AaZFP3, a Novel CCCH-Type Zinc Finger Protein from Adonis amurensis, Promotes Early Flowering in Arabidopsis by Regulating the Expression of Flowering-Related Genes

CCCH-type zinc finger proteins (ZFP) are a large family of proteins that play various important roles in plant growth and development; however, the functions of most proteins in this family are uncharacterized. In this study, a CCCH-type ZFP, AaZFP3, was identified in the floral organ of Adonis amurensis. Quantitative real-time PCR (qPCR) analysis revealed that AaZFP3 was widely expressed in the flowers of A. amurensis. Subcellular localization analysis showed that the AaZFP3 protein was mainly localized to the cytoplasm in tobacco and Arabidopsis. Furthermore, the overexpression of AaZFP3 promoted early flowering in Arabidopsis under both normal and relatively low-temperature conditions. RNA-sequencing and qPCR analyses revealed that the expression of multiple key flowering-time genes was altered in transgenic Arabidopsis overexpressing AaZFP3 compared to wild-type. Of these genes, FLOWERING LOCUS T (AtFT) expression was most significantly up-regulated, whereas FLOWERING LOCUS C (AtFLC) was significantly down-regulated. These results suggest that the overexpression of AaZFP3 promotes early flowering in Arabidopsis by affecting the expression of flowering-time genes. Overall, our study indicates that AaZFP3 may be involved in flowering regulation in A. amurensis and may represent an important genetic resource for improving flowering-time control in other ornamental plants or crops.


Introduction
Zinc finger proteins (ZFPs) are a superfamily involved in many important biological processes in plant growth, development, and stress responses [1]. The zinc finger motif is a local peptide structure stabilized by the coordination of zinc ions by cysteines and histidines; this motif is conserved across ZFPs and functions as part of the DNA or RNA binding domain or protein-protein interaction domain [2]. CCCH-type ZFPs (also known as C3Htype ZFPs) are a group of ZFPs that usually contain one to six tandemly arranged CCCHtype zinc finger motifs comprising three cysteine ("CCC") residues and one subsequent histidine ("H") residue [3,4]. A consensus sequence for CCCH-type motifs was defined as CX 4-17 -CX 4-6 -CX 3 -H (where X represents any amino acid) based on whole-genome analysis of CCCH-type ZFP proteins in Arabidopsis thaliana and Oryza sativa [4]. To date, 68, 67, 68, 69, and 53 CCCH-type ZFP proteins have been identified in Arabidopsis, O. sativa, Zea mays, Vitis vinifera, and Hordeum vulgare, respectively [4][5][6][7]. However, the characterization of their functions is very limited.
In the model plant Arabidopsis, the functions of several members of the CCCH-type ZFP family have been reported [8]. For example, Arabidopsis AtHUA1, a CCCH-type ZFP with six tandem zinc-finger (TZF) motifs, has been identified as an RNA-binding protein that may be involved in flower development [9]. The CCCH-type ZFP AtC3H23/AtTZF1 Int. J. Mol. Sci. 2022, 23, 8166 2 of 13 traffics between the nucleus and cytoplasmic foci and can bind both DNA and RNA and is likely involved in gibberellin/abscisic acid-mediated developmental and environmental responses [10]. AtC3H20/AtTZF2 and AtC3H49/AtTZF3 were reported to be involved in abscisic acid and jasmonic acid responses by affecting mRNA turnover processes [11]. AtC3H14 and AtC3H15 play roles in the regulation of secondary wall thickening and anther development by participating in both transcriptional and post-transcriptional processes [12]. AtC3H36/AtKHZ1 [CCCH zinc-finger and K homology (KH) domain proteins] and AtC3H52/AtKHZ2 both possess transactivation activities and RNA-binding abilities and have redundant roles in the regulation of flowering and senescence in Arabidopsis [13]. AtC3H17, a non-tandem CCCH-type ZFP, functions as a nuclear transcriptional activator and has pleiotropic effects on vegetative, flowering, seed development, and salt stress responses [14,15]. Taken together, these studies indicate the importance of CCCH-type ZFPs for a variety of processes in Arabidopsis; however, the functions of most members of this ZFP family remain uncharacterized.
In addition, the functions of only a few members of the CCCH-type ZFP family have been studied in other plants. For example, the O. sativa CCCH-type ZFP, OsDOS, was reported to be involved in delaying leaf senescence [16]. GhZFP1 from Gossypium hirsutum was found to enhance salt stress tolerance and fungal disease resistance in transgenic tobacco [17]. IbC3H18 from Ipomoea batatas functions as a nuclear transcriptional activator and enhances abiotic stress tolerance [18]. These studies suggest broad roles for CCCH-type ZFPs in plant growth and development.
In both Arabidopsis and V. vinifera, CCCH-type ZFP3 is a member of a ZFP family that contains five TZF motifs [4,5]; however, regarding its function, it is completely unknown. In our previous study, a CCCH-type ZFP member, AaZFP3, was identified from the floral organ of Adonis amurensis [19], suggesting that it may play an important role in flowering regulation. A. amurensis is a perennial herbaceous flower in the family Ranunculaceae, which is naturally distributed in northeast China. A. amurensis can blossom naturally under extremely low temperatures (day/night temperatures between −15 and 10 • C) [19]. Therefore, it is the ideal plant species to study flowering control at extremely low temperatures. In this study, the function of AaZFP3 in flowering regulation was further investigated in Arabidopsis through transgenic experiments. Our results revealed that overexpression of AaZFP3 promotes early flowering in Arabidopsis under both normal (22 • C) and relatively low temperature (18 • C) conditions, which may be related to the altered expression of flowering time-related genes.

Sequence and Expression Analysis of AaZFP3
The full-length open reading frame (ORF) of AaZFP3 consisted of 1188 bp, encoding 395 amino acids. Sequence analysis showed that AaZFP3 contains five conserved C-x8-C-x5-C-x3-H motifs ( Figure 1A). The phylogenetic and conserved motif analyses revealed that AaZFP3 was more similar to NnZFP3 in Nelumbo nucifera ( Figure 1B,C). In the Arabidopsis CCCH-type ZFP family, it is most similar to AtC3H3 ( Figure 1B,C). In terms of its expression profile, AaZFP3 exhibited the highest expression in A. amurensis flowers, followed by leaves and stems, with the lowest expression in roots (Figure 2A). During the six stages of flower development, AaZFP3 expression was higher in flowers at the visible color alabastrum (VCA) and the early flowering stage (EFS) ( Figure 2B). Moreover, AaZFP3 expression was higher in the stamens and calyx than in the petals and pistil in the floral organs of A. amurensis ( Figure 2C). The subcellular localization of AaZFP3 was investigated using transient and stable expression methods involving green fluorescent protein (GFP) fusion. In tobacco leaf epidermal cells, the AaZFP3-GFP fluorescence signal was similar to the control GFP fluorescence signal ( Figure 2D,E). In the stem cells of transgenic Arabidopsis, the AaZFP3-GFP signal was mainly distributed in the cytoplasm and did not appear to overlap with the nuclear dye DAPI ( Figure 2F). These results suggest that AaZFP3 is primarily localized in the cytoplasm. was similar to the control GFP fluorescence signal ( Figure 2D,E). In the stem cells of transgenic Arabidopsis, the AaZFP3-GFP signal was mainly distributed in the cytoplasm and did not appear to overlap with the nuclear dye DAPI ( Figure 2F). These results suggest that AaZFP3 is primarily localized in the cytoplasm.

Effect of AaZFP3 Overexpression on Flowering Time in Arabidopsis
To investigate the function of AaZFP3, we generated transgenic Arabidopsis overexpressing AaZFP3 driven by the 35 S promoter ( Figure 3A). Soil-grown, mature transgenic

Effect of AaZFP3 Overexpression on Flowering Time in Arabidopsis
To investigate the function of AaZFP3, we generated transgenic Arabidopsis overexpressing AaZFP3 driven by the 35 S promoter ( Figure 3A). Soil-grown, mature transgenic

Effect of ZFP3 Overexpression on the Expression of Flowering-Related Genes in Arabidopsis
RNA-sequencing (RNA-seq) was performed to identify differentially expressed genes (DEGs) between WT and transgenic Arabidopsis plants. Four samples from each condition were sequenced. The correlation matrix and principal component analysis based on gene expression demonstrated that the samples clustered by condition, as expected ( Figure 5A,B). DEGs were screened using the transcripts per million (TPM) method with a false discovery rate (FDR) threshold of 0.05 and an absolute log2 ratio of 2. In total, we identified 1364 DEGs, of which 903 were down-regulated and 461 were up-regulated in transgenic Arabidopsis compared to WT ( Figure 5C). Gene Ontology (GO) enrichment analysis of these DEGs identified eight over-represented flower-development-related GO terms, which contained a total of 25 genes related to flowering or flower development ( Figure 5D; Table S1). Furthermore, the majority of DEGs included under the "flower development" or "positive regulation of flower development" GO terms exhibited differential expression profiles in WT versus transgenic Arabidopsis plants ( Figure 6A,B). Under the "positive regulation of flower development" GO term, four DEGs were identified, including FLOWERING LOCUS T (FT), flowering promoting factor 1 (FPK1), AGAMOUS-LIKE6 (AGL6), and CAULIFLOWER (CAL) ( Figure 6B). Quantitative real-time PCR (qPCR) confirmed the reliability of the RNA-seq data ( Figure 6C). Interestingly, both RNA-seq

Effect of ZFP3 Overexpression on the Expression of Flowering-Related Genes in Arabidopsis
RNA-sequencing (RNA-seq) was performed to identify differentially expressed genes (DEGs) between WT and transgenic Arabidopsis plants. Four samples from each condition were sequenced. The correlation matrix and principal component analysis based on gene expression demonstrated that the samples clustered by condition, as expected ( Figure 5A,B). DEGs were screened using the transcripts per million (TPM) method with a false discovery rate (FDR) threshold of 0.05 and an absolute log2 ratio of 2. In total, we identified 1364 DEGs, of which 903 were down-regulated and 461 were up-regulated in transgenic Arabidopsis compared to WT ( Figure 5C). Gene Ontology (GO) enrichment analysis of these DEGs identified eight over-represented flower-development-related GO terms, which contained a total of 25 genes related to flowering or flower development ( Figure 5D; Table S1). Furthermore, the majority of DEGs included under the "flower development" or "positive regulation of flower development" GO terms exhibited differential expression profiles in WT versus transgenic Arabidopsis plants ( Figure 6A,B). Under the "positive regulation of flower development" GO term, four DEGs were identified, including FLOWERING LOCUS T (FT), flowering promoting factor 1 (FPK1), AGAMOUS-LIKE6 (AGL6), and CAULIFLOWER (CAL) ( Figure 6B). Quantitative real-time PCR (qPCR) confirmed the reliability of the RNAseq data ( Figure 6C). Interestingly, both RNA-seq and qPCR detected significantly higher (20 folds) expression of FT in transgenic Arabidopsis plants than in WT plants, and the fold change of FT expression was significantly higher than that of other DEGs ( Figure 7A). We also detected up-regulation (2.5 folds) of SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (AtSOC1) (Figure 7A), an integrator of the Arabidopsis flowering pathway. In addition, significantly lower expression of two flowering suppression-related genes, FLOWERING LOCUS T (FLC) and AT-hook motif nuclear localized 22 (AHL22), was observed in transgenic Arabidopsis plants compared to WT plants ( Figure 7B). These results suggest that the overexpression of AaZFP3 affects the expression of flowering-related genes, which may contribute to the early flowering phenotype observed in AaZFP3-overexpressing Arabidopsis.  Figure 7A). We also detected up-regulation (2.5 folds) of SUP-PRESSOR OF OVEREXPRESSION OF CONSTANS1 (AtSOC1) ( Figure 7A), an integrator of the Arabidopsis flowering pathway. In addition, significantly lower expression of two flowering suppression-related genes, FLOWERING LOCUS T (FLC) and AT-hook motif nuclear localized 22 (AHL22), was observed in transgenic Arabidopsis plants compared to WT plants ( Figure 7B). These results suggest that the overexpression of AaZFP3 affects the expression of flowering-related genes, which may contribute to the early flowering phenotype observed in AaZFP3-overexpressing Arabidopsis.    In addition, whether AaZFP3 binds to the promoter (Pro) of AtFT was investigat using yeast one hybrid (Y1H). Y1H assays showed that when fused pAbAi-ProAtFT w coexpressed with pGADT7-AaZFP3 in yeast, the yeast strains could not grow on SD Ura/-Leu plates with AbA ( Figure 8). This result suggests that AaZFP3 does not activa the promoter of the AtFT gene, and that it may affect AtFT expression in other ways.  In addition, whether AaZFP3 binds to the promoter (Pro) of AtFT was investigated using yeast one hybrid (Y1H). Y1H assays showed that when fused pAbAi-ProAtFT was coexpressed with pGADT7-AaZFP3 in yeast, the yeast strains could not grow on SD/-Ura/-Leu plates with AbA ( Figure 8). This result suggests that AaZFP3 does not activate the promoter of the AtFT gene, and that it may affect AtFT expression in other ways. In addition, whether AaZFP3 binds to the promoter (Pro) of AtFT was investigated using yeast one hybrid (Y1H). Y1H assays showed that when fused pAbAi-ProAtFT was coexpressed with pGADT7-AaZFP3 in yeast, the yeast strains could not grow on SD/-Ura/-Leu plates with AbA ( Figure 8). This result suggests that AaZFP3 does not activate the promoter of the AtFT gene, and that it may affect AtFT expression in other ways.
In addition, whether AaZFP3 binds to the promoter (Pro) of AtFT was investigat using yeast one hybrid (Y1H). Y1H assays showed that when fused pAbAi-ProAtFT w coexpressed with pGADT7-AaZFP3 in yeast, the yeast strains could not grow on SD Ura/-Leu plates with AbA ( Figure 8). This result suggests that AaZFP3 does not activa the promoter of the AtFT gene, and that it may affect AtFT expression in other ways. Figure 8. Yeast one hybrid (Y1H) assay of AaZFP3 binding to AtFT promoter. The full lengths of AaZFP3 and AtFT promoter (1907 bp) were constructed pGADT7 and pAbAi vectors, respectively, and they were subsequently cotransformed with yeast strain Y1HGold. Transformed yeast was grown in SD/-Ura-Leu media with or without AbA (0.6 mg L −1 ). The pGAD-p53 + p53-pAbAi was used as a positive control. Pro: promoter.

Discussion
Several studies have demonstrated important roles for CCCH-type ZFPs in flowering regulation. For example, the overexpression of AtC3H17, AtC3H36/AtKHZ1, and AtC3H52/AtKHZ2 promoted early flowering in Arabidopsis [13,14], while the overexpression of AtC3H23/AtTZF1 delayed flowering [20,21]. Notably, AtC3H17, AtC3H36/AtKHZ1, and AtC3H52/AtKHZ2 were found to be specifically localized to the nucleus, whereas AtC3H23/AtTZF1 was found to traffic between the nucleus and cytoplasmic foci [10,13,14]. The localization of CCCH-type ZFPs in the nucleus or cytoplasm may be related to their propensity to bind DNA or RNA. These studies suggest that different members of the CCCH-type ZFP family may regulate flowering regulation via different mechanisms, including transcriptional and post-transcriptional regulation through DNA or RNA binding.
AtFLC and AtFT are two key genes involved in flowering regulation in Arabidopsis [22]. AtFLC directly represses AtFT expression during vegetative phases to inhibit the transition to flowering [23,24], and AtFT represses AtFLC expression in seedlings and leaves to promote flowering [25,26]. In our study, the overexpression of AaZFP3 promoted early flowering in Arabidopsis, and the expression of AtFT in transgenic Arabidopsis plants was significantly higher than in WT plants, whereas AtFLC expression was significantly lower. Thus, we propose that the early flowering phenotype caused by AaZFP3 overexpression may be driven by the increased AtFT expression and/or decreased AtFLC expression ( Figure 9). This is similar to previous studies that showed that the overexpression of two CCCH-type ZFPs, AtTZF1 and MsZFN, from Arabidopsis and Medicago sativa, respectively, delayed flowering in transgenic Arabidopsis by enhancing AtFLC expression and decreasing AtFT expression [20,27]. Furthermore, recent reports indicate that AtC3H36/AtKHZ1 and AtC3H52/AtKHZ2 can promote flowering by repressing the splicing efficiency of AtFLC pre-mRNA [28]. These findings suggest that some members of CCCH-type ZFPs are involved in flowering regulation by altering flowering-time gene expression via DNA or RNA binding.
In addition to AtFT, AtFLC suppresses AtSOC1 expression by directly binding to its promoter [29]. In contrast to the significant up-regulation of AtFT, AtSOC1 expression was only weakly up-regulated in transgenic Arabidopsis overexpressing AaZFP3. Furthermore, the repressor of AtFT expression, AtAHL22 [30], was significantly suppressed in transgenic Arabidopsis ( Figure 7B). Taken together, these results suggest that the overexpression of AaZFP3 likely promotes early flowering in Arabidopsis primarily by enhancing AtFT expression rather than by inhibiting AtFLC expression. Enhanced expression of AtFT may further affect the expression of genes involved in flower development regulation, such as SHOOT MERISTEMLESS (AtSTM; regulates stem meristem development [31]), SHATTER-PROOF (AtSHP; controls fruit development and seed dispersal in Arabidopsis [32,33]), and the ABCDE model genes (APETALA1 (AtAP1), AtAP3, PISTILLATA (AtPI), SEPALLATA1 (AtSEP1), AtSEP2, and AtSEP3; control the development of Arabidopsis floral organs [34]) ( Figure 9). In addition to AtFT, AtFLC suppresses AtSOC1 expression by directly binding to its promoter [29]. In contrast to the significant up-regulation of AtFT, AtSOC1 expression was only weakly up-regulated in transgenic Arabidopsis overexpressing AaZFP3. Furthermore, the repressor of AtFT expression, AtAHL22 [30], was significantly suppressed in transgenic Arabidopsis ( Figure 7B). Taken together, these results suggest that the overexpression of AaZFP3 likely promotes early flowering in Arabidopsis primarily by enhancing AtFT expression rather than by inhibiting AtFLC expression. Enhanced expression of AtFT may further affect the expression of genes involved in flower development regulation, such as SHOOT MERISTEMLESS (AtSTM; regulates stem meristem development [31]), SHAT-TERPROOF (AtSHP; controls fruit development and seed dispersal in Arabidopsis [32,33]), and the ABCDE model genes (APETALA1 (AtAP1), AtAP3, PISTILLATA (AtPI), SEP-ALLATA1 (AtSEP1), AtSEP2, and AtSEP3; control the development of Arabidopsis floral organs [34]) ( Figure 9).
In our study, AaZFP3 was mainly localized to the cytoplasm in tobacco and Arabidopsis by transient and stable expression experiments ( Figure 2E,F). This result is highly similar to the localization of AtC3H49/AtTZF3 and AtC3H20/AtTZF2 in tobacco [11]. AtC3H49/AtTZF3 and AtC3H20/AtTZF2 contain two TZF motifs, and their recombinant proteins displayed Rnase activity in vitro, suggesting that they may be involved in the mRNA turnover process [11]. Furthermore, the Y1H assays showed that AaZFP3 does not activate the promoter of AtFT (Figure 8). Together with our results, these findings suggest that AaZFP3 is most likely an RNA-binding protein localized in the cytoplasm. However, whether AaZFP3 binds to the pre-mRNA or mRNA of AtFT requires further investigation. In our study, AaZFP3 was mainly localized to the cytoplasm in tobacco and Arabidopsis by transient and stable expression experiments ( Figure 2E,F). This result is highly similar to the localization of AtC3H49/AtTZF3 and AtC3H20/AtTZF2 in tobacco [11]. AtC3H49/AtTZF3 and AtC3H20/AtTZF2 contain two TZF motifs, and their recombinant proteins displayed Rnase activity in vitro, suggesting that they may be involved in the mRNA turnover process [11]. Furthermore, the Y1H assays showed that AaZFP3 does not activate the promoter of AtFT (Figure 8). Together with our results, these findings suggest that AaZFP3 is most likely an RNA-binding protein localized in the cytoplasm. However, whether AaZFP3 binds to the pre-mRNA or mRNA of AtFT requires further investigation.

Quantitative Real-Time PCR Analysis
Total RNA extraction of the plant samples was performed using TRIzol (9108, TaKaRa, Kusatsu, Japan), and reverse transcription was performed using the PrimeScript RT Kit with gDNA Eraser (RR047A, TaKaRa). qPCR was performed using a CFX96 real-time PCR detection system (Bio-Rad, Hercules, CA, USA) and SYBR Green PCR Master Mix (RR420A, TaKaRa) according to the manufacturer's instructions. AaActin or AtActin2 (At3g18780) was used as a reference gene. Three biological and three technical replicates were performed for each sample. The primers used in this study are listed in Supplementary Table S2.

Gene Cloning, Vector Construction, and Plant Transformation
The ORF of AaZFP3 (GenBank: ON464743) was amplified by PCR and cloned into pBI121 or pBI121-GFP vectors using XbaI and SacI or XbaI and KpnI sites, respectively (with-out the stop codon). The primers used in this study are listed in Supplementary Table S2. The above constructs were confirmed by sequencing and transformed into Agrobacterium tumefaciens strain EHA105 for plant transformation. Transient expression of the pBI121-AaZFP3-GFP construct in tobacco (Nicotiana benthamiana) leaves was performed as previously described [35]. Arabidopsis (Columbia ecotype) transformation of the pBI121-AaZFP3-GFP and pBI121-AaZFP3 constructs was performed using the floral dip method [36]. Transgenic Arabidopsis plants were selected on 1/2 Murashige and Skoog (MS) medium containing 30 µg mL −1 of kanamycin. The expression of AaZFP3 in transgenic Arabidopsis plants was assessed by semi-quantitative reverse transcript PCR analyses. T3-generation Arabidopsis seeds were treated at 4 • C for 2 days and then grown on 1 2 MS medium or soil under a 12 h light/12 h dark photoperiod at 22 or 18 • C for phenotypic analyses. The light intensity of the growth chambers was 150 µE m −2 s −1 .

RNA-Sequencing and Differential Gene Expression Analysis
Total RNA was extracted from both wild-type (WT) and transgenic Arabidopsis flowering plants grown on the 1/2 MS medium using TRIzol reagent (9108, TaKaRa). Four plants were used as biological replicates for each condition. Sequencing was conducted using an Illumina-Seq system using the BGISEQ-500 system (Beijing Genomic Institute, Shenzhen, China). RNA-seq data processing and DEG analysis were conducted as previously described [19]. DEGs were screened using the TPM method with an FDR threshold of 0.05 and an absolute log 2 ratio of 2. Pearson correlation coefficients were calculated between two samples using the cor function in R software. Principle components analysis (PCA) was performed using the princomp function in R software. All DEGs were mapped in GO biological process terms, and significantly over-represented terms were identified based on a corrected p ≤ 0.05. GO enrichment analysis of the DEGs was performed by the GOseq R packages based on Wallenius non-central hyper-geometric distribution as previously described [37].

Y1H Assays
The promoter of AtFT (1907 bp) was PCR amplified and inserted into a pAbAi-BR yeast integrating vector (bait-reporter; Clontech, Palo Alto, Santa Clara, CA, USA) and the AaZFP3 gene was cloned into the pGADT7-rec vector (Clontech). The pAbAi-ProAtFT and pGADT7-AaZFP3 vectors were cotransformed into the yeast Y1HGold strain using a Matchmaker One-Hybrid Library Construction and Screening Kit (Clontech). Cotransformed yeast cells were selected on SD/-Ura-Leu (a synthetic dropout medium lacking Ura and Leu) with or without AbA (0.6 mg L −1 ; Sigma-Aldrich, St. Louis, MO, USA), and were then incubated for 3-5 days at 30 • C. The positive control (pGAD-p53 + p53-pAbAi) was carried out in the same manner. The primers used for these analyses are listed in Supplementary Table S2.

Conclusions
In summary, we identified a CCCH-type ZFP gene, AaZFP3, from the floral organs of A. amurensis. AaZFP3 was mainly localized to the cytoplasm. Overexpression of AaZFP3 promoted early flowering in Arabidopsis, which may be caused by the altered expression of flowering-time genes, especially AtFT. Our results suggest that AaZFP3 may play an important role in regulating flowering in A. amurensis. Furthermore, this gene may represent