Regulation of micoRNA2111 and its target IbFBK in sweet potato on wounding

Journal Pre-proof Regulation of micoRNA2111 and its target IbFBK in sweet potato on wounding Shiau-Ting Weng, Yun-Wen Kuo, Yu-Chi King, Hsin-Hung Lin, Pin-Yang Tu, Kuei-Shu Tung, Shih-Tong Jeng

PII:

S0168-9452(19)31564-X

DOI:

https://doi.org/10.1016/j.plantsci.2019.110391

Reference:

PSL 110391

To appear in:

Plant Science

Received Date:

6 August 2019

Revised Date:

25 November 2019

Accepted Date:

24 December 2019

Please cite this article as: Weng S-Ting, Kuo Y-Wen, King Y-Chi, Lin H-Hung, Tu P-Yang, Tung K-Shu, Jeng S-Tong, Regulation of micoRNA2111 and its target IbFBK in sweet potato on wounding, Plant Science (2019), doi: https://doi.org/10.1016/j.plantsci.2019.110391

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. 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. © 2019 Published by Elsevier.

Title: Regulation of micoRNA2111 and its target IbFBK in sweet potato on wounding

Shiau-Ting Weng1#, Yun-Wen Kuo1#, Yu-Chi King 1#, Hsin-Hung Lin2#, Pin-Yang Tu1, Kuei-Shu Tung3, Shih-Tong Jeng1*

1

Institute of Plant Biology and Department of Life Science, National Taiwan

2

ro of

University, Taipei 10617, Taiwan

Department of Horticulture and Biotechnology, Chinese Culture University, Taipei

11114, Taiwan

Institute of Molecular and Cellular Biology and Department of Life Science,

-p

3

National Taiwan University, Taipei 10617, Taiwan These authors contributed equally to this work.

re

# *

lP

Corresponding author, tel: 886-2-33662538

Shiau-Ting Weng: [email protected]; Yun-Wen Kuo: [email protected];

na

Yu-Chi King: [email protected]; Hsin-Hung Lin: [email protected]; Pin-Yang Tu: [email protected]; Kuei-Shu Tung: [email protected]; Shih-

Jo

ur

Tong Jeng: [email protected]

1

Highlights 

IbFBK is the target of miR2111 and down-regulated in sweet potato after wounding.



IbFBK interacts with IbSKP1 through its F-box domain to form SCF complex while interacts with IbCNR8 through its kelch-repeat domain.



miR2111 and IbFBK participate in the wounding response of sweet potato by

ro of

regulating the degradation of IbCNR8.

Abstract

Plant microRNAs (miRNAs) are non-coding RNAs, which are composed of 20-24

-p

nucleotides. MiRNAs play important roles in plant growth and responses to biotic and abiotic stresses. Wounding is one of the most serious stresses for plants; however, the

re

regulation of miRNAs in plants upon wounding is not well studied. In this study,

lP

miR2111, a wound-repressed miRNA, identified previously in sweet potato (Ipomoea batatas cv Tainung 57) by small RNA deep sequencing was chosen for further analysis. Based on sweet potato transcriptome database, F-box/kelch repeat protein

na

(IbFBK), a target gene of miR2111, was identified. IbFBK is a wound-inducible gene, and the miR2111-induced cleavage site in IbFBK mRNA is between the 10th and 11th

ur

nucleotides of miR2111. IbFBK is a component of the E3 ligase SCF (SKP1-Cullin-

Jo

F-box) complex participating in protein ubiquitination and degradation. The results of yeast two-hybrid and bimolecular fluorescence complementation assays demonstrate that IbFBK was conjugated with IbSKP1 through the F-box domain in IbFBK Nterminus to form SCF complex, and interacted with IbCNR8 through the kelch-repeat domain in IbFBK C-terminus. The interaction of IbFBK and IbCNR8 may lead to the ubiquitination and degradation of IbCNR8. In conclusion, the suppression of miR2111 resulted in the increase of IbFBK, and may regulate protein degradation of IbCNR8 in 2

sweet potato responding to wounding.

Keywords: sweet potato, wounding, small RNA deep sequencing, miR2111, Fbox/kelch repeat protein (FBK), SCF complex

1. Introduction Plants are not motile and have evolved complex survival mechanisms to respond to

ro of

abiotic and biotic stresses. Biotic stresses are caused by pathogen infection, while abiotic stresses result from environmental factors including salt, cold, drought, and

wounding [1]. After wounding, the signal transduction related to defense mechanism

-p

quickly occurs in plants. Besides the well-known signal molecules including jasmonic acid (JA) and systemin, those defense-related genes such as Pathogen Related genes

re

(PR genes) are activated. It is also shown that microRNAs (miRNAs) may play a crucial role in the post-transcriptional regulation of gene expression during wounding

lP

[2-6].

The miRNAs are 21-24 nucleotide noncoding RNAs encoded by MIR genes,

na

most of which are located on the intergenic region of genome [7]. In plants, the primary miRNAs (pri-miRNAs) are processed into the precursor miRNAs (pre-

ur

miRNAs), which contain stem-loop structure recognized by Dicer-like ribonuclease, especially Dicer-like 1 (DCL1). Pre-miRNAs are subsequently processed into

Jo

miRNA/miRNA* duplexes and methylated by HUA ENHANCER 1 (HEN-1) before translocating into cytoplasm where mature miRNAs are released [8-11]. Mature miRNAs in cytoplasm are further associated with RNA-induced silencing complexes (RISC) containing Argonaute 1 (AGO1) to recognize target mRNAs with complementary sequences and to result in gene silencing via mRNA degradation or translational repression [12-16]. 3

Many plant miRNAs are highly conserved and their targets contain transcription factors, indicating that miRNAs are important for plants growth, development, metabolism and stress responses [15, 17]. For example, the miR156/SPLs and miR172/AP2-like transcription factors modules regulate Arabidopsis phase transitions and flower development [17]. MiRNAs participate in plants growth and development since seed germination. In Arabidopsis, miR156 and miRNA159 regulate seed growth and germination [18]. MiR160 targets ARF10/ARF16/ARF17 to regulate root

ro of

development and growth, miR164 targets NAC1 for lateral root emergence, and

miR390 cleaves the non-coding TAS3 precursor RNA to produce tasiRNA-ARF, which further cleaves ARF3/ARF4 to control lateral root development [7]. In addition,

-p

miR164, miR167, miR169, miR171 and miR172 regulate flower development and

flowering process [7]. Recent researches showed that miRNAs play important roles in

re

plants stress responses. In tobacco, topping stress induces the expression of miR164 in

lP

root to regulate plant defense genes [19]. In wheat, miR398 is inhibited after wounding to protect plants from oxidative stress [20]. In sweet potato, miR828 is induced upon wounding, and regulates the biogenesis of lignin and hydrogen peroxide

na

(H2O2) to enhance defense ability [6].

MiR2111 was initially identified in Arabidopsis under phosphorus deficient

ur

stress, and its target gene At3g27150 encodes F-box/kelch repeat protein (FBK) [21].

Jo

Moreover, miR2111 is also induced under topping stress in tobacco, and further regulates FBK genes to affect circadian clock and flowering time [22, 23]. In contrast, miR2111 is inhibited two days after wounding in Aquilaria sinensis [24]. These indicate that the expression of miR2111 is induced or inhibited during wounding in different plant species. Ubiquitin proteasome pathway is a well-known protein degradation pathway of the post-translational regulation in multicellular organisms. Ubiquitin proteasome 4

pathway is based on three key enzymes: E1 (ubiquitin-activating enzyme), E2 (ubiquitin -conjugating enzyme) and E3 (ubiquitin -ligase enzyme) [25]. Among these E3 enzymes, Skp1-Cullin1-F-box protein (SCF) complex is widely studied. SCF complex is mainly base on four proteins, which are S-phase Kinase associated 1 (SKP1), cullin 1 (CUL1), RING-BOX1 (RBX1), and F-box protein. F-box proteins are categorized into different families according to the protein interaction domain in C-terminus, including leucine-rich repeats (LRRs), WD40 repeats, Kelch repeats, etc.

ro of

[26]. F-box proteins play a role in recognizing target proteins to regulate different physiological responses containing flowering time, circadian clock, signal transduction of plant hormones, and defense responses [27-39].

-p

Although one of the target gene of miR2111, FBK, was confirmed in Arabidopsis [21], the relationship among miR2111, FBK and wounding is not clear in sweet

re

potato. In this study, miR2111 was identified as a wounding-repression miRNA in

lP

sweet potato. The relationship between miR2111 and FBK was confirmed, and the possible downstream protein, cell number regulator 8 (CNR8), of FBK was identified.

Jo

ur

na

The roles of miR2111 and FBK were suggested in sweet potato upon wounding.

5

2. Materials and Methods

2.1. Plant materials and wounding treatments Sweet potato (Ipomoea batatas cv Tainung 57) plants were grown in a controlled environment (16 h/25 oC day; 8 h/22oC night; humidity, 70%; light intensity, 40 mol photons m-2s-1). Sweet potato plants with six to eight fully expanded leaves were used. The third and fourth fully expanded leaves counted from the terminal bud were

ro of

excised, and their petiole cuts were immersed in water for 16 h to reduce background. Then, the leaves were mechanically wounded by forceps pressure and were collected

at 15 minutes, 30 minutes, 45 minutes, 60 minutes, or 240 minutes. Leaves were used

-p

for gene isolation and real-time PCR analyses.

Tobacco (Nicotiana tabacum L. cv W38 and Nicotiana benthamiana) plants were

re

maintained in growth chamber (16 h/25 oC day; 8 h/22oC night; humidity, 70%; light

lP

intensity, 40 mol photons m-2s-1). The four- to five-week-old tobacco plants that contain at least five fully expanded leaves were used. The third to five leaves counted from the terminal bud were used for agroinfiltration transient expression and

na

bimolecular fluorescence complementation (BiFC) assays. Arabidopsis thaliana (Col0) plants were grown at 22 oC under a 16 h light/8 h dark photoperiod with light at

Jo

ur

100 µmol photons m-2 s-1, and 15-day-old plants were used for BiFC assays.

2.2. Analysis of gene expression Total RNAs were isolated from leaves according to the manufacture’s instruction of Trizol regent (Invirogen, CA, USA). The isolated RNAs were then treated with RNase-free Turbo DNase (Thermo Fisher, MA, USA). For target genes and miR2111 precursors, the isolated RNAs were reverse-transcribed by using MMLV transcriptase (Invitrogen, CA, USA) with primer T25VN (Supplementary Table S1). For mature 6

form of miRNAs, the isolated RNAs were polyadenylated by poly(A) polymerase ([40], New England BioLabs, MA, USA), and the products were reverse-transcribed into cDNA by using MultiScribeTM Reverse Transcriptase (Applied Biosystems, MA, USA). The expression levels of the target genes and the precursors and mature forms of miRNAs were detected by quantitative real-time RT-PCR with primer pairs (Supplementary Table S1). All real-time RT PCR data were represented as means  SD (n=3). The qPCR procedure was followed by the instruction of Bio-Rad (Bio-Rad,

ro of

CA, USA).

2.3. Plasmid construction

-p

For sweet potato transformation, the full length cDNA fragment of sweet potato miR2111 precursor (pre-miR2111) was obtained by PCR with BamHI-

re

premiR2111F/SacI-premiR2111R primer sets (Supplementary Table S1). The

lP

fragment was then inserted into yT&A vector (T&A™ Cloning Kit, Biotech), and further inserted into the region between 35S promoter and terminator in pBI221 vector. The 35S- pre-miR2111-terminator fragment was further cloned into

na

pCAMBIA2300 vector and transformed into Agrobacterium tumefaciens strain 15834 for sweet potato transformation. For GFP localization experiment, the DNA fragments

ur

of IbFBK and IbSKP1 were amplified by PCR using primer sets, NcoI-FBK-F/FBK-

Jo

BamHI-R and XbaI-SKP-F/NcoI-SKP-nostopR (Supplementary Table S1), respectively. Products were then inserted into pBI221 for further analysis. For BiFC assay, the DNA fragments of IbFBK, IbSKP1, IbCNR8, and IbFBKΔF-box were amplified by PCR with primer sets, NcoI-FBK-F/FBK-BamHI-R, XbaI-SKP-F/NcoISKP-nostopR, CNR-8-F/CNR8-FL-nostop and XbaI-kelch-F/FBK-BamHI-R (Supplementary Table S1), individually, and cloned into PCR8®/GW/TOPO® vector (invitrogen). These DNA fragments were further cloned into pEarlyGate201, 7

pEarlyGate202, or pEarlyGate103 vectors by using Gateway® LR ClonaseTM II Enzyme Mix (Invitrogen, CA, USA). For yeast two-hybrid assays of target screening, sweet potato cDNA library was cloned into pGADT7 vector as prey, while the full length IbFBK was introduced into pGBKT7 vector as bait. For yeast two-hybrid assays of target recognition, the full length cDNA of IbSKP1, isolated from PCR with primer pairs EcoRI-SKP1-F/ XmaI-SKP1-R (Supplementary Table S1), and IbCNR8 were cloned into pGADT7 vectors as prey. While the full length IbFBK and IbFBKΔFisolated from PCR with primer pairs NcoI-kelch-F/ FBK-BamHI-R

ro of

box,

(Supplementary Table S1), was introduced into pGBKT7 vectors as bait.

-p

2.4. Isolation of target gene and recognition of cleavage site

5’RACE was used to isolate the target gene of miR2111. The BD SMARTTM RACE

re

cDNA Amplification Kit (Clontech, CA, USA, http://www.clontech.com/) was used to obtain the 5’ ends of potential miR2111 target. Then, cDNA was amplified by PCR

lP

with UPM-L/5’RLM F-box-L and UPM-S/5’RLM F-box-S primer sets (Supplementary Table S1), and its products were cloned and sequenced to obtain the

na

potential target of miR2111. The 5’RLM-RACE was used to determine the cleavage site in target gene induced by miR2111. The total RNA were extracted and incubated

ur

with 5-RLM adaptor (Supplementary Table S1) and RNA ligase at 16oC for 16 h. Its products were amplified by PCR with 5-RLM adaptor/5’RLM F-box-L and 5-RLM

Jo

adaptor/5’RLM F-box-S primer sets (Supplementary Table S1). The PCR fragments were cloned and sequenced, and the cleavage sites in the target gene induced by miR2111 can be estimated.

2.5. BiFC assays and GFP localization experiments Plasmids, including IbFBK- pEarleyGate201, IbSKP1- pEarleyGate202, IbCNR88

pEarleyGate202, IbCNR8- pEarleyGate103, IbFBK-GFP-pBI221, and IbSKP1-GFPpBI221, used in Arabidopsis protoplast transfection were purified by Midi Plasmid Kit (Geneaid, Taipei, Taiwan) according to manufacturer’s instruction. The preparation and transfection procedures of Arabidopsis protoplasts were based on Yoo et al. (Yoo et al., 2007). The florescence signals in Arabidopsis protoplasts were observed by confocal laser-scanning microscope (Leica TCS SP5 Spectral Confocal).

controls.

2.6. Agrobacteria-mediated transient expression

ro of

The protoplasts transfected with plasmid encoding YN and YC were used as negative

-p

Tobacco leaves (Nicotiana benthamiana) from 5-week-old plants were used for

observing the influence of MG132, a 26S proteasome inhibitor, in the interaction of

re

IbFBK and IbCNR8. The MG132 treatment was based on the procedure as described

lP

[41]. In brief, third to five tobacco leaves counted from the terminal bud were agroinfiltrated by Agrobacterium tumefaciens strain LBA4404. After 16 hours dark treatment, the florescence of tobacco plants was observed by florescence microscope

na

(Olympus BX51). The excitation wavelength for observation of GFP and YFP are set

ur

at 488 nm and 514 nm, respectively.

Jo

2.7. Yeast two-hybrid assay The cDNA library of sweet potato was synthesized by Make Your Own Mate Plate Library System (Clontech, CA, USA). Sweet potato’s total RNA was extracted and reverse transcribed to produce first strand cDNA by using CDS III primer and SMART III-modified oligo (Supplementary Table S1). The cDNA was then amplified by PCR with 5’ PCR Primer/ 3’ PCR Primer sets (Supplementary Table S1), and its products were purified by using CHROMA SPIN TE-400 column. The yeast two9

hybrid assay was performed according to the procedure as described [42]. YeastmakerTM Yeast Transformation System 2 (Clontech, CA, USA) was used for the yeast two-hybrid assays in this study. Competent yeast (strain Gold yeast) cells were mixed with 100 ng plasmid DNA (IbSKP1- pGADT7, IbCNR8- pGADT7, IbFBKpGBKT7, or IbFBKΔF-box- pGBKT7), and 5 L carrier DNA. The solution was inversed gently with 1 mL PEG/LiAc, and incubated at 30oC for 30 minutes. After incubation, 40 L DMSO was added and the yeast solution was heat shocked at 42oC

ro of

for 15 minutes. The yeast solution was centrifuged, and the pellets were restored by YPDA at 30oC for 90 minutes. The yeast solution was centrifuged and washed two

times by sterile water, and the pellets were restored in 1 mL NaCl (0.9% W/V). Yeast

-p

solution (200 L) was then incubated on SD/-Leu-Trp, SD/-Leu-Trp-His or SD/-Leu-

re

Trp-His-Ade plate at 30oC for three days.

lP

2.8. Generation of transgenic sweet potato plant

The generation of transgenic sweet potato plant was based the procedure as described (Lin et al., 2012). Agrobacterium rhizogenes strain 15834 was transformed with

na

pCAMBIA2300 carrying pre-miR2111 by electrophoresis. The cultured sweet potato leaves were infected by the transformed Agrobacterium rhizogenes strain 15834 for

ur

hairy root induction. The induced roots were further selected by 30 ppm kanamycin

Jo

for 14 days, and the plants generated from the transgenic hairy roots were used for further analysis.

10

3. Results

3.1. Identification of wounding responsive miRNAs in sweet potato In order to study the wounding responsive miRNAs, the small RNA deep sequencing by Illumina Genome Analyzer IIx platform were performed to identify the expression of miRNAs affected by wounding for 30 minutes. Compared to the data from the wounded and un-wounded leaves, miR2111 was chosen for further analysis. The

ro of

values of miR2111 reads before and after wounding were 2153 and 1757,

respectively, indicating that miR2111 was repressed after wounding (Supplementary Table S2). To confirm the data from the small RNA deep sequencing, the expression

-p

of miR2111 was determined by real-time RT-PCR in the sweet potato leaves after

wounding. It showed that the mature form miR2111 was repressed at 30 minutes after

re

wounding, and the ratio of wounding to un-wounding treatment was 0.52±0.03 (Fig.

lP

1A).

The sweet potato transcriptome data was established by De-Novo sequencing. Then, the miR2111 precursor (pre-miR2111) was predicted by the program MirScore

na

[43]. The penalty score of MirScore is 1 when there is one mismatch between two fragments and is 0.5 when a G:U wobble exists. Compared to the sequence of

ur

miR2111 and sweet potato transcriptome data, a fragment comp18073_c0_seq1,

Jo

whose penalty score was 0, was found (Table 1). After blasted in NCBI, this fragment was not matched with any gene. This suggested that comp18073_c0_seq1 might be the precursor of miR2111. Previous research indicated that the structures of miRNA precursors contain stem-loop structures, and there are several common features of them. First, the miRNA and its miRNA* (miRNA star) were derived from the opposite stem-arms, which formed duplex, with two-nucleotide overhangs. Second, the mismatch of base 11

pairing between the miRNA and its miRNA* should be no more than four bases [44]. Based on these criteria, miR2111* was identified from the small RNA deep sequencing dataset, and the predicted miR2111 precursor could form a stem-loop structure by using mfold (http://mfold.bioinfo.rpi.edu/), a website to predict the secondary structure of miRNAs (Fig. 1B). Moreover, the read values of miR2111* were much lower than those of miR2111 before or after wounding, suggesting that miR2111* was unstable (Supplementary Table S2). Just like mature form miR2111,

ro of

the real-time RT-PCR showed that the expression of miR2111 precursor was also repressed after wounding (Fig. 1C). These data demonstrated that this fragment,

comp18073_c0_seq1, was the precursor of miR2111. Besides, the sequence of pre-

-p

miR2111 from sweet potato was aligned to those of grape (Vitis vinifera) and

Arabidopsis, showing that the sequence similarity of precursors among different

re

species was low and the most conservative region was located in the sequence of

lP

miR2111 mature form (Supplementary Fig. S1).

3.2. Regulation of miR2111 and its targets after wounding

na

The sequence of miR2111 was compared to those from sweet potato transcriptome data. Penalty score less than 4 was selected, indicating five possible targets of

ur

miR2111 were obtained (Table 1). The criteria to further filter the targets of miR2111 were based on the previous studies [43]. First, the mismatch of base-pairing in the

Jo

miRNA 5’ region, the first to eighth nucleotides from the miRNA 5’ end, between miR2111 and its targets is no more than one base. Second, any mismatch of basepairing in the center region, the ninth to eleventh nucleotides from the miRNA 5’ end, between miR2111 and its targets is not allowed. Third, the mismatched base-pairing in the 3’ region of miRNA is allowed as long as the penalty score is no more than four. Based on these criteria, there is a mismatch in center region of contig 12

comp72427_c0_seq1, comp317294_c0_seq1, and comp42439_c0_seq1 and there is more than one mismatch in the 5’ region of contig comp124356_c0_seq1. Hence, only contig comp82662_c0_seq1 was totally conformed, and was annotated as an Fbox/kelch-repeat protein At3g27150-like (FBK) gene, which was known as a target of miR2111 in Arabidopsis [21]. According to these results, IbFBK was chosen for

3.3. Relationship between miR2111 and IbFBK

ro of

further analysis.

To confirm the possibility that IbFBK was the target of miR2111, the expression

pattern of IbFBK after wounding was detected by real-time RT-PCR, indicating the

-p

expression of IbFBK was induced after wounding. Hence, the expression pattern of

IbFBK was opposite to that of miR2111 (Fig. 2). To further analyze the relationship

re

between miR2111 and IbFBK, the cleavage site in IbFBK mRNA caused by miR2111

lP

was determined by a modified 5’ RLM-RACE method [45]. It indicated that the cleavage sites induced by miR2111 in IbFBK mRNA was mainly located on the center region, the tenth nucleotides counted from the 5’ end of miR2111 (Fig. 3A). It was a

na

typical cleavage site of miRNAs in target mRNA, and the same cleavage sites in FBK by miR2111 was also found in Arabidopsis [21].

ur

To further confirm the relationship between miR2111 and IbFBK in plant, the

Jo

transient expression experiment was performed. The full length cDNA of IbFBK and the pre-miR2111 were separately introduced into vectors with CaMV 35S promoter. Tobacco leaves (Nicotiana tabacum L. cv W38) were first infected by Agrobacteria transformed with IbFBK, pre-miR2111, or empty vector for three days, and their total RNAs were isolated for analysis. The real-time RT-PCR results showed that the expression of IbFBK was much less in the presence than in the absence of miR2111 precursor (Fig. 3B and 3C), demonstrating that miR2111 could repress the expression 13

of IbFBK in vivo. Furthermore, transgenic sweet potato plants overexpressing pre-miR2111 were created to confirm the effect of miR2111 on IbFBK in sweet potato. Although the expression levels of pre-miR2111 were significantly higher in sweet potato overexpressing pre-miR2111 than that in wild type without wounding (Supplementary Fig. S2A), the expressing levels of mature form miR2111 in the overexpressing lines were lower than that in wild type (Supplementary Fig. S2B). Transgenic sweet

ro of

potatoes also showed the higher expression of IbFBK than wild type (Supplementary Fig. S2C). However, after wounding for 240 minutes, the expression of not only the

pre-miR2111 but also the mature form miR2111 was significantly higher in transgenic

-p

sweet potatoes, 2111OE-2 and 2111OE-5, than those in wild type (Fig. 4A and B).

Moreover, the expression levels of IbFBK were lower in transgenic sweet potatoes

re

than that in wild type (Fig. 4C). These results may indicate that the wounding signal

lP

was required for the production of the mature miR2111 from its precursor form, and that overexpression of miR2111 after wounding repressed the expression of IbFBK,

na

the target of miR2111, in sweet potato.

3.4. Identification of proteins interacting with IbFBK

ur

IbFBK contains an F-box domain in the N-terminus and three kelch repeat domains in

Jo

the C-terminus, and it is possible that IbFBK could recognize and interact with other proteins. Hence, yeast two-hybrid assay was performed to screen proteins interacting with IbFBK after wounding. The full coding region of IbFBK inserted into the pGBKT7 vector was used as bait, and the cDNA library from sweet potato treated with wounding for 30 minutes was used as prey. After transformation of yeast, 18 possible targets were present in SD/-His-Trp-Leu plates and only one colony existed in SD/-His-Trp-Leu-Ade plates. After sequencing, this clone carried the gene S-phase 14

kinase-associated protein 1-like protein (IbSKP1). The amino acid sequence of IbSKP1 was similar to those of two Arabidopsis Skpi-like genes (ASKs), and its similarity to AtASK1 was 78% and AtASK2 was 74% (Supplementary Fig. S3). To further confirm the interaction between IbSKP1 and IbFBK, the full length cDNA of IbSKP1 was isolated by RACE and inserted into pGADT7 vector as prey. On the other hand, the full length cDNA of IbFBK and IbFBK lacking F-box domain (IbFBKF-BOX, Fig. 5A) were individually inserted into pGBKT7 as baits. After

ro of

performing yeast two-hybrid assay, yeasts transformed with IbSKP1 and IbFBK

normally produced colonies on SD/-His-Trp-Leu-Ade plate, while those transformed

with IbSKP1 and IbFBKF-BOX showed no colony (Fig. 5B). These results proved that

-p

IbSKP1 interacted with IbFBK directly in yeast and the F-box domain of IbFBK was essential for the interaction between IbSKP1 and IbFBK.

re

Subcellular localization assays indicated that IbFBK localized in nucleus and

lP

IbSKP1 localized in both nucleus and cytoplasm in Arabidopsis protoplasts (Fig. 5C). Moreover, bimolecular fluorescence complementation (BiFC) was performed to detect the interaction between IbFBK and IbSKP1 in plant. The full length cDNAs of

na

IbFBK and IbSKP1 were inserted into pEarleyGate201YFPN (IbFBK-YN) and pEarleyGate202YFPC (IbSKP1-YC) vectors, respectively, and introduced into

ur

Arabidopsis protoplasts. After observation by confocal microscope, the image showed

Jo

that only protoplasts with both IbFBK-YN and IbSKP1-YC possessed fluorescence signal (Fig. 5D). These results suggested that IbFBK and IbSKP1 interacted with each other in plant.

As described above, IbSKP1 interacted with IbFBK through the F-box domain localized in the N-terminus of IbFBK; however, proteins interacted with IbFBK through the kelch-repeat domain localized in the C-terminal of IbFBK were unclear. Previous studies indicated that the kelch-repeat domain interacting with specific 15

proteins may result in the degradation of target proteins by ubiquitin proteasome pathway [46]. To identify other proteins interacting with IbFBK, yeast two-hybrid assay was performed again. To avoid the expression levels of target proteins were influenced by wounding, cDNA library was extracted from the sweet potato leaves treated with wounding for 15 minutes, the early stage of wounding. The full coding region of IbFBK inserted into pGBKT7 vector was used as bait, and cDNA library from sweet potato treated with wounding for 15 minutes was used as prey. After

ro of

transformation, 26 possible targets existed in SD/-His-Trp-Leu plates and only one colony was present in SD/-His-Trp-Leu-Ade plates. After sequencing, this clone contained the gene Cell Number Regulator 8 (IbCNR8). To further confirm the

-p

interaction between IbCNR8 and IbFBK, the full length cDNA of IbCNR8 was

isolated by RACE and inserted into pGADT7 vector as prey, and, on the other hand,

re

the full length cDNA of IbFBK and IbFBK lacking F-box domain (IbFBKF-BOX, Fig.

lP

5A) were individually inserted into pGBKT7 as baits. After yeast two-hybrid assay, yeast transformed with IbCNR8 and IbFBK normally produced colonies on SD/-HisTrp-Leu-Ade plate. Moreover, yeast transformed with IbCNR8 and IbFBKF-BOX also

na

produced colonies (Fig. 6A). These results proved that IbCNR8 interacted with IbFBK directly in yeast and the F-box domain of IbFBK was unnecessary for this

ur

interaction.

Jo

Previous studies indicated that CNRs are transmembrane proteins [47]. To further confirm the subcellular localization of IbCNR8, IbCNR8-GFP was introduced into tobacco leaves (Nicotiana benthamiana), showing that IbCNR8 was located in the membrane (Fig. 7). Moreover, BiFC was performed to detect the interaction between IbFBK and IbCNR8 in plant. IbFBK and IbCNR8 was inserted into pEarleyGate201YFPN (IbFBK-YN) and pEarleyGate202YFPC (IbCNR8-YC) vectors, respectively, and introduced into Arabidopsis protoplasts. After investigation by 16

confocal microscope, however, no fluorescence signal was observed in the presence of both IbFBK-YN and IbCNR8-YC (Fig. 6B). In order to avoid Arabidopsis protoplasts were too small to transform with large vectors, BiFC was further performed in tobacco leaves (Nicotiana benthamiana). Although the IbFBK-YN / IbSKP1-YC group showed YFP signal, there was still no fluorescence signal observed in IbFBK-YN / IbCNR8-YC group (Supplementary Fig. S4). Furthermore, to protect IbCNR8 form degrading by proteasome, a 26S proteasome inhibitor MG132 and

ro of

IbFBKF-box-YC, lacking F-box domain to prohibit the SCF complex formation, were used. The co-expression of IbCNR8-GFP and IbFBK-YN showed no fluorescence

signal. However, when they were under the treatment of MG132 or using IbFBKFrather than IbFBK-YN for the co-expression with IbCNR8-GFP, the

-p

C box-Y

fluorescence signal was present (Fig. 7). These results may indicate that IbCNR8 was

re

affected by IbFBK, an E3 ligase, to direct IbCNR8 to be degraded by 26S

Jo

ur

na

IbFBK occurred (Fig. 7).

lP

proteasome. Hence, these results point out that the interaction between IbCNR8 and

17

4. Discussion

4.1. Regulation of miR2111 and its target gene Recent studies indicate that miRNAs participate in many physiological processes including growth, development, and biotic and abiotic stress defenses in plants [7, 48, 49]. The roles of miRNAs in plants under different stresses were analyzed via the small RNA deep sequencing [19, 23, 24, 50, 51]. In tobacco, topping results in the

ro of

increase of miR479, miR2111, miR160a, miR396, and miR399a and the decrease of miR156, miR159, miR397, miR166, and miR171f [22, 23]. Besides, in Aquilaria

sinensis, miR159, miR168, and miR393 are induced and miR156, miR162, miR164,

-p

miR396, miR160, and miR2111 are repressed after wounding [24]. These studies

indicate that the expression patterns of miR2111 are various in different plant species

re

upon wounding.

lP

MiRNAs and transcriptome databases of sweet potato upon wounding were analyzed by RNA deep sequencing in our previous studies [52]. MiR2111 was chosen for further study due to its high expression levels in sweet potato with or without

na

wounding treatment. In addition, no isomiR of miR2111 was found in sweet potato. These advantages make the expression of miR2111 was easily detected by real-time

ur

PCR. Furthermore, the existence of miR2111* was identified by the small RNA deep

Jo

sequencing. The expression levels of miR2111 were decreased by only 0.81 folds in sweet potato upon wounding compared to un-wounding (Supplementary Table S2), indicating the subtle change of miRNA expression levels is sufficient to regulate target genes. Similarly, the expression level of nta-miR159 in tobacco is decreased by only 0.8 times after wounding, and the expression of its target genes MYB63 and MYB65 significantly increases [23]. In sweet potato, the decrease of miR2111 precursor expression after wounding (Fig. 1C) results in the increase of target gene 18

FBK expression (Fig. 2).

4.2. Relationship between miR2111 and IbFBK In plants, miRNAs recognize target genes by complementary sequences, and regulate plant responses via gene silencing. To analyze the relationship between miRNA and its target gene, the opposite gene expression patterns of miRNA and target gene has to be revealed, and the target gene cleavage site caused by miRNA should be confirmed.

ro of

Most cleavage sites in miRNA’s target genes localize at the open reading frame

(ORF); however, few of them are found at the 5’-UTR, 3’-UTR or non-coding regions of RNA [53, 54]. The complementary sequences between miRNA and target gene are

-p

divided into three parts. The first to eighth nucleotides from the 5’ end of miRNAs are named the 5’ region, the ninth to eleventh nucleotides belong to the central region

re

where AGOs recognizes, and the twelfth and remaining nucleotides are in the 3’ region [43]. In plants, the base pairings at the 5’ and central region are critical for

lP

miRNA-mediated target repression while the mismatches at the miRNA 3’ region are much less harmful than those at the 5’ and central regions [43]. To confirm the

na

cleavage site between miR2111 and IbFBK, a modified 5’ RLM-RACE was performed, indicating the cleavage site of IbFBK localized between the tenth and

ur

eleventh nucleotides (Fig. 3A), which are the typical sites for AGOs recognition. In

Jo

addition, the cleavage site identified in sweet potato (Fig. 3A) is same as that of miR2111 in Arabidopsis [21]. Moreover, Agrobacterium tumefaciens-mediated transient assay was performed to confirm the relationship between miR2111 and IbFBK (Fig. 3B), indicating overexpression of miR2111 precursor resulted in the repression of IbFBK expression. In transgenic sweet potato overexpressing miR2111 precursor, the expression levels of mature form miR2111 were lower than that in wild type without wounding 19

treatment (Supplementary Fig. S2B). However, the expression levels of mature miR2111 were dramatically higher in transgenic plants than that in wild type after wounding (Fig. 4B). These results suggested that wounding may decrease the expression of pre-miR2111 by repressing the promoter, however, wounding may also trigger the signal to activate DCL1 to modify pre-miR2111 RNA. Besides, the higher expression of miR2111 resulted in the lower expression of IbFBK (Fig. 4C),

ro of

indicating that IbFBK is one of the targets of miR2111.

4.3. Localization and interaction of IbFBK and IbSKP1

FBK is one of the F-box proteins, which conjugate with SKP1, RBX1, CUL1 to form

-p

SCF complex [55]. The F-box motif localized at the N-terminus of FBK interacts with SKP1, and protein-protein interaction domain localized at the C-terminus recognizes

re

specific proteins for degradation [56]. In this study, IbSKP1 was identified by the

lP

yeast two-hybrid assay to interact with the F-box of IbFBK (Fig. 5B). In Arabidopsis, 21 SKP1 homolog genes are identified. Arabidopsis SKP1-Like 1 (ASK1), which interacts with the different F-box proteins including TIR1 and COI1, was widely

na

studied [26, 57]. Besides, proteins interacting with ASK1 participate in many physiology processes including photomorphogenesis, post-translational modification

ur

and stress responses [58]. Arabidopsis ASK2, whose amino acid sequence is similar to

Jo

ASK1, play a role in male meiosis [59]. After alignment, IbSKP1 showed high similarity to ASK1 and ASK2 (Supplementary Fig. S3), suggesting that IbSKP1 with IbFBK may participate in different physiology processes including stress responses. AtFBK encoded by At3g27150, which is homologous to IbFBK, localizes in the third chromosome of Arabidopsis, and expresses in nucleus [60, 61]. Besides, AtASK1 was observed to express in both nucleus and cytoplasm [62]. In this study, IbFBK expressed in nucleus and IbSKP1 expressed in both nucleus and cytoplasm (Fig. 5C), 20

and their localizations are similar to those of AtFBK and AtASK1. Previous studies indicated that F-box proteins interact with different SKP1 to form various SCF complexes under physiological regulation [56, 62]. Moreover, SKP1 could also interact with different F-box proteins to cause various localizations [56, 62]. BiFC assay in this study proved that IbFBK interacted with IbSKP1 in both nucleus and cytoplasm in Arabisopsis protoplasts (Fig. 5D), suggesting that IbFBK interacted with IbSKP1 to form SCF complex. However, AtFBK encoded by At3g27150 did not

ro of

interact with ASK1 [60]. These results may indicate that the interaction between FBK and SKP1 depended on the different species and condition such as wounding in this

-p

study.

4.4. Localization and interaction of IbFBK and IbCNR8

re

IbCNR8 identified by yeast two-hybrid assay can bind to IbFBK. Previous studies

lP

indicate that FBK interact with specific target proteins through kelch repeat domain [31, 35, 36]. Our results confirmed that IbCNR8 could interact with both IbFBK and IbFBKF-BOX (Fig. 6A), demonstrating that IbCNR8 is one of the targets of IbFBK.

na

BiFC was also performed to confirm the interaction between IbFBK and IbCNR8. However, there was no fluorescence signal observed in neither Arabidopsis

ur

protoplasts nor tobacco leaves (Fig.s 6B and S4). The conflict results from yeast two-

Jo

hybrid and BiFC may result from the characteristics of SCF complexes, which add ubiquitins to target proteins for degradation. Hence, once IbFBK and IbCNR8 were introduced to Arabidopsis or tobacco leaves, IbFBK could interact with IbSKP1 to form SCF complex and further activate the degradation of IbCNR8 so that the fluorescence signal could not be observed. On the contrary, there may be no functional SKP1 for plant SCF complex in yeast, and the interaction between IbFBK and IbCNR8 was observed in yeast two-hybrid assay (Fig. 6A). To verify this 21

hypothesis, a 26S proteasome inhibitor MG132 was used. MG132 protect proteins from degradation by inhibiting the proteolytic activity of 26S proteasome [63]. The fluorescence of IbCNR8-GFP was observed under the presence of MG132 (Fig. 7). Furthermore, the co-expression of IbCNR8-GFP with IbFBKF-box-YN showed GFP fluorescence signal, while no fluorescence was found when the co-expression of IbCNR8-GFP with IbFBK-YN (Fig. 7). These results demonstrated that IbFBK and IbCNR8 interacted with each other in plant and the degradation of IbCNR8 depends

ro of

on the F-box domain in IbFBK.

CNRs were identified in 2010 by Guo et al. for screening orthologs of fw2.2, which regulates fruit size in tomato and in maize [64, 65]. There are 13 CNRs in

-p

maize, and CNRs are negative regulators. Among these 13 CNRs, the revolutionary

relationship among CNR1, CNR2 and fw2.2 is close. CNRs regulate organ and plant

re

sizes by altering cell numbers. Plants overexpressing CNR1 and CNR2 showed

lP

smaller organ and plant size phenotype. On the contrary, the larger organ and plant sizes were observed when plants inhibit the expression of CNR1 and CNR2 [64]. CNRs and fw2.2 are all transmembrane proteins [47, 64]. In this study, IbCNR8 was

na

transient expressed in tobacco leaves (Fig. 7), showing that the similar results with the previous studies [47, 64] that IbCNR8 was mainly on membrane. Moreover, results

ur

from two transmembrane protein prediction websites,

Jo

http://www.cbs.dtu.dk/services/TMHMM/ and http://topcons.cbr.su.se/, demonstrate that IbCNR8 may be a transmembrane protein and the transmembrane domain are during the 131st to 158th amino residues (data not shown). The potential target proteins of AtFBK (At3g27150) were not found, however, in our study, IbCNR8 was found to interact with IbFBK.

5. Conclusion 22

In sweet potato, the repression of miR2111 expression under wounding resulted in the increase of IbFBK expression. IbFBK is an E3 ligase, and it contains F-box domain in the N-terminus and kelch repeat domain in the C-terminus. IbFBK interacts with IbSKP1 through F-box domain to form SCF complex and interacts with IbCNR8 through kelch repeat domain to regulate the degradation of IbCNR8. Hence, miR2111 and IbFBK participate in sweet potato wounding responses by regulating the

ro of

degradation of IbCNR8 (Fig. 8).

Authors’ contributions

S-T W, Y-W K and Y-C K carried out conception of the research, and wrote the

-p

manuscript. S-T W and P-Y T performed the experiments. Y-C K, H-H L and K-S T

re

gave the critical suggestion of this study and revised the manuscript. S-T W, Y-W K, Y-C K and H-H L contribute equally to this work. S-T J supervised the entire study.

lP

All authors have read and approved the final manuscript.

ur

na

Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Jo

Acknowledgements

This work was supported by the Ministry of Science and Technology, R.O.C 1052313-B-002-052-MY3 and 105-2311-B-002-018 and by the National Taiwan University under grants 105R104509, 106R891504, 108L893103, and 108L8807 to Shih-Tong Jeng.

23

Reference

[1] M. Fujita, Y. Fujita, Y. Noutoshi, F. Takahashi, Y. Narusaka, K. YamaguchiShinozaki, K. Shinozaki, Crosstalk between abiotic and biotic stress responses: a current view from the points of convergence in the stress signaling networks, Curr Opin Plant Biol, 9 (2006) 436-442. [2] A.L. Schilmiller, G.A. Howe, Systemic signaling in the wound response, Curr Opin Plant Biol, 8 (2005) 369-377. [3] G.A. Howe, Jasmonates as signals in the wound response, J Plant Growth Regul, 23 (2004) 223-237.

ro of

[4] J.C. Carrington, V. Ambros, Role of microRNAs in plant and animal development, Science, 301 (2003) 336-338. [5] B.H. Zhang, X.P. Pan, G.P. Cobb, T.A. Anderson, Plant microRNA: A small

lP

re

-p

regulatory molecule with big impact, Dev Biol, 289 (2006) 3-16. [6] J.S. Lin, C.C. Lin, H.H. Lin, Y.C. Chen, S.T. Jeng, MicroR828 regulates lignin and H2O2 accumulation in sweet potato on wounding, New Phytol, 196 (2012) 427-440. [7] V. Eldem, S. Okay, T. Unver, Plant microRNAs: new players in functional genomics, Turk J Agric For, 37 (2013) 1-21. [8] G.L. Tang, B.J. Reinhart, D.P. Bartel, P.D. Zamore, A biochemical framework for RNA silencing in plants, Gene Dev, 17 (2003) 49-63. [9] Y. Kurihara, Y. Watanabe, Arabidopsis micro-RNA biogenesis through Dicer-like 1

Jo

ur

na

protein functions, P Natl Acad Sci USA, 101 (2004) 12753-12758. [10] Z.Y. Yang, Y.W. Ebright, B. Yu, X.M. Chen, HEN1 recognizes 21-24 nt small RNA duplexes and deposits a methyl group onto the 2 ' OH of the 3 ' terminal nucleotide, Nucleic Acids Research, 34 (2006) 667-675. [11] M.Y. Park, G. Wu, A. Gonzalez-Sulser, H. Vaucheret, R.S. Poethig, Nuclear processing and export of microRNAs in Arabidopsis, P Natl Acad Sci USA, 102 (2005) 3691-3696. [12] D.P. Bartel, MicroRNAs: Genomics, biogenesis, mechanism, and function, Cell, 116 (2004) 281-297. [13] A.C. Mallory, T. Elmayan, H. Vaucheret, MicroRNA maturation and action - the expanding roles of ARGONAUTEs, Curr Opin Plant Biol, 11 (2008) 560-566. [14] O. Voinnet, Origin, Biogenesis, and Activity of Plant MicroRNAs, Cell, 136 (2009) 669-687. [15] Y.J. Meng, C.G. Shao, H.Z. Wang, M. Chen, The Regulatory Activities of Plant MicroRNAs: A More Dynamic Perspective, Plant Physiology, 157 (2011) 1583-1595. [16] J. Martinez, A. Patkaniowska, H. Urlaub, R. Luhrmann, T. Tuschl, Single24

stranded antisense siRNAs guide target RNA cleavage in RNAi, Cell, 110 (2002) 563574. [17] P. Huijser, M. Schmid, The control of developmental phase transitions in plants, Development, 138 (2011) 4117-4129. [18] G. Shumin, D. Yanfei, Z. Cheng, [Role of miRNA in plant seed development], Yi Chuan, 37 (2015) 554-560. [19] S. Tang, Y. Wang, Z.F. Li, Y.J. Gui, B.G. Xiao, J.H. Xie, Q.H. Zhu, L.J. Fan, Identification of wounding and topping responsive small RNAs in tobacco (Nicotiana tabacum), Bmc Plant Biol, 12 (2012). [20] B. Wang, Y.F. Sun, N. Song, J.P. Wei, X.J. Wang, H. Feng, Z.Y. Yin, Z.S. Kang, MicroRNAs involving in cold, wounding and salt stresses in Triticum aestivum L.,

ro of

Plant Physiol Bioch, 80 (2014) 90-96. [21] L.C. Hsieh, S.I. Lin, A.C. Shih, J.W. Chen, W.Y. Lin, C.Y. Tseng, W.H. Li, T.J. Chiou, Uncovering small RNA-mediated responses to phosphate deficiency in

lP

re

-p

Arabidopsis by deep sequencing, Plant Physiol, 151 (2009) 2120-2132. [22] H.X. Guo, Y.C. Kan, W.Q. Liu, Differential Expression of miRNAs in Response to Topping in Flue-Cured Tobacco (Nicotiana tabacum) Roots, Plos One, 6 (2011). [23] Y.C. Qi, H.X. Guo, K. Li, W.Q. Liu, Comprehensive analysis of differential genes and miRNA profiles for discovery of topping-responsive genes in flue-cured tobacco roots, Febs J, 279 (2012) 1054-1070. [24] Z.H. Gao, Y. Yang, Z. Zhang, W.T. Zhao, H. Meng, Y. Jin, J.Q. Huang, Y.H. Xu, L.Z. Zhao, J. Liu, J.H. Wei, Profiling of MicroRNAs under Wound Treatment in

Jo

ur

na

Aquilaria sinensis to Identify Possible MicroRNAs Involved in Agarwood Formation, Int J Biol Sci, 10 (2014) 500-510. [25] R.D. Vierstra, The ubiquitin/26S proteasome pathway, the complex last chapter in the life of many plant proteins, Trends Plant Sci, 8 (2003) 135-142. [26] H. Kuroda, N. Takahashi, H. Shimada, M. Seki, K. Shinozaki, M. Matsui, Classification and expression analysis of Arabidopsis F-box-containing protein genes, Plant Cell Physiol, 43 (2002) 1073-1085. [27] J.C. del Pozo, M. Estelle, F-box proteins and protein degradation: An emerging theme in cellular regulation, Plant Mol Biol, 44 (2000) 123-128. [28] E. Lechner, P. Achard, A. Vansiri, T. Potuschak, P. Genschik, F-box proteins everywhere, Curr Opin Plant Biol, 9 (2006) 631-638. [29] J.B. Song, Y.X. Wang, H.B. Li, B.W. Li, Z.S. Zhou, S. Gao, Z.M. Yang, The Fbox family genes as key elements in response to salt, heavy mental, and drought stresses in Medicago truncatula, Funct Integr Genomic, 15 (2015) 495-507. [30] S. Paquis, F. Mazeyrat-Gourbeyre, O. Fernandez, J. Crouzet, C. Clement, F. Baillieul, S. Dorey, Characterization of a F-box gene up-regulated by phytohormones 25

and upon biotic and abiotic stresses in grapevine, Mol Biol Rep, 38 (2011) 33273337. [31] Y. Chen, Y. Xu, W. Luo, W. Li, N. Chen, D. Zhang, K. Chong, The F-box protein OsFBK12 targets OsSAMS1 for degradation and affects pleiotropic phenotypes, including leaf senescence, in rice, Plant Physiol, 163 (2013) 1673-1685. [32] A. Franciosini, B. Lombardi, S. Iafrate, V. Pecce, G. Mele, L. Lupacchini, G. Rinaldi, Y. Kondou, G. Gusmaroli, S. Aki, T. Tsuge, X.W. Deng, M. Matsui, P. Vittorioso, P. Costantino, G. Serino, The Arabidopsis COP9 SIGNALOSOME INTERACTING F-BOX KELCH 1 protein forms an SCF ubiquitin ligase and regulates hypocotyl elongation, Mol Plant, 6 (2013) 1616-1629. [33] W.Y. Kim, S. Fujiwara, S.S. Suh, J. Kim, Y. Kim, L. Han, K. David, J. Putterill,

ro of

H.G. Nam, D.E. Somers, ZEITLUPE is a circadian photoreceptor stabilized by GIGANTEA in blue light, Nature, 449 (2007) 356-360. [34] M. Sawa, D.A. Nusinow, S.A. Kay, T. Imaizumi, FKF1 and GIGANTEA

lP

re

-p

complex formation is required for day-length measurement in Arabidopsis, Science, 318 (2007) 261-265. [35] X. Zhang, M. Gou, C. Guo, H. Yang, C.J. Liu, Down-regulation of Kelch domain-containing F-box protein in Arabidopsis enhances the production of (poly)phenols and tolerance to ultraviolet radiation, Plant Physiol, 167 (2015) 337350. [36] X. Zhang, M. Gou, C.J. Liu, Arabidopsis Kelch repeat F-box proteins regulate phenylpropanoid biosynthesis via controlling the turnover of phenylalanine ammonia-

Jo

ur

na

lyase, Plant Cell, 25 (2013) 4994-5010. [37] Y.H. Song, D.A. Estrada, R.S. Johnson, S.K. Kim, S.Y. Lee, M.J. MacCoss, T. Imaizumi, Distinct roles of FKF1, Gigantea, and Zeitlupe proteins in the regulation of Constans stability in Arabidopsis photoperiodic flowering, Proc Natl Acad Sci U S A, 111 (2014) 17672-17677. [38] S.H. Han, S.C. Yoo, B.D. Lee, G. An, N.C. Paek, Rice FLAVIN-BINDING, KELCH REPEAT, F-BOX 1 (OsFKF1) promotes flowering independent of photoperiod, Plant Cell Environ, 38 (2015) 2527-2540. [39] E. Demarsy, C. Fankhauser, Higher plants use LOV to perceive blue light, Curr Opin Plant Biol, 12 (2009) 69-74. [40] R. Shi, V.L. Chiang, Facile means for quantifying microRNA expression by realtime PCR, Biotechniques, 39 (2005) 519-525. [41] L. Camacho, A.P. Smertenko, J. Perez-Gomez, P.J. Hussey, I. Moore, Arabidopsis Rab-E GTPases exhibit a novel interaction with a plasma-membrane phosphatidylinositol-4-phosphate 5-kinase, J Cell Sci, 122 (2009) 4383-4392. [42] J. Liu, H.C. Zhang, X.P. Lian, R. Converse, L.Q. Zhu, Identification of 26

Interacting Motifs Between Armadillo Repeat Containing 1 (ARC1) and Exocyst 70 A1 (Exo70A1) Proteins in Brassica oleracea, Protein J, 35 (2016) 34-43. [43] Q. Liu, F. Wang, M.J. Axtell, Analysis of complementarity requirements for plant microRNA targeting using a Nicotiana benthamiana quantitative transient assay, Plant Cell, 26 (2014) 741-753. [44] B.C. Meyers, M.J. Axtell, B. Bartel, D.P. Bartel, D. Baulcombe, J.L. Bowman, X. Cao, J.C. Carrington, X.M. Chen, P.J. Green, S. Griffiths-Jones, S.E. Jacobsen, A.C. Mallory, R.A. Martienssen, R.S. Poethig, Y.J. Qi, H. Vaucheret, O. Voinnet, Y. Watanabe, D. Weigel, J.K. Zhui, Criteria for Annotation of Plant MicroRNAs, Plant Cell, 20 (2008) 3186-3190. [45] X. Sun, Y.P. Zhang, X.D. Zhu, N.K. Korir, R. Tao, C. Wang, J.G. Fang, Advances

ro of

in identification and validation of plant microRNAs and their target genes, Physiol Plantarum, 152 (2014) 203-218. [46] L. Chen, H. Hellmann, Plant E3 ligases: flexible enzymes in a sessile world, Mol

lP

re

-p

Plant, 6 (2013) 1388-1404. [47] B. Cong, S.D. Tanksley, FW2.2 and cell cycle control in developing tomato fruit: a possible example of gene co-option in the evolution of a novel organ, Plant Mol Biol, 62 (2006) 867-880. [48] A.C. Mallory, H. Vaucheret, Functions of microRNAs and related small RNAs in plants, Nat Genet, 38 Suppl (2006) S31-36. [49] Q. Liu, Y.Q. Chen, Insights into the mechanism of plant development: Interactions of miRNAs pathway with phytohormone response, Biochem Bioph Res

Jo

ur

na

Co, 384 (2009) 1-5. [50] T.A. Bozorov, I.T. Baldwin, S.G. Kim, Identification and profiling of miRNAs during herbivory reveals jasmonate-dependent and -independent patterns of accumulation in Nicotiana attenuata, Bmc Plant Biol, 12 (2012) 209. [51] X.B. Liu, L. Ma, A.H. Zhang, Y.H. Zhang, J. Jiang, W. Ma, L.M. Zhang, W.C. Ren, X.J. Kong, High-throughput analysis and characterization of Astragalus membranaceus transcriptome using 454 GS FLX, PLoS One, 9 (2014) e95831. [52] M.-Y. Jhu, Identification and functional characterization of wounding-responsive miRNAs in sweet potato (Ipomoea batatas cv. Tainung 57), Graduate Institute of Plant Biology, College of Life Sciense, National Taiwan University, (2014). [53] L. Beauclair, A. Yu, N. Bouche, microRNA-directed cleavage and translational repression of the copper chaperone for superoxide dismutase mRNA in Arabidopsis, Plant J, 62 (2010) 454-462. [54] C. Brousse, Q.K. Liu, L. Beauclair, A. Deremetz, M.J. Axtell, N. Bouche, A noncanonical plant microRNA target site, Nucleic Acids Research, 42 (2014) 5270-5279. [55] C.D. Nezames, X.W. Deng, The COP9 signalosome: its regulation of cullin27

based E3 ubiquitin ligases and role in photomorphogenesis, Plant Physiol, 160 (2012) 38-46. [56] H. Kuroda, Y. Yanagawa, N. Takahashi, Y. Horii, M. Matsui, A Comprehensive Analysis of Interaction and Localization of Arabidopsis SKP1-LIKE (ASK) and FBox (FBX) Proteins, Plos One, 7 (2012). [57] N. Takahashi, H. Kuroda, T. Kuromori, T. Hirayama, M. Seki, K. Shinozaki, H. Shimada, M. Matsui, Expression and interaction analysis of Arabidopsis SKP1-related genes, Plant Cell Physiol, 45 (2004) S177-S177. [58] X.F. Wang, W.M. Ni, X.C. Ge, J.J. Zhang, H. Ma, K.M. Cao, Proteomic identification of potential target proteins regulated by an ASK1-mediated proteolysis pathway, Cell Res, 16 (2006) 489-498.

ro of

[59] D.H. Zhao, T.F. Han, E. Risseeuw, W.L. Crosby, H. Ma, Conservation and divergence of ASK1 and ASK2 gene functions during male meiosis in Arabidopsis thaliana, Plant Mol Biol, 53 (2003) 163-173.

lP

re

-p

[60] N. Schumann, A. Navarro-Quezada, K. Ullrich, C. Kuhl, M. Quint, Molecular Evolution and Selection Patterns of Plant F-Box Proteins with C-Terminal Kelch Repeats, Plant Physiology, 155 (2011) 835-850. [61] Y.J. Sun, X.F. Zhou, H. Ma, Genome-wide analysis of Kelch repeat-containing Fbox family, J Integr Plant Biol, 49 (2007) 940-952. [62] M.H. Dezfulian, D.M. Soulliere, R.K. Dhaliwal, M. Sareen, W.L. Crosby, The SKP1-Like Gene Family of Arabidopsis Exhibits a High Degree of Differential Gene Expression and Gene Product Interaction during Development, Plos One, 7 (2012).

Jo

ur

na

[63] Y.H. Han, H.J. Moon, B.R. You, W.H. Park, The effect of MG132, a proteasome inhibitor on HeLa cells in relation to cell growth, reactive oxygen species and GSH, Oncol Rep, 22 (2009) 215-221. [64] M. Guo, M.A. Rupe, J.A. Dieter, J.J. Zou, D. Spielbauer, K.E. Duncan, R.J. Howard, Z.L. Hou, C.R. Simmons, Cell Number Regulator1 Affects Plant and Organ Size in Maize: Implications for Crop Yield Enhancement and Heterosis, Plant Cell, 22 (2010) 1057-1073. [65] M. Guo, C.R. Simmons, Cell number counts - The fw2.2 and CNR genes and implications for controlling plant fruit and organ size, Plant Sci, 181 (2011) 1-7. [66] K. Iwabuchi, B. Li, P. Bartel, S. Fields, Use of the two-hybrid system to identify the domain of p53 involved in oligomerization, Oncogene, 8 (1993) 1693-1696. [67] B. Li, S. Fields, Identification of mutations in p53 that affect its binding to SV40 large T antigen by using the yeast two-hybrid system, FASEB J, 7 (1993) 957-963.

28

Table1 Prediction of precursor and targets of miR2111 by Mir Score. Contig_Name

Penalty_Score1

5'region2

3'region

Center_region

Annotation

0

0//0

0//0

0//0

non

comp18073_c0_seq1

PREDICTED: F-box/kelchcomp82662_c0_seq1

3

0//0

2//2

0//0

repeat protein At3g27150-

ro of

like [Solanum tuberosum] PREDICTED:

uncharacterized protein

comp72427_c0_seq1

3

1//0

1//1

0//1

3.5

0//1

1//1

0//1

3.5

0//1

1//1

Jo

comp124356_c0_seq1

lycopersicum] PREDICTED: abscisic acid 8'-hydroxylase 3-like [Solanum lycopersicum] PREDICTED: ubiquitin carboxyl-terminal hydrolase

0//1 14-like [Solanum tuberosum]

ur

comp42439_c0_seq1

na

lP

comp317294_c0_seq1

re

-p

LOC101257921 [Solanum

PREDICTED: uncharacterized protein

3.5

1//1

0//2

0//0 LOC102594483 isoform X1 [Solanum tuberosum]

1

To predict the miR2111 precursor gene, a program Mir Score, based on Liu et al., was established

[43]. The penalty score of Mir Score is 1 when there is one mismatch between two fragments and is 29

0.5 when there was a G:U wobble.

ur

na

lP

re

-p

ro of

The number of G-U wobble//the number of mismatch

Jo

2

30

Figure Legends

Fig. 1. Expression levels of the mature and precursor forms of miR2111 in WT

Jo

ur

na

lP

re

-p

ro of

sweet potato after wounding treatment.

(A) Expression patterns of mature form miR2111 after wounding. The expression levels of 5.8S rRNA were used as internal controls. (B) The predicted miR2111 precursor forms stem-loop structure by using mfold (http://mfold.bioinfo.rpi.edu/), a website to predict the secondary structure of RNA. The lines indicate the sequences of miR2111 with yellow background and miR2111*. (C) Expression patterns of 31

precursor form miR2111 (pre-miR2111) after wounding. The expression levels of IbActin were used as internal controls. The third fully-expanded leaves of WT sweet potato were harvested with petiole, and immersed in ddH2O for 16 hours. Leaves were then wounded for the indicated time, and the leaves without wounding were used as controls (W-). Total RNAs were extracted and analyzed by real-time RT-PCR. The expression levels of genes in W- were treated as one to normalize those treated with wounding. Data were represented as means ± SD (n=3). Asterisk indicated

Jo

ur

na

lP

re

-p

ro of

significant difference to W- (p<0.05; t-test).

32

ro of

Fig. 2. Expression levels of IbFBK in WT sweet potato after wounding treatment.

The third fully-expanded leaves of WT sweet potato were harvested with petiole and

-p

immersed in ddH2O for 16 hours. Leaves were then wounded for the indicated time,

and leaves without wounding were used as controls (W-). Total RNAs were extracted

re

and analyzed by real-time RT-PCR. The expression levels of IbFBK in W- were treated as one to normalize those treated with wounding. The expression levels of

lP

IbActin were used as internal controls. Data were represented as means  SD (n=3).

Jo

ur

na

Asterisk indicated significant difference to W- (p<0.05; t-test).

33

ro of

Fig. 3. Interaction between miR2111 and IbFBK

-p

(A) Schematic representation of the sequence complementary between miR2111 and IbFBK. The arrow indicated the miR2111-induced cleavage site, confirmed by 5’-

re

RLM RACE, in IbFBK mRNA. The cleavage site was between the tenth and eleventh nucleotides from the 5’ end of miR2111. The numbers above the arrow represent

lP

independent experimental results from 5’ RLM-RACE (same results/ total results). (B) and (C) Agro-infiltration of miR2111 and IbFBK in tobacco (Nicotiana Tabacum

na

cv W38). Tobacco leaves were infiltrated with agrobacteria carrying vectors containing 35S:FBK (IbFBK) and 35S:pre-miR2111 (pre-miR2111) or an empty vector

ur

(EV2300). After three days, the total RNAs of these leaves were extracted for real-

Jo

time RT-PCR analysis. The expression levels of NPTII and IbActin were used as internal controls. The expression levels of IbFBK or pre-miR2111 in EV2300/IbFBK group were treated as one to normalize pre-miR2111/IbFBK group. Data were represented as means  SD (n=3). Asterisks indicate significant difference between pre-miR2111/IbFBK and EV2300/IbFBK ( p<0.05; t-test).

34

Fig. 4. Expression levels of pre-miR2111, miR2111 and IbFBK after wounding in

ro of

transgenic sweet potatoes overexpressing pre-miR2111.

-p

(A) The expression of pre-miR2111 in WT and transgenic sweet potatoes, OE-2 and OE-5, after wounding for four hours. The expression levels of IbActin were used as

re

internal controls. (B) The expression of miR2111 in WT, OE-2, and OE-5 after

lP

wounding for four hours. The expression levels of 5.8S were used as internal controls. (C) The expression of IbFBK in WT, OE-2, and OE-5 after wounding for four hours. The expression levels of IbActin were used as internal controls. The third fully-

na

expanded leaves of WT and transgenic sweet potatoes were wounded for four hours. Total RNAs were extracted and analyzed by real-time RT-PCR. The expression levels

ur

of genes in WT were treated as one to normalize transgenic plants. Data were

Jo

represented as means  SD (n=3). Asterisks indicated significant difference between transgenic plants and WT (p<0.05; t-test).

35

ro of

Fig. 5. Interaction of IbFBK and IbSKP1.

(A) Schematic representation of the full length IbFBK and IbFBK lacking F-box

-p

domain (IbFBKF-box). The open square indicates the F-box domain and the closed square is the kelch-repeat domain. (B) Yeast two-hybrid assays to analyze the

re

interaction of IbFBK and IbSKP1. BD: empty bait vector; IbFBK: bait vector inserted

lP

with the full length IbFBK; IbFBKF-box: bait vector inserted with IbFBKF-box; AD: empty prey vector; IbSKP1: prey vector inserted with the full length IbSKP1. Yeasts were cultured on SD/-Leu-Trp, SD/-Leu-Trp-His and SD/-Leu-Trp-His-Ade plates,

na

respectively. 10-1 indicates the concentration of yeast was ten times diluted from 100, and 10-2 indicates one hundred times diluted from 100. Murine p53 and SV40 large T-

ur

antigen were used as positive controls [66, 67]. (C) Subcellular localization of IbFBK

Jo

and IbSKP1. GFP-IbFBK and IbSKP1-GFP expressed transiently in Arabidopsis protoplasts, and the fluorescence of GFP was observed by confocal microscope. NLSmCherry indicates the localization of nucleus. (D) BiFC assay to detect the interaction of IbFBK and IbSKP1 in Arabidopsis protoplasts. IbFBK-YN: pEarleyGate201 inserted with IbFBK; IbSKP1-YC: pEarleyGate202 inserted with IbSKP1; YN: empty pEarleyGate201; YC: empty pEarleyGate202. IbFBK-YN and/or IbSKP1-YC 36

expressed transiently in Arabidopsis protoplasts and the fluorescence of YFP was

Jo

ur

na

lP

re

-p

ro of

observed by confocal microscope. NLS-mCherry indicates the localization of nucleus.

37

ro of

Fig. 6. Interaction of IbFBK and IbCNR8.

(A) Yeast two-hybrid assay to analyze the interaction of IbFBK and IbCNR8. BD:

box:

-p

empty bait vector; IbFBK: bait vector inserted with the full length IbFBK; IbFBKFbait vector inserted with IbFBKF-box; AD: empty prey vector; IbCNR8: prey

re

vector inserted with the full length IbCNR8. Yeasts were cultured on SD/-Leu-Trp, SD/-Leu-Trp-His and SD/-Leu-Trp-His-Ade plates, respectively. 10-1 indicates the

lP

concentration of yeast was ten times diluted from 100, and 10-2 indicates one hundred times diluted from 100. Murine p53 and SV40 large T-antigen were used as positive

na

controls [66, 67]. (B) BiFC assay to detect the interaction of IbFBK and IbCNR8 in Arabidopsis protoplasts. IbFBK-YN: pEarleyGate201 inserted with IbFBK; IbCNR8-

ur

YC: pEarleyGate202 inserted with IbCNR8; YN: empty pEarleyGate201; YC: empty

Jo

pEarleyGate202. IbFBK-YN and IbCNR8-YC expressed transiently in Arabidopsis protoplasts and the fluorescence of YFP was observed by confocal microscope. NLSmCherry indicates the localization of nucleus.

38

Fig. 7. Subcellular localization of IbCNR8 and the interaction of IbFBK and

ro of

IbCNR8 in tobacco leaves.

BiFC assay to detect the interaction of IbFBK and IbCNR8 in tobacco leaves.

-p

IbCNR8-GFP: subcellular localization of IbCNR8 in tobacco leaves (Nicotiana

re

benthamiana). IbFBK-YN: pEarleyGate201 inserted with IbFBK; IbFBKF-box-YN: pEarleyGate201 inserted with IbFBKF-box; MG132: a 26S proteasome inhibitor used

lP

to prohibit the degradation of IbCNR8-GFP. IbFBK-YN and IbCNR8-GFP coexpressed transiently in tobacco leaves by agroinfiltration with or without MG132

na

treatment, and IbFBKF-box-YN and IbCNR8-GFP co-expressed transiently in tobacco leaves without MG132. The fluorescence of GFP was observed by fluorescence

Jo

ur

microscope. NLS-mCherry indicates the localization of nucleus.

39

Fig. 8. Proposed model of miR2111-dependent signaling pathway upon

-p

ro of

wounding.

re

Wounding repressed the expression of miR2111 precursor (pre-miR2111), and induced

lP

the processing of pre-miR2111 to become miR2111. The decrease of miR2111 resulted in the increase of IbFBK transcripts. IbFBK interacted with IbSKP1 by the F-box of IbFBK to form part of SCF complex, and recognized IbCNR8 through the kelch-

na

repeat domain. The interaction of SCF complex and IbCNR8 caused the degradation of IbCNR8 by adding ubiquitin (ub). MG132 is a 26S proteasome inhibitor. The SCF

Jo

ur

complex model was modified from the previous study in Arabidopsis [55].

40