Study on the Interaction of F-box Protein FKF1 and Transcription Factor FUL in Regulating Flowering in Arabidopsis

doi:10.13560/j.cnki.biotech.bull.1985.2021-0499

Abstract

Abstract:

F-box protein FLAVIN-BINDING KELCH REPEAT F-BOX 1（FKF1）is involved in regulating photoperiod flowering in Arabidopsis;however,the underling molecular mechanism is not fully understood yet. Here,we confirmed that FKF1 interacted with transcription factor FRUITFULL（FUL）in vivo and in vitro. qRT-PCR and Western blot results indicated that FKF1 positively regulated the FUL expression in transcriptional level,but had no influence on the stability of FUL protein. Genetic analyses indicated that the flowering phenotype of 35S-FKF1-Myc/ful-8 double mutants and the transcription level of flowering integrator gene FLOWERING LOCUS T（FT）in them were consistent to that in the ful-8 mutant. Collectively,these findings indicate that FKF1 interacts with FUL and functions the upstream of FUL to promote FT expression,thereby promoting flowering.

Key words: flowering, FKF1, FUL, Arabidopsis

ZHOU Juan, YAN Jin-dong, LI Xin-mei, LIU Xue-qing, ZHAO Qiang, ZHAO Xiao-ying. Study on the Interaction of F-box Protein FKF1 and Transcription Factor FUL in Regulating Flowering in Arabidopsis[J]. Biotechnology Bulletin, 2022, 38(3): 1-8.

Figures/Tables 6

Table 1 Candidate interaction proteins of FKF1

基因座Locus	蛋白名称Name of protein	分值Score	功能Function
AT1G22770	Gigantea protein（GI）	0.954	Long day pathway
AT1G04400	Cryptochrome 2（CRY2）	0.952	Light perception
AT5G60910	AGAMOUS-like 8（FUL）	0.946	Floral promoter
AT3G42830	RING/U-box superfamily protein（RBX1b）	0.938
AT2G45660	AGAMOUS-like 20（SOC1）	0.9	Floral promoter
AT5G20570	RING-box 1（RBX1a）	0.876
AT5G15840	B-box type zinc finger protein with CCT domain（CO/BBX1）	0.87	Long day pathway
AT1G10940	Protein kinase superfamily protein（SNRK1A）	0.766
AT2G32950	Transducin/WD40 repeat-like superfamily protein（COP1）	0.726
AT1G09570	Phytochrome A（PhyA）	0.614	Light perception
AT2G18790	Phytochrome B（PhyB）	0.57	Light perception
AT5G11260	Basic-leucine zipper（bZIP）transcription factor family protein（HY5）	0.512
AT4G16250	Phytochrome D（PhyD）	0.87	Light perception
AT2G25930	Hydroxyproline-rich glycoprotein family protein（ELF3）	0.878	Circadian clock
AT2G46830	Circadian clock associated 1（CCA1）	0.608	Circadian clock
AT1G09530	Phytochrome interacting factor 3（PIF3）	0.6	Light signaling
AT5G61380	CCT motif-containing response regulator protein（TOC1）	0.888	Circadian clock

Fig.1 Yeast two-hybrid assay showing the interactions of FKF1 with FUL and GI

Fig. 2 BiFC and Co-IP assays showing FKF1 interaction with FUL A：BiFC assay showing the interaction of FKF1 with FUL in nucleus in Arabidopsis protoplast. Bar = 50μm. B：Co-IP assay showing the interaction of FKF1 with FUL

Fig. 3 Effect of FKF1 on FUL protein stability A：Semi-in vivo stability analysis of FUL protein. Escherichia coli-purified GST-FUL protein was mixed with total protein from the 12 d seedlings of wild-type Col,fkf1-1 or fkf1-t in equal proportion,and incubated for the given time. Sampled,and anti-GST antibody was detected by Western blotting. Ponceau staining was used as an internal standard. Three independent experiments were conducted,showing similar results. B：Protein level of GST-FUL in（A）. GST-FUL protein level was normalized to Ponceau. The value of the starting point was set to 1. Bars refer to the standard deviations of three biological replicates. C：In vivo stability analysis of FUL protein. The FUL-Flag was co-expressed with increasing amounts of FKF1-Myc in tobacco leaves,and the total protein was extracted. The anti-Flag and anti-Myc proteins were detected by Western blotting,respectively. Numbers indicate the ratios of the concentrations of Agrobacteria used in co-infiltration. Ponceau staining was used as an internal standard. Three independent experiments were conducted,showing similar results

Fig. 4 FUL mRNA expression regulated by FKF1 A：mRNA expression of FUL in the wild-type Col and fkf1-1 mutant. B：mRNA expression of FKF1 in the wild-type Col and ful-8 mutant. Plants grew on 1/2 MS solid medium under long-day conditions（LDs）,and aboveground seedlings were collected for quantitative real-time PCR（qRT-PCR）analysis. ACTIN2 served as the internal control. Error bars refer to the standard deviations of three biological replicates. Significant differences are indicated：**P<0.01,***P<0.001（Tukey's least significant difference test）

Fig. 5 Flowering phenotype of Col,35S-FKF1-Myc,ful-8 and 35S-FKF1-Myc / ful-8 plants A：Images of 40 d plants grown in soil under LDs. B：The days of plant sowed to bolting and blossoming of the respective genotypes. C：Number of rosette leaves at bolting of the respective genotypes. D：mRNA expression levels of FT in the respective genotypes. Plants grew in 1/2MS solid medium under LDs,samples were collected at every 4 h on day 17 for qRT-PCR analysis. ACTIN2 served as the internal control. Error bars refers to the standard deviations of three biological replicates. The white/black bars refer to light/dark phases. The time of collecting first sample is set as 0 h. Error bar refers to standard deviations（n≥10）. Significant differences are indicated：*P<0.05 and **P<0.01（Tukey's least significant difference test）

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