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A tandem CCCH type zinc finger protein gene CpC3H3 from Chimonanthus praecox promotes flowering and enhances drought tolerance in Arabidopsis



CCCH-type zinc finger proteins play important roles in plant development and biotic/abiotic stress responses. Wintersweet (Chimonanthus praecox) is a popular ornamental plant with strong resistance to various stresses, which is a good material for exploring gene resource for stress response. In this study, we isolated a CCCH type zinc finger protein gene CpC3H3 (MZ964860) from flower of wintersweet and performed functional analysis with a purpose of identifying gene resource for floral transition and stress tolerance.


CpC3H3 was predicted a CCCH type zinc finger protein gene encoding a protein containing 446 amino acids with five conserved C-X8-C-X5-C-X3-H motifs. CpC3H3 was localized in the cell membrane but with a nuclear export signal at the N-terminal. Transcripts of CpC3H3 were significantly accumulated in flower buds at floral meristem formation stage, and were induced by polyethylene glycol. Overexpression of CpC3H3 promoted flowering, and enhanced drought tolerance in transgenic A. thaliana. CpC3H3 overexpression affects the expression level of genes involved in flower inducement and stress responses. Further comparative studies on physiological indices showed the contents of proline and soluble sugar, activity of peroxidase and the rates of electrolyte leakage were significantly increased and the content of malondialdehyde and osmotic potential was significantly reduced in transgenic A. thaliana under PEG stress.


Overall, CpC3H3 plays a role in flowering inducement and drought tolerance in transgenic A. thaliana. The CpC3H3 gene has the potential to be used to promote flowering and enhance drought tolerance in plants.

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Zinc finger protein is a superfamily with five types (C2H2, C3H, C3HC4, C2HC5 and C3H2C3) of proteins. Typical CCCH type zinc finger protein was defined as the proteins contain 1-6 copy C-X6–14-C-X4–5-C-X3-H type zinc finger motifs. CCCH type zinc finger protein gene was widely exist in plant species, studies have revealed that there are 68 C3H proteins in A. thaliana, 67 in rice, 68 in Zea mays, 34 in Medicago truncatula and 91 in Populus trichocarpa. Functional characterizations showed CCCH type zinc finger proteins play roles in various progresses of plant growth and stress responses.

The transition of vegetative growth to flowering is a key developmental progress in flowering plants which is important not only for environmental adaptation, but also for agricultural productivity. Studies on molecular mechanism and genetic basis of the transition in the model plant A. thaliana found that the transition is regulated by elaborate genetic pathways, namely the photoperiod, vernalization, gibberellic acid (GA), age, autonomous, and ambient temperature signaling pathways, in response to endogenous (age, GA), and environmental (day length, temperature, ambient temperature and stress) stimuli [1, 2]. More than 180 genes participate in floral transition [2], such as genes from various pathways, CONSTANS 1 from photoperiod pathway, SHORT VEGETATIVE PHASE from ambient temperature pathway, VERNALIZATION INSENSITIVE (VINs) from vernalization pathway, The GIBBERELLIN 20 OXIDASE (GA20ox) from gibberellic acid pathway, and signal integrators, FLOWERING LOCUS T (FT), SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1), AGAMOUSLIKE 24 (AGL24), and floral meristem identity genes, APETALA1 (AP1), APETALA2 (AP2), LEAFY (LFY) [3,4,5]. CCCH type zinc finger proteins were also found play roles in floral transition, MsZFN (Medicago sativa zinc finger protein) a gene induced by dark from alfalfa delayed flowering in A. thaliana [6], and AtC3H17 promotes flowering in A. thaliana [7], Ehd4 (Early heading date 4) acts as a critical regulator promoting flowing in rice in photoperiod pathway [8].

As one of the most severe natural stresses, drought affects the productivity and quality of plants [9]. Plants have evolved multifaceted adaptation strategies to recognize and adapt the drought stress at molecular, biochemical, physiological and morphological levels [10]. Numerous stress-induced genes are involved in the recognition and adaption of drought stress. According to the functional annotation, these genes can be divided into two groups, the first class involves in drought response through signal transduction, including the protein kinase genes (MAP kinase, CDB kinase), transcription factors (MYB, NAC, MYC and DREB). The second class involves in drought tolerance as function genes, such as late embryogenesis abundant (LEA) proteins, osmotin proteins, water channel proteins, stomatal movement proteins, sugar and proline transporters, oxidative enzyme, and various proteases [11]. CCCH type zinc finger protein genes were also found play roles in stress response, such as OsC3H10, OsTZF5 (Oryza sativa CCCH-tandem zinc finger protein 5) improve drought tolerance in rice [12, 13], AtSZF1 (Arabidopsis thaliana salt-inducible zinc finger 1), AtSZF2 (Arabidopsis thaliana salt-inducible zinc finger 2), GhZFP1 (Gossypium hirsutum putative CCCH-type zinc finger transcription factor) play role in salt stress response [14, 15].

Few CCCH type zinc finger protein genes have multiple functions in stress response and floral transition. Overexpression of AtZFP1 (Arabidopsis thaliana Zinc finger protein 1) delays floral transition and enhances salt tolerance in A. thaliana [16], and overexpression of AtTZF1 (Arabidopsis thaliana tandem zinc finger protein 1) delays flowering and enhanced cold and drought tolerance [17].

Wintersweet (Chimonanthus praecox) is a popular Chinese endemic shrub with bright yellow flowers and attractive fragrance. It blossoms in hard winter and shows strong stress resistance to drought, cold, heat and waterlogging [18, 19]. It is a good material for exploring stress response genes, but fewer genes from wintersweet were characterized [19,20,21,22]. We obtained a novel tandem CCCH type zinc finger protein gene CpC3H3 (accession number: MZ964860) from flower of wintersweet previously, but is function was still unknown. We aimed to characterize the function of CpC3H3 for identifying stress resistance gene resource.


CpC3H3 isolation and sequence analysis

The CpC3H3 CDS contained 1341 bp that encoded a 446 amino-acid protein with a calculated molecular mass of 48.43 KDa. Sequence analysis showed that five putative conserved C-X8-C-X5-C-X3-H motifs were detected in CpC3H3 (Fig. 1). Multiple sequence alignments performed among CpC3H3 and its homologues from model plants (Arabidopsis thaliana, Oryza sativa, Populus trichocarpa, etc) and other species (Cinnamomum micranthum, Nelumbo nucifera, etc.) showed that they were highly conserved and shared the same numbers and types of the CCCH motifs which were important for their functions (Fig. 1).

Fig. 1
figure 1

Multiple sequence alignment of CpC3H3 and its homologues from A. thaliana, Zea mays, Populus trichocarpa, Cinnamomum micranthum, Nelumbo nucifera, Vitis vinifera, Glycine max, and Camellia sinensis. The blank lines indicate the position of the C-X8-C-X5-C-X3-H type motifs; Red box, white character indicates the strict identity of the residues, red character indicates the similarity residues in a group and the blue frame indicates the similarity residues across groups

The phylogenetic analysis of CpC3H3 and other homologues from various plants showed that the C3H3 proteins were divided into two main branches, proteins from lower plants like Selaginella moelledndorffii, Marchantia polymorpha, and Physcomitrella patents and Monocotyledonous plant, such as Zea mays, Oryza sativa were clustered within one clade, and the Dicotyledonous plant C3H3 proteins were in the other branch where CpC3H3 was belonged to (Fig. 2). This finding indicated that the C3H3 proteins of Dicotyledonous plants may share some distance from that of Monocotyledonous and lower plants which may ultimately lead to the function difference among these plants.

Fig. 2
figure 2

Phylogenetic analysis of CpC3H3 and its homologues from lower plants Selaginella moellendorffii, Physcomitrella patens, Marchantia polymorpha, Monocotyledonous plants Oryza sativa, Zea mays, and Dicotyledonous plants Arabidopsis thaliana, Populus trichocarpa, Cinnamomum micranthum, Nelumbo nucifera, Vitis vinifera, Glycine max, Camellia sinensis, Ricinus comunis, Hevea brasiliensis, Manihot esculenta, Thalictrum thalictroides, Ziziphus jujuba, and Papacer somniferum

Cis-acting elements prediction revealed that 12 hormone responsive elements, including 2 ABRE, 4 MYC ABA responsive elements, 4 MeJA responsive elements, 1 Ethylene responsive elements and 1 Auxin responsive elements, and 10 stress responsive elements, including 2 TC-rich repeats (Cis-acting element involved in defense and stress responsiveness), 1 MBS (myb binding site involved in drought-inducibility), 4 MYB binding site, 1MYBHv1 biding site and 2 ARE (Anaerobic induction) elements and 16 light responsive cis-elements were predicted in the 2138 bp upstream sequence of CpC3H3 (supplementary table S2).

Subcellular localization analysis

The subcellular localization of CpC3H3 was first predicted by online tool, result showed that CpC3H3 was predicted localizing to cell membrane. And to further examine the subcellular localization of CpC3H3, the roots of the transgenic A. thaliana plants carrying 35S:GFP and 35S:CpC3H3-GFP were used to detect the GFP signal respectively. GFP observation n revealed that the GFP signal generated by CpC3H3-GFP was observed in cell membrane which was co-localized with the RFP generated by membrane marker FM4-64 while the control 35S:GFP was in the nucleus and cytoplasm (Fig. 3A), this result indicated that CpC3H3 is mainly located in cell membrane. But Nuclear export signal (NES) prediction showed that there was a putative nuclear export signal (NES: between amino acids 30 to 37) at the N-terminal of CpC3H3 (Fig. 3B). Few other CCCH proteins with NES signal can shuttle from nucleus to other organelles under certain conditions. We assume CpC3H3 might be a shuttling protein under drought stress, so we also detected the subcellular localization of CpC3H3 after PEG treatment, but no significant difference of GFP signal was observed (Fig. 3A).

Fig. 3
figure 3

Subcellular localization analysis of the CpC3H3. A Subcellular localization of the CpC3H3 protein in root of transgenic A. thaliana. 35S-GFP was served as the control. Fluorescence of 35S-GFP was detected throughout the cell while the 35S-CpC3H3-GFP was co-localized with RFP of FM 4-64 in the cell membrane. B Nuclear export signal (NES) was predicted by online tool of NetNES 1.1, putative NES sequence was found between amino acids 30 to 37. Bars = 20 μm

Expression patterns of CpC3H3 in C. praecox

Tissue specific expression analysis in cotyledons, roots, stems, young leaves, mature leaves, petals, stamens and pistils showed that CpC3H3 was widely expressed in tissues of wintersweet, but its expression level is higher in flower organs than in vegetative organs (Fig. 4A). The dynamic expression of CpC3H3 in flower at different developmental stages showed that CpC3H3 was more abundantly expressed in floral meristem formation stages than in other stages (Fig. 4B). The results of induced expression analysis showed that CpC3H3 was induced by PEG, and its expression reached a peak 12 hours after treatment with an expression level 4 times that of untreated. (Fig. 4C).

Fig. 4
figure 4

Expression pattern of CpC3H3 in wintersweet. A, relative expression of CpC3H3 in various tissues of wintersweet; B, expression pattern of CpC3H3 in flower developmental stages; C, relative expression of CpC3H3 in leaves in response to 20% PEG after 0 h, 2 h, 6 h, 12 h, 24 h. FMS, flower meristem formation stage; SDS, sepal primordium deferential stage; PDS, petal primordium deferential stage; StDS, sepal primordium deferential stage, PiDS, Pistil primordium deferential stage; AtDS, anther developmental stage; OvDS, ovule developmental stage; S1, flower bud stage; Stage 2, petal-display; Stage 3, initiating bloom; Stage 4, bloom; Stage 5, early-withering, and Stage 6, late-withering. The * and ** in fig. 4C indicate a significant difference from 0 h at p < 0.05 and p < 0.01, respectively, as determined by the Student t-test. Mean values followed by the same letter are not significantly different according to LSD multiple range test in fig. 4A and Games-Howell test in fig. 4B (P ≤ 0.01) respectively. The value of each bar represents mean ± SE (n = 3) of three replicates

Overexpressing of CpC3H3 promotes flowering in A. thaliana

To investigate the functions of CpC3H3, the CDS of CpC3H3 was introduced into A. thaliana under the control of the 35S promoter. Transgenic lines were confirmed by PCR and qRT-PCR using the DNA and cDNA as the template (supplementary Fig. S1-S2), and the line 10, 14 and 18 were then chosen for further phenotype observation and related gene expression analysis.

To observe the phenotype of the CpC3H3 overexpression (OE) plants, the OE plants and WT plants were raised in a greenhouse under long day condition. We found that, the OE plants required an average of 23.7 days to bolting and 25.7 days from germination to flowering, while WT plants needed 27.1 days and 29.8 days respectively (Fig. 5A, B,D), and the rosette leaves of OE plants were less than that of WT (Fig. 5C). We also detected the expression levels of the key genes involved in flowering, results showed that the expression of the flowering promoter AP1, FT, LFY and SOC1 were upregulated in transgenic plants while the flowering repressor FLC was downregulated (Fig. 5E). These results indicated that CpC3H3 has a role in flowering inducement in A. thaliana.

Fig. 5
figure 5

Overexpression of CpC3H3 promote floral transition. WT, wild type; OE-18, overexpression line-18; OE-10, overexpression line-10; OE-14, overexpression line-14; the error bars represent the standard deviation per triplicate. The * and ** indicate a significant difference from WT at p < 0.05 and p < 0.01, respectively, as determined by the Student’ t-test. Bars = 2 cm

Overexpressing of CpC3H3 enhances drought tolerance in A. thaliana

The expression of CpC3H3 was induced by PEG, CpC3H3 was deemed to play some roles in drought tolerance. Four-week-old WT and transgenic A. thaliana plants grown in soil were treated with 20% PEG6000, 150 mmol/L mannitol and planted without watering to evaluate the drought tolerance. The leaves of wild type plants were severely withered after the treatment, while that of overexpression lines were still robust (Fig. 6A-C).

Fig. 6
figure 6

Overexpression of CpC3H3 enhance drought tolerance. A-C, transgenic and WT A. thaliana plants treated with PEG, natural water deficit and mannitol; D-G, The content of soluble sugar, proline, MDA, activity of POD, electrolyte leakage rates and osmotic potentials in the transgenic A. thaliana plants and WT. The OE-plants and WT plants were treated with PEG after 24 h. OE-18, overexpression line-18; OE-10, overexpression line-10; OE-14, overexpression line-14. the error bars represent the standard deviation per triplicate. The * and ** indicate a significant difference from WT at p < 0.05 and p < 0.01, respectively, as determined by the student’ t-test. Bars = 2 cm

Several physiological indices widely used to evaluate the plant stress response were determined in WT and transgenic plants under normal and drought condition. The content of soluble sugar showed little difference under normal condition but increased more significantly in overexpression strains than in wild type A. thaliana under drought stress (Fig. 6D), and the contents of proline were higher in transgenic plants under both normal and drought condition (Fig. 6E). As a marker for lipid peroxidation, content of MDA reflects the resistance ability of plants to stress, as shown in Fig. 6F, MDA content was higher in WT plants both in normal and drought conditions. As an important ROS scavenging antioxidant enzyme, activity of POD was significantly higher in the transgenic plants in both normal and drought conditions (Fig. 6G). The electrolyte leakage rates were almost the same in both transgenic and WT plants under normal condition, but were significantly lower in CpC3H3-overexpression A. thaliana plants after PEG stress treatments (Fig. 6H). The osmotic potentials showed no significant difference in WT and transgenic plants in normal condition, but significantly higher in transgenic plants after treated with PEG (Fig. 6I). The significant difference of proline, MDA and POD in normal condition, and the higher content of soluble sugar, proline, osmotic potential, activity of POD and lower REL and MDA under drought condition indicated that overexpression of CpC3H3 enhanced the drought tolerance of transgenic A. thaliana.

Identification of genes involved in the CpC3H3-regulated flowering and drought tolerance

To explore the regulatory mechanisms of CpC3H3 mediated floral transition and drought tolerance, RNA sequencing was performed with twenty-day-old WT and OE plants. Genes with more than 2-fold differential expression levels in WT and overexpression lines are identified to be regulated by CpC3H3 overexpression. 79 up- and 44 down-regulated genes were identified in the transgenic plants (Table S3). Since CpC3H3 was mainly expressed in wintersweet flowers and induced by PEG6000, and its overexpressors exhibited early flowering and drought tolerance, we attempted to focus on the genes which were both regulated by CpC3H3 overexpression and play roles in the floral transition or (and) stress tolerance. In terms of flowering, 15 genes, including the GATA transcription factors, bHLH transcription factors, etc., were annotated playing roles in floral transition according to Tair. 7 out of 15 were predicted components of the photoperiod pathway, and 8 of which were hormone related proteins (Table.1). And in the aspect of drought tolerance, 26 protein genes, including transcription factors (e.g.,MYB, APETALA2/ERF, NAC), oxidation-reduction process-related protein (e.g. cytochrome P450 family proteins), protein kinases (e.g.MAP 3 K), Dehydration-responsive protein DREB2A, compatible solute-related protein mannose-binding-lectin1 (MNB1), carbohydrate metabolism–related proteins UDP-glycosyltransferase, E3 ubiquitin-protein ligase ATDIP2 (DNA Binding Protein Interacting Protein 2), cell wall formation related protein pectin methylesterase inhibitor 11 and 13 (PMEI11, PMEI13), transport related protein genes (e.g. PHO1) and stomatal movement protein expansin1 (EXPA1), were annotated corresponding to drought tolerance (Table 1).

Table 1 Differential expression genes related to flowering and drought tolerance

We further confirmed the expression patterns of several genes, Isopentenyltransferase 3 (IPT3), HOMOLOG of BEE2 INTERACTING WITH IBH 1 (HBI1), Arabidopsis Thaliana Response Regulator 5 and 7 (ARR5, ARR7), and Expansin-A1 (EXPA1), randomly picked from the candidates identified in WT and OE plants by qRT-PCR. The analysis showed their expression patterns were similar to the expression data derived from RNA sequencing analysis (Fig. 7).

Fig. 7
figure 7

Expression validation of the genes randomly picked from the RNA-sequencing profile. WT, wild type; OE-18, overexpression line-18; OE-10, overexpression line-10; OE-14, overexpression line-14; the error bars represent the standard deviation per triplicate. The * and ** indicate a significant difference from WT at p < 0.05 and p < 0.01, respectively, as determined by the Student t-test


CpC3H3 is a tandem CCCH zinc finger protein

The C-X8-C-X5-C-X3-H type zinc finger motif was found in most of the CCCH type zinc finger proteins, 44 of 68 A. thaliana CCCH type zinc finger proteins, and 36 of 67 rice CCCH proteins contain the C-X8-C-X5-C-X3-H motif [23], and 82% of Populus CCCH proteins contain the conventional C-X7-8-C-X5-C-X3-H motifs [24]. The C-X8-C-X5-C-X3-H motif may be an ancient CCCH motif. Since CpC3H3 contains 5 tandem C-X8-C-X5-C-X3-H motifs, and results of multiple sequence alignment and phylogenetic analysis showed that CpC3H3 shared high conservation with its homologues, even the homologues from the lower plant physcomitrella patent, Selaginella moelledndorffi, CpC3H3 might be an ancient gene with conserved functions.

CpC3H3 might be a shuttling protein

CCCH-type zinc finger proteins can localize to different position of cells, some proteins can localize to nucleus with transcriptional activities [7, 15, 25], some can localize to plasma membrane [26], some can localize to cytoplasmic [13, 27], and some of them are shuttling proteins [12, 28,29,30]. In our study, subcellular localization analysis showed CpC3H3 was localized in plasma membrane, but NES sequence prediction showed CpC3H3 contained a NES sequence at the N-terminal which showed some similarities with other shuttling CCCH proteins. GhZFP1 which function in salt resistance was located in nuclear but with a NES peptide [15]; AtTZF1 and AtTZF9 (Arabidopsis thaliana tandem zinc finger protein 9) function in stress response can traffic between the nucleus and cytoplasm [31]. These implied that CpC3H3 might be a shuttling protein like other CCCH shuttling proteins, therefore, we detected the subcellular location of CpC3H3 after PEG treatment, but no significant difference was observed, this result showed CpC3H3 can’t shuttling under PEG stress. The subcellular localization property of CpC3H3 is still need to be further explored.

CpC3H3 promote floral transition in A. thaliana

The functions of CCCH proteins are closely related to their expression patterns [12, 13, 31], AtC3H3 is preferentially expressed in vascular tissue, and highly expressed in the secondary wall forming tissues, has a function in cell wall elongation [32], CpCZF1 and CpCZF2 (Chimonanthus praecox C3H-type zinc finger protein gene 1 and 2), which was expressed highly in stamen primordium differentiation stage, affect stamen development [25]. In this study, tissue specific expression analysis revealed that CpC3H3 was highly expressed in the flower organs, and further temporal expression analysis also showed that CpC3H3 was mainly expressed in floral meristem formation stage rather than other flower development stages (Fig. 4). The expression patterns indicate that CpC3H3 could play some roles in floral transition or development. Further functional characterization performed by overexpressing CpC3H3 in A. thaliana showed that the OE lines bolted and flowered earlier and had less rosette leaves, showed upregulated flower promoters and downregulated flower repressor (Fig. 5C), these results implied that overexpression of CpC3H3 promote flowering in A. thaliana. This is highly consistent with the former expression analysis, and shares some similarities with other CCCH-type zinc finger proteins. MsZFN from alfalfa delays flowering in A. thaliana with its transcripts increased under continuous dark conditions [6]. Ehd4, a CCCH type zinc finger protein from rice, showed a diurnal expression pattern which accumulates after dusk and reaching a peak at dawn, and then damping rapidly, regulate flowering in photoperiod pathway [8]. All in all, CpC3H3 which expressed highly in flower of floral meristem formation stage played a role in flowering transition.

CpC3H3 may promote flowering through photoperiod and hormone signal pathways

To explore the regulation pathway that CpC3H3 regulate flowering, transcriptome profile in WT plants and OE plants were analyzed. The transcriptome analysis revealed among the 123 DEGs, 15 genes were functionally associated with flowering (Table 1). Some genes are participating in flowering in Photoperiod pathway, such as Early Phytochrome Responsive1 (EPR1), Brassinosteroid Enhanced Expression1 (BEE1), Arabidopsis Thaliana Response Regulator 4 (ARR4), HBI1. EPR1 is regulated by phytochrom A and phytochrom B, and its overexpressors delayed flowering in A. thaliana [33]; BEE1 is a positive regulator of photoperiod flowering, promote flowering by directly binding to the floral integrator FT [34]; ARR4 is critical for proper circadian period [10]; HBI1, a basic helix-loop-helix protein, was regulated by light and affected flowering when overexpressed in A. thaliana [35]; Nuclear factor YC protein 4 (NFYC4) is required for CONSTANS-mediated, photoperiod-dependent flowering in A. thaliana [36]. And others, such as IPT3, ARRs and GA-STIMULATED ARABIDOPSIS 6 (GASA6), are involved in flowering in hormone signal pathways. Cytokinin (CK) play role in flowering by activating the Twin Sister of FT (TSF) [37]. CK biosynthesis gene IPT3 and receptor gene ARR5, ARR6, ARR7, ARR9, ARR15 were upregulated in overexpression lines. GASA6 is a GA-inducible and ABA-repressible gene which accelerated flowering when overexpressed in A. thaliana [38]. Flowering regulation mechanism of these DEGs implied CpC3H3 may promote flowering through Photoperiod and hormone signal pathways, but further verification is needed.

CpC3H3 enhances drought tolerance in A. thaliana

Cis-acting elements such as ABRE, ERE, MBS and TC-rich repeats are commonly associate with stress response [20, 39], their occurring in the promoter regions implied that CpC3H3 may have some role in stress response. Expression analysis figured out CpC3H3 was induced by PEG, and functional analysis performed by overexpression CpC3H3 in A. thaliana also showed that the OE plants are more tolerance than the WT plants when treated with PEG, this was similar to a lot of drought response genes, such as IbZFP1, RICE CENTRORADIALIS 1 (RCN1), RhEXPA4 [40,41,42]. Content of osmolytes, lipid peroxidation, ROS scavenging ability, electrolyte leakage rates are important reflections of plant stress resistance. Soluble sugar and proline are important osmolytes, osmotic potential could directly reflect the ability of plants to resist drought, MDA is a maker for lipid peroxidation, POD is one of an important ROS scavenging antioxidant enzyme, and electrolyte leakage rate is a reflection of cell death. These physiological indices have been widely used to evaluate the plant stress response. Contents of proline, MDA, activity of POD were significantly different under normal condition which indicated overexpression of CpC3H3 affected these indices and thus enhanced drought tolerance. Further studies also showed contents of proline, soluble sugar and activity of POD were higher, and the content of MDA, osmotic potential and REL was lower in transgenic plants under drought condition. We also tested the osmotic potential of leaves of wintersweet under normal and PEG treatment, the osmotic potential was − 19.5 bars in normal condition, but decreased to − 22 bars after PEG treatment, which showed the same trend with the osmotic potential in transgenic plants. These results implied CpC3H3 plays role in drought tolerance.

CpC3H3 may enhance drought tolerance by regulating the stress responsive genes

In response to drought stress, plants tend to alleviate the drought stress by activating the expression of the regulative and functional genes involved in shutting up stoma, developing roots, osmotic regulation, scavenging of reactive oxygen species, etc. In this study, 26 out of 123 differential expression genes are annotated stress-associated. 15 genes, including MYBs, NAC DOMAIN CONTAINING PROTEIN 90 (NAC90), ERF DOMAIN PROTEIN 9 (ERF9), ETHYLENE-RESPONSIVE ELEMENT BINDING PROTEIN (AtEBP), CYTOKININ RESPONSE FACTOR 2 (CRF2), CALCIUM-DEPENDENT PROTEIN KINASE 29 (CDBK 29), MITOGEN-ACTIVATED PROTEIN KINASE KINASE KINASE 14 (MAP 3 K), PBS1-LIKE PROTEIN 20, DEHYDRATION-RESPONSIVE ELEMENT BINDING PROTEIN 2 (DREB2A), TREHALOSE-6-PHOSPHATE SYNTHASE 11 (AtTPS11), Calcium-transporting ATPase 4, argininosuccinate lyase, DBP-INTERACTING PROTEIN 2 (AtDIP2), were function in stress-related signal transduction [43,44,45]. PHO1, MNB1, and GRANULE BOUND STARCH SYNTHASE 1 (GBSS1) were function in osmotic equilibrium [46, 47]. PMEI11, PMEI13, AtDIP2 play roles in plant cell protection [48, 49]. And the four CYPs were involved in membrane lipid antioxidation. AtEXPA1 is a stomatal opening rate controller of A. thaliana [50]. Functional annotation of these genes indicates that overexpression of CpC3H3 may enhance drought tolerance through regulating the stoma closing, osmotic balance, ROS scavenging and plant cell protecting. Additionally, the results of the determination of the physiological indices strengthened this conjecture.


In conclusion, our results demonstrate that CpC3H3, a tandem CCCH type zinc finger protein with 5 C-X8-C-X5-C-X3-H motifs from wintersweet, was conserved with its homologues from other plants. Subcellular localization analysis showed CpC3H3 was located in cell membrane, but with a NES at the N-terminal which implied CpC3H3 might be a shuttling protein but could not shuttle under PEG treatment. CpC3H3 was expressed highly in flower organs and floral meristem formation stage of wintersweet, and induced by PEG. Overexpression of CpC3H3 caused early flowering and enhanced drought tolerance in A. thaliana. Transcriptome analysis revealed 15 and 26 of 123 DEGs were associated with flowering and stress response respectively. The content of MDA, proline, soluble sugar, osmotic potential, the activity of POD, and the rate of electronic leakage were significantly different in WT and transgenic A. thaliana under drought stress. All the results indicated that overexpression of CpC3H3 promotes floral transition and enhances drought tolerance in A. thaliana. These findings not only extend our understanding of function of the CCCH-type zinc finger proteins, but also provide useful gene resource that can regulate flowering and drought tolerance. Moreover, CpC3H3 is the first functional studied C3H3s, the characterization of CpC3H3 also provide valuable reference for the study of C3H3 gene function in other species.


Plant materials and growth condition

Roots, stems, cotyledons, young leaves, mature leaves, flower organs (petal, stamen, pistil) were collected for detecting the expression level of CpC3H3 in wintersweet. Seedlings of wintersweet were raised in the greenhouse with a relative humidity of 70%, a photoperiod of 16-h light (120 umol m− 2 s− 1, 25 °C) /8-h dark (20 °C) and the adult plants were planted in the campus of southwest university of China. Wintersweet plants were identified and owned by Shunzhao Sui from southwest University of China.

The Arabidopsis thaliana (Col-0) was used for plant transformation and phenotype comparison, and the tobacco (Nicotiana benthamiana) was planted for subcellular localization analysis. The seeds of A. thaliana and tobacco were stored at 4 °C, germinated on the Murashige and Skoog (MS) medium with 3% sucrose and 0.7% agar, then the plants with 4 leaves were transferred to sterile soil and cultivated in a greenhouse with a relative humidity of 70%, a photoperiod of 16-h light (120 umol m− 2 s− 1, 22 °C) /8-h dark (20 °C).

Cloning and sequence analysis

Total RNA was extracted by Trizol reagent (Thermofisher, CN) from the wintersweet flower following the RNA extraction protocol. The cDNA was synthesized with a PrimeScript® II First Strand cDNA Synthesis Kit (TaKaRa, Japan). The cDNA sequence of CpC3H3 was obtained from the transcriptome database of wintersweet flower [51]. The promoter sequence was validated based on the genome sequence of Chimonanthus salicifolius [52]. Primers for CpC3H3-cDNA, CpC3H3-CDS and promoter of CpC3H3 amplifying were designed with primer primer 6.0 based on the transcriptome and genome sequence of CpC3H3 (Table S1).

A BLASTX was performed to search the NCBI database for the homologues of CpC3H3 from the model plants, such as Arabidopsis thaliana, Glycine max, Zea mays, Populus trichocarpa, and several other plants like Cinnamomum micranthum, Nelumbo nucifera, Vitis vinifera, Camellia sinensis. Multiple Sequence Alignment was conducted using online MSA tool ( A neighbor-joining phylogenetic tree was constructed by using the MEGA 6.0 software with a bootstrap of 1000 replicates. Putative cis-acting elements were predicted by the online tool PlantCARE (

Expression analysis

Total RNA extracted from various tissues was reverse-transcribed following the instructions of the primerscript RT reagent kit (Takara, Japan) to generate cDNA samples. Then quantitative real time PCR amplifications were conducted with the specific primers and cDNA template. Wintersweet CpTublin, CpActin genes, and A. thaliana AtActin gene were used as internal control for the expression analysis respectively. The gene expression was quantified by the comparative CT method as previously used [25]. Total RNA extracted from various tissues (cotyledons, roots, stems, young leaves, mature leaves, petals, stamens and pistils) and from flower bud at floral meristem formation stage, flower organ primordium formation stages (sepal, petal, stamen, and pistil) and flowering stages [25] (Stage 1-6: bud-display-initiating bloom-bloom-early withering-late withering) were used to analyze the spatiotemporal expression of CpC3H3 in wintersweet. Total RNA extracted from 8-week wintersweet seedlings at 0 h, 2 h, 6 h, 12 h, 24 h after treated with 20% polyethylene glycol (PEG) 6000 were used to analyze the induced expression pattern. Total RNA of twenty-day-old A. thaliana seedlings were used to analysis the expression pattern of the differential expression genes.

Vector construction and plant transformation

The CpC3H3 CDS without the stop codon was cloned into the modified vector pCAMBIA1300 which contained a 35S promoter and a GFP reporter gene to generate the plant expression vector 35S: CpC3H3-GFP. Then the vector 35S:CpC3H3-GFP and 35S:GFP were transformed into the Agrobacterium tumefaciens strain GV3101 respectively. The 35S:CpC3H3-GFP was then transformed to A. thaliana plants under the instruction of the floral dip method. Transgenic plants were selected on MS medium with 25 mg/L hygromycin to obtain homozygous transgenic plants, and then confirmed by PCR and qPCR amplifying.

Subcellular localization

The subcellular localization of CpC3H3 was first predicted by the online tool WoLF PSORT (WoLF PSORT: Protein Subcellular Localization Prediction Tool ( And then roots of the 35S:CpC3H3-GFP and 35S:GFP overexpression plants were used to analyze the subcellular localization. DAPI was used to mark the location of nucleus and FM 4-64 was used to mark the position of the cell membrane. Roots of transgenic plant treated with 20% PEG 6000 for 24 h were used to analyzed the shuttling of CpC3H3. Fluorescent protein was observed by confocal microscopy (Olympus, FV-10-ASW, Japan).

Drought tolerance assay

Transgenic A. thaliana T3 and WT seedlings were grown in pots containing a mixture of Peat: Perlite (2:1, v/v) for 4 weeks, and then 30 plants of each line were treated with 50 mL of 20% PEG6000 solution for 2 days, 50 mL of 150 mmol/L mannitol for 2 days, and planted without watering for 10 days respectively to detect the drought tolerance.

RNA sequencing analysis

The total RNA extracted from twenty-day-old WT and T3 transgenic plants were used to construct the cDNA libraries by following the user’ instruction of TIANSeq Stranded RNA-Seq Kit (Illumina) (YAD, China). The libraries were sequenced with an Illumina Hiseq system at Beijing Genomics institution. Single-end sequences were first obtained, and adaptor sequences were removed from the raw sequences, and then the lower quality sequences (<Q20) were removed, all the reads were mapped to the reference genome TAIR 10. The differentially expressed genes (DEGs) were identified with a cut-off change more than 2 folds between WT and transgenic plants and an independent T-test p < 0.05. Three biological replicates of WT and overexpression line 18, 10, 14 (each line was served as a replicate of overexpression plants) were analyzed. The RNA-sequencing data set can be obtained from SRA database with an accession number of PRJNA779571.

Determination of physiological indices

The content of proline, soluble sugar, malondialdehyde (MDA), activities of peroxidase (POD) and electrolyte leakage rates in WT and the transgenic A. thaliana plants those were treated with 20%PEG solution for 24 h were determined according to the method previously described [36, 53,54,55,56]. The 8th and 9th leaves of each line were collected for the determination. The osmotic potentials of the leaves of 1-year-old wintersweet, 7-days-old WT and transgenic A. thaliana those were treated with 20% PEG solution for 24 h were tested by following the method descripted by Vijay Paul [57].

Availability of data and materials

The datasets analysed during the current study are available in the SRA ( repository with a with an accession number of PRJNA779571.


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Study protocol in this study complied with relevant institutional, national, and international guidelines and legislation.


This work was supported by Natural Science Foundation Project of Chongqing (No. cstc2019jcyj-msxmX0687) and High-Level Talent Introduction Program of Chengdu Normal University (YJRC2020—22).

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HL, SX, SS, RH and XL designed and conducted the experiments. HL, SX, HW and XW performed the experiments. HL conducted the data and wrote the manuscript. SS contributed to discussion and manuscript revision. All the authors were involved in the discussion of the data and approved the final manuscript.

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Correspondence to Xia Liu.

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Liu, H., Xiao, S., Sui, S. et al. A tandem CCCH type zinc finger protein gene CpC3H3 from Chimonanthus praecox promotes flowering and enhances drought tolerance in Arabidopsis. BMC Plant Biol 22, 506 (2022).

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