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seedlings were grown in a nutrient solution with (control) or without Zn, Fe, Mn, or

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Cu for 1 week or under 200nmol/L Cd stress for 1 week. The relative expression

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levels were determined by quantitative real-time RT-PCR. OsUBC was used as the

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internal control. Data are expressed as means ±SD of at least three biological

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replicates.

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Fig. S5 The construction of over-expression vector and the expression level of

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representative transgenic lines. A: Schematic diagram showing the structure of the

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pTCK303 binary transformation vector carrying OsSPL9.B: Relative expression of

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OsSPL9 in WT and OsSPL9-overexpression lines. Total RNA was isolated from

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7-day-old WT and transgenic rice plants, reverse-transcribed into cDNA, and

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ACCEPTED MANUSCRIPT analyzed by real-time RT-PCR. OsUBC was used as the internal control. Data are

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expressed as means ±SD of at least three biological replicates.

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Fig. S6 Mineral element concentrations in the roots and shoots of WT and

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OsSPL9-overexpression plants. Concentration of Fe (A andD), Mn (B andE), and Zn

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(C and F) in seedling roots and shoots, respectively. Both WT ‘Nipponbare’ and

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OsSPL9-overexpression lines were grown in a nutrient solution containing essential

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elements for four weeks. The roots and shoots were harvested and subjected to

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elemental analysis by ICP-OES. Data are means ± SD of at least three biological

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replicates.

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Fig. S7 Uptake and translocation of Cu in WT and OsSPL9-overexpression plants. Cu

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concentrations in the roots (A) and shoots (B), the translocation rate of Cu from roots

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to shoots (C) and total Cu uptake (D)are shown. Both WT ‘Nipponbare’ and

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transgenic lines overexpressing OsSPL9 were grown in a nutrient solution containing

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50 nmol/L Cu2+ for four weeks. The roots and shoots were harvested and subjected to

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Cu concentration analysis by ICP-OES. Data are means ± SD of at least three

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biological replicates. Asterisks indicate significant differences from the WT at *P<

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0.05 and **P < 0.01 by Student’s t test.

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Fig. S8 Cu concentration in tissues of WT and OsSPL9-overexpression plants. The

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WT ‘Nipponbare’ and OsSPL9-overexpression lines were grown in a paddy field until

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ripening, and different tissues were then subjected to Cu concentration analysis by

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ICP-OES. Data are means ± SD of at least three biological replicates.

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Fig. S9 Mineral element concentrations in WT and OsSPL9-overexpression plants.

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Concentrations of Fe (A), Mn (B), and Zn (C) were measured in brown rice. The WT

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‘Nipponbare’ and OsSPL9-overexpression lines were grown in a paddy field until

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ripening, and the harvested grain and different tissues were then subjected to mineral

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element concentration analysis by ICP-OES. Data are means±SD of at least three

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biological replicates.

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Fig. S10 Response of OsSPL9-overexpression and WT plants to Cu treatment. Both

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WT rice and the overexpression lines were cultivated hydroponically in a normal

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ACCEPTED MANUSCRIPT nutrient solution for two weeks, after which they were transferred to nutrient solutions

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containing 0, 0.1, 10, 50, 100, 200, or 400 µmol/L CuSO4·5H2O. After two weeks, the

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roots (A) and shoots (B) were harvested and measured and the dry weights (C) were

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determined. The insets in each panel show the plant responses at very lowCu

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concentrations including 0 and 0.1µmol/L CuSO4·5H2O.The data are presented as

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means±SD of three independent biological replicates.

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Fig. S11 Expression of Cu transporter genes in OsSPL9-overexpression plants and

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WT plants. The relative expression of COPT3(A), COPT4(B), and COPT7(C) in

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OsSPL9-overexpression seedlings and WT seedlings were measured in qRT-PCR

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assays using OsUBC as the internal control. Data are means ±SD of at least three

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biological replicates, and at least 10 seedlings were included in each tissue mixture.

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Fig. S12 Schematic representation of the DNA constructs used for transient

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expression assays. The reporter construct consists of the corresponding promoters, a

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nuclear localization sequence (NLS) fused with the GFP coding sequence, and a

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poly(A) terminator. Effector constructs express OsSPL9 under the control of the

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cauliflower mosaic virus (CaMV) 35S promoter.

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Fig.

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double-overexpression

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OsSPL9-overexpression seedlings and WT seedlings. B: Cu concentration in brown

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rice in miR408-overexpression lines and the WT. C: Cu concentration in brown rice in

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miR528-overexpression lines and WT. D:Cu concentration in brown rice in miR408

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and miR528 double-overexpression lines and WT. The WT rice line ‘Nipponbare’ and

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the OsSPL9-overexpression lines were grown in a paddy field until ripening. Error

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bars represent means ± SD of three independent biological replicates.

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Table S1 Homology genes of AtSPL7 in the Arabidopsis genome and 7 homology

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genes in the rice genome.

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Table S2 Brown rice Cu concentration in wild-type and OsSPL9 overexpression lines

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in different places.

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Table S3 Chemical composition (g/kg, DW basis) of the two fermentation substrates.

Cu

concentration

lines.A:

analysis

of

Expression

miR408 of

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miR408

miR528 and

single

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miR528

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substrates.

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Table S5 Oligonucleotide primers used in this study.

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ACCEPTED MANUSCRIPT Materials and Methods Plasmid construction and transformation of rice To generate the OsSPL9-overexpression plants, the coding region of OsSPL9 was amplified from first-strand cDNA synthesized from total RNA using gene-specific

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primer pairs (Table S5), and the DNA fragment was then cloned between the Kpn I and Sac I sites of the binary expression vector pTCK303 under the control of the ubiquitin promoter. After sequencing the insert to confirm the identity of OsSPL9, the construct

was

transformed

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Agrobacterium-mediated transformation. RT-PCR and quantitative PCR analysis

Oryza

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‘Nipponbare’ by

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Total RNA was isolated from rice tissues using TRIzol® reagent (Invitrogen, Carlsbad, CA, USA). First-strand cDNA was synthesized from 2 µg samples of total RNA using SuperSript™II Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA) and an oligo (dT) primer. The qRT-PCR assays were performed using gene-specific primers (Table S5) in a total volume of 20 µL containing 10 µL SYBR Premix Ex

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Taq™ Perfect Time (TaKaRa, Japan), 0.4 µL ROX reference dye, 4 µL primer mix (1:1 mix of forward and reverse primers at 2.5 µmol/µL each), and 5.6 µL of a 3-fold dilution of the cDNAs as template. The PCR reaction conditions were: 30s at 95°C

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followed by 40 cycles of 30s at 95°C, and 30s at 60°C. The rice UBC gene was used as an internal expression standard. The mRNA relative expression levels were calculated by the comparative Ct method. At least three independent biological

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replicates were carried out for each gene. Transcriptional activation analysis in yeast The transactivation experiment was carried out as described in the Yeast Protocols Handbook (Clontech, USA). The PCR fragments of the full ORF and truncated fragments (all confirmed by sequencing) were fused in-frame with the GAL4 DNA binding domain in pGBKT7 to make constructs pGBKT7-OsSPL9 a to g (for primers, see Table S5). pGBKT7 was used as a negative control. These constructs were transformed into the yeast strain Y2HGold using the lithium acetate-method. The resulting transformants were streaked on SD/Trp- and SD/Trp-/His-/Ade- medium. 1

ACCEPTED MANUSCRIPT After incubation at 28oC for three days, the growth state of each transformant was evaluated. The β-galactosidase filter assay was carried out according to the manufacturer’s instructions (Clontech). Plasmid construction and transient expression assays

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Genomic DNA from the japonica rice ‘Nipponbare’ was used as the template for amplification of the upstream regulatory promoter sequences of COPT1, COPT5, COPT6, COPT7, miR528 and miR408. The NLS and GFP fusion was kindly provided by Dr. Diqiu Yu, and was amplified before inserting into pCAMBIA1301 digested

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with Hind III and BstE II. After sequence verification, reporters were constructed acquired by inserting all the promoter sequences into the new pCAMBIA1301 vector,

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which was digested with EcoR I and BamH I. For the effector, the OsSPL9 coding sequences was inserted between the CaMV35S promoter and the poly(A) of the binary vector pEZR_(K)-LN after EcoR I and Xba I digestion. Primers used for these constructs are listed in Table S5. Plasmids were transformed into Agrobacterium tumefaciens strain EHA105 and then infiltrated into leaves of Nicotiana benthamiana

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in infiltration buffer (0.2 mmol/L acetosyringone and 10 mmol/L MES, pH 5.6). After 48h incubation, GFP fluorescence was observed with a confocal laser scanning microscope.

Determination of heavy metal concentrations

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Rice tissues were dried overnight at 80oC to reach a constant weight. Dried plant tissues (50-100 mg roots; 100-200 mg shoots; 2 g brown rice) were weighed precisely

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and digested with a mixture of 12 mL of HNO3 (80%) and 3 mL of HClO4. The digested tissues were washed with deionized water and diluted to a constant volume, and then filtered into a fresh acid cleaned polypropylene tube. The filtered samples were analyzed by Inductively Coupled Plasma-Optical Emission Spectroscopy (ICP-OES). Blank tests for the procedure were also performed. In vitro ruminal fermentation assay The

experiment

was

conducted

in

a

comparative

trial

arrangement.

OsSPL9-overexpression and WT grains were used as substrates in an in vitro ruminal fermentation in this study. The grains were dried at 65oC for 24 h, and then ground to 2

ACCEPTED MANUSCRIPT pass through a 1 mm sieve and stored in plastic bags for the assay. Culture solutions, i.e., macroelement solution, buffered solution, and reducing solution used for in vitro ruminal fermentation were prepared to form artificial saliva according to the procedures modified by Tang et al. (2006). The artificial saliva was kept anaerobic by

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continuously pumping carbon dioxide through it for 2 h. Rumen fluids were obtained from three rumen-cannulated Xiangdong black goats (a local breed in South China) fed ad libitum a mixed diet of rice straw and concentrate (60:40, w/w) offered twice daily at 07:00 and 19:00. The goats were managed

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of Sciences, Changsha, China. Rumen contents of each goat were obtained from various locations within the rumen immediately before the morning feeding, mixed and strained through four layers of cheesecloth under a continuous CO2 stream. The collected rumen fluids were then anaerobically combined with artificial saliva in a ratio of 1 to 9 at 39oC.

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Samples of 500.0 mg OsSPL9-overexpression or WT grains were accurately weighed into 100 mL fermentation bottles (Wanhong Glass Instrument Factory, China) and prewarmed at 39oC, and 50 mL of the mixed fluids (artificial saliva plus rumen fluids)

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was then introduced into each bottle using a dispenser (Varispenser 4960000.060, Eppendorf, Germany). Blanks containing only mixed fluids were incubated together with the treated bottles. Gas production and sampling, in vitro dry matter

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disappearance (IVDMD) determination, and VFA content determination were done as described (Wang et al., 2016).

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the Arabidopsis MANUSCRIPT genome and 7 homology genes in the rice genome Table S1 Homology genes of AtSPL7 in ACCEPTED Gene locusProtein locus Symbols description Score(bits) E Value AT5G18830 AT5G18830.3 SPL7 squamosa promoter 1618binding protein-like 0 7 AT5G18830.1 SPL7, ATSPL7 squamosa promoter 1575binding protein-like 0 7 AT5G18830.2 SPL7 squamosa promoter 1509binding protein-like 0 7 AT2G47070 AT2G47070.1 SPL1 squamosa promoter 119binding 1E-26 protein-like 1 AT1G76580 AT1G76580.1 SPL16 squamosa promoter 111binding 2E-24 protein-like 16 AT1G20980 AT1G20980.1 SPL14, FBR6, squamosa SPL1R2, promoter ATSPL14 110binding 3E-24 protein-like14 AT3G60030 AT3G60030.1 SPL12 squamosa promoter 106binding 6E-23 protein-like12 AT2G33810 AT2G33810.1 SPL3 squamosa promoter98binding 2E-20 protein-like 3 AT1G69170 AT1G69170.1 SPL6 squamosa promoter97binding 3E-20 protein-like 6 AT1G02065 AT1G02065.1 SPL8 squamosa promoter97binding 4E-20 protein-like 8 AT3G57920 AT3G57920.1 SPL15 squamosa promoter96binding 1E-19 protein-like 15 AT1G53160 AT1G53160.1 SPL4 squamosa promoter95binding 2E-19 protein-like 4 AT1G53160.2 SPL4 squamosa promoter95binding 2E-19 protein-like 4 AT1G27360 AT1G27360.4 SPL11 squamosa promoter-like 94 4E-19 11 AT1G27360.3 SPL11 squamosa promoter-like 94 4E-19 11 AT1G27360.2 SPL11 squamosa promoter-like 94 4E-19 11 AT1G27360.1 SPL11 squamosa promoter-like 94 4E-19 11 AT5G50570 AT5G50570.2 SPL13A, SPL13 Squamosa promoter-binding 93 9E-19 protein-like(SBP domain) transcription factor f AT5G50670.1 SPL13B, SPL13 Squamosa promoter-binding 93 9E-19 protein-like(SBP domain) transcription factor f AT5G50570.1 SPL13A, SPL13 Squamosa promoter-binding 93 9E-19 protein-like(SBP domain) transcription factor f AT1G27370.4 SPL10 squamosa promoter92binding 1E-18 protein-like 10 AT1G27370 AT1G27370.3 SPL10 squamosa promoter92binding 1E-18 protein-like 10 AT1G27370.2 SPL10 squamosa promoter92binding 1E-18 protein-like 10 AT1G27370.1 SPL10 squamosa promoter92binding 1E-18 protein-like 10 AT3G15270 AT3G15270.1 SPL5 squamosa promoter89binding 9E-18 protein-like 5 AT2G42200 AT2G42200.1 SPL9, AtSPL9 squamosa promoter89binding 1E-17 protein-like 9 AT5G43270.1 SPL2 squamosa promoter88binding 2E-17 protein-like 2 AT5G43270.3SPL2 squamosa promoter88binding 2E-17 protein-like 2 AT5G43270.2 SPL2 squamosa promoter88binding 2E-17 protein-like 2 AT1G02065 AT1G02065.2 SPL8 squamosa promoter62binding 2E-09 protein-like 8 Os08g39890LOC_Os08g39890.1 OsSPL14 protein|OsSPL14248 - SBP-box 7E-22 gene family member, expressed Os01g69830LOC_Os01g69830.1 OsSPL2 protein|OsSPL2 -233 SBP-box 5E-20 gene family member, expressed Os05g33810LOC_Os05g33810.1 OsSPL9 protein|OsSPL91295 - SBP-box 1E-166 gene family member, expressed Os08g41940LOC_Os08g41940.1 OsSPL16 protein|OsSPL16231 - SBP-box 1E-19 gene family member, expressed Os03g61760LOC_Os03g61760.1 OsSPL6 protein|OsSPL6 -265 SBP-box 4E-23 gene family member, expressed Os01g18850LOC_Os01g18850.2 OsSPL1 protein|OsSPL1 -458 SBP-box 9E-47 gene family member, expressed LOC_Os01g18850.4 OsSPL1 protein|OsSPL1 -458 SBP-box 9E-47 gene family member, expressed LOC_Os01g18850.3 OsSPL1 protein|OsSPL1 -458 SBP-box 9E-47 gene family member, expressed Os01g18850LOC_Os01g18850.1 OsSPL1 protein|OsSPL1 -458 SBP-box 9E-47 gene family member, expressed Os08g40260LOC_Os08g40260.1 OsSPL15 protein|OsSPL15311 - SBP-box 1E-28 gene family member, expressed

ACCEPTED Table S2 Brown rice Cu concentration in wild-type andMANUSCRIPT OsSPL9 overexpression lines in different places a

2.31±0.01

2015BS2 a

3.63±0.29

2015CS1 a

1.91±0.24a

WT

3.62±0.1

OE2

4.28±0.27a

3.68±0.67b

4.54±0.03b

2.49±1.18a

OE19

5.55±0.6b

3.96±0.52b

5.77±0.01b

2.84±0.77a

OE23

5.23±0.45b

3.33±0.83a

5.97±0b

2.38±0.28b

OE30

5.49±0.96b

4.47±0.53b

4.52±0.04b

3.09±0.8a

OE32

4.77±0.28b

3.22±0.25b

4.35±0.07a

2.25±0.4a

a,b

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2015BS1

2014BS

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Table S3 Chemical composition (g/kg, DW basis) of the two fermentation substrates Substrates Items WT OE Dry matter (DM) 910.77 916.31 Ash 36.15 34.71 Crude protein (CP) 93.54 92.52 Crude fat (CF) 25.76 28.31 Acid detergent fiber (ADF) 28.82 28.04

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a,b

0.023

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pH 6.63a 6.55b SEM, standard error of the mean; TVFA = total short chain fatty acids; A:P = ratio of acetate to propionate;

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Table S4 In vitro ruminal fermentation parameters for the two fermentation substrates Substrates Items SEM P WT OE Acetate (mmol/L) 0.495 0.015 13.52b 15.47a Propionate (mmol/L) 6.03 7.11 0.436 0.104 a b Butyrate (mmol/L) 0.153 <0.0001 4.67 2.72 Isobutyrate (mmol/L) 0.38 0.37 0.013 0.471 Valerate (mmol/L) 0.32 0.29 0.011 0.148 Isovalerate (mmol/L) 0.34 0.33 0.012 0.293 TVFA 25.27 27.13 0.863 0.153 a b A/P 0.038 <0.0001 2.24 1.92

ACCEPTED Table S5 Oligonucleotide primers used in this study

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Sequence CATGGAGGCCGAATTCATGGACGCCCCCGGCGGCGGCG TAGTTATGCGGCCGCTGCAGGGCGGCGTGCGCGCAGCGCAA TAGTTATGCGGCCGCTGCAGCACAGGTGTTTCCCTGTCCAA TAGTTATGCGGCCGCTGCAGGACATGGATAAAGAAGGCCCCT TAGTTATGCGGCCGCTGCAGGGTAATATTTATCCGGAATATT TAGTTATGCGGCCGCTGCAGCTATGATGAGTAGTTCCTAGAC TAGTTATGCGGCCGCTGCAGCGGGTCGCGCTTCCTCACGCG CATGGAGGCCGAATTCCGGCTGGTTTGCCCGAACTACC CTAGAGGATCCCCGGGTACCATGGACGCCCCCGGCG GATCGGGGAAATTCGAGCTCCTATGATGAGTAGTTCCTAG TATGTCCGCCTCACAACCAGGCA AACTCTGTGGCCACGTCCTCGA ACTTGGATGATGGCATATGCAGCAGC CCGTTTGTAGAGCCATAATTGCA AGGTTGCCTGAGTCACAGTTAAGTG AATGCAGCTTCTGGAGAGGA AGGCAGCGTTAAGCCATCTA CATGGGCGCCATGAAGTC GTGAAGAGCACCTCCGAGTTCT TGCGGCGTGCTGCTAGA CAAGAGCAGATCCGCACTCA ACGGGCATGTCCTTCACCT GAGGAGGAGGCAGAGGAAGT TGCACATGACCTTCTTCTGG AGCACGAAGAGGAGGCAGAG GCTGTCTCGCTCGTCATGGT CGCACACACAAAACATCAACAA CGAGCCCCGCCACGAC ATGCTGGCCGTCATGTCGTT GCCTAGGGTTTGGCTTTGC ACAAGATCGGGAAACCAAACA

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Primer name OsSPL9-AF OsSPL9-AR OsSPL9-BR OsSPL9-CR OsSPL9-DR OsSPL9-ER OsSPL9-FR OsSPL9-GF OsSPL9 overIF-F OsSPL9 overIF-R osSPL9-R1 osSPL9-R2 UbiF2 OsUBC-F OsUBC-R OsSPL9 real-F OsSPL9 real-R OsCOPT1-F OsCOPT1-R OsCOPT2-F OsCOPT2-R OsCOPT3-F OsCOPT3-R OsCOPT4-F OsCOPT4-R OsCOPT5-F OsCOPT5-R OsCOPT6-F OsCOPT6-R OsCOPT7-F OsCOPT7-R

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