Genomic Identification of CCCH-Type Zinc Finger Protein Genes Reveals the Role of HuTZF3 in Tolerance of Heat and Salt Stress of Pitaya (Hylocereus polyrhizus)

Pitaya (Hylocereus polyrhizus) is cultivated in a broad ecological range, due to its tolerance to drought, heat, and poor soil. The zinc finger proteins regulate gene expression at the transcriptional and post-transcriptional levels, by interacting with DNA, RNA, and proteins, to play roles in plant growth and development, and stress response. Here, a total of 81 CCCH-type zinc finger protein genes were identified from the pitaya genome. Transcriptomic analysis showed that nine of them, including HuTZF3, responded to both salt and heat stress. RT-qPCR results showed that HuTZF3 is expressed in all tested organs of pitaya, with a high level in the roots and stems, and confirmed that expression of HuTZF3 is induced by salt and heat stress. Subcellular localization showed that HuTZF3 is targeted in the processing bodies (PBs) and stress granules (SGs). Heterologous expression of HuTZF3 could improve both salt and heat tolerance in Arabidopsis, reduce oxidative stress, and improve the activity of catalase and peroxidase. Therefore, HuTZF3 may be involved in post-transcriptional regulation via localizing to PBs and SGs, contributing to both salt and heat tolerance in pitaya.


Introduction
Soil salinity and increasing temperature caused by human activities, are two major environmental factors affecting plant growth and production [1,2]. To face salt and heat stress, plants initiate a series of physiological and biochemical reactions, to reduce damage and maintain adequate growth [3]. Pitaya (dragon fruit) is one of the tropical fruits belonging to the genus Hylocereus, in the Cactaceae family, with high commercial and medical value [4]. Pitaya is cultivated in a broad ecological range, due to its tolerance to drought, heat, and poor soil [5,6]. Pitaya performs crassulacean acid metabolism (CAM)type photosynthesis [7], and its stems have spines and no leaves. Pitaya, like other CAM plants, has high water-use efficiency, due to the unique stomatal regulation pattern. The stomata of CAM plants open for CO 2 uptake and fixing at night with lower temperature and higher humidity, and close for reducing water loss in the daytime when the stored CO 2 is re-fixed by Rubisco for sugar synthesis [8][9][10]. So, CAM plants can endure various stresses including drought, salinity, and heat, and successfully survival in various ecological habitats, from deserts to forests [11].
There are many studies at transcriptomic and proteomic levels exploring the molecular mechanism in pitaya response to abiotic stresses including drought, salt, cold, and heat [5,6,12,13]. The pitaya catalase gene HuCAT3, is induced by H 2 O 2 , cold, drought, and

Identification of the CCCH-Type Zinc Finger Protein Genes from Pitaya
To identify the CCCH-type zinc finger protein genes from pitaya, the Pitaya Genomic Database was searched, and the CCCH zinc finger domain was confirmed in SMART, NCBI conserved domain search tools, and the Pfam database. A total of 81 CCCH-type zinc finger protein genes (HuCCCHs) were obtained (Figure 1 and Supplementary Table S1), and they were randomly distributed on the 11 chromosomes of pitaya ( Figure 1A). These HuCCCHs were named from HuCCCH1 to HuCCCH81, based on their chromosomal location, and their information is listed in Supplementary Table S1. In order to investigate their phylogenetic relationship, we constructed a phylogenetic tree with the CCCH-type zinc finger proteins from pitaya and Arabidopsis ( Figure 1B). The pitaya HuCCCH proteins could be divided into 11 subfamilies, like those of Arabidopsis.
These HuCCCHs were named from HuCCCH1 to HuCCCH81, based on their chromosomal location, and their information is listed in Supplementary Table S1. In order to investigate their phylogenetic relationship, we constructed a phylogenetic tree with the CCCH-type zinc finger proteins from pitaya and Arabidopsis ( Figure 1B). The pitaya HuCCCH proteins could be divided into 11 subfamilies, like those of Arabidopsis. Figure 1. Systematic identification of the CCCH-type zinc finger protein genes from pitaya. (A) Chromosomal locations of pitaya CCCH genes. The chromosomal locations of HuCCCH genes were mapped by TBtools [32]. Names of the HuCCCH genes are shown on the right of the chromosomes. The scale of the genome size is shown on the left. (B) Phylogenetic tree of the CCCH proteins from pitaya and Arabidopsis. The 81 HuCCCH proteins from pitaya and the 68 AtCCCH proteins from Arabidopsis were used to construct the phylogenetic tree by MAG11 and visualized by iTOL. The red star indicates HuTZF3.
Since HuTZF3 belongs to subfamily IX of the HuCCCH family ( Figure 1B), we analyzed the conserved motif, conserved domain, cis-elements in promoter, and gene structure of the 16 TZF-type zinc finger protein genes in the subfamily IX (Supplementary  Figures S1 and Table S3). The promoter cis-element analysis showed that there are many cis-elements in the promoter of HuTZFs, such as plant growth and development, hormone, and abiotic stress-related cis-elements (Supplementary Figure S1B). According to the phylogenetic tree, conserved motif, and conserved domain, the 16 pitaya TZF proteins were divided into two groups: the RR-TZF group, including HuTZF1-10, and the ANK-RR-TZF group, including HuTZF11-16, similar to that of Arabidopsis (Supplementary Figure S2). All HuTZFs of the subfamily IX have the RR-TZF domain, that the RR (arginine-rich) domain localized in front of the TZF domain (Supplementary Figure S2A). HuTZF11-16 contains the ANK (Ankyrin repeat) domain and the RR-TZF domain belongs to the ANK-RR-TZF group (Supplementary Figure S2B).

Identification of HuCCCHs Response to Heat and Salt Stress
To screen which HuCCCHs respond to both heat and salt stress, we analyzed our previous transcriptomic data of pitaya seedlings under salt and heat treatment [5,6]. We found that sixteen of the HuCCCHs responded to heat treatment (Figure 2A), and 26 genes responded to salt treatment ( Figure 2B). Among them, nine genes were induced by both salt and heat stress, and they are HuTZFs3/4/12 and C3H31/32/39/40/54/67. To verify this result, the expression pattern of HuTZF3 was analyzed in different tissues and response Since HuTZF3 belongs to subfamily IX of the HuCCCH family ( Figure 1B), we analyzed the conserved motif, conserved domain, cis-elements in promoter, and gene structure of the 16 TZF-type zinc finger protein genes in the subfamily IX (Supplementary Figure S1 and Table S3). The promoter cis-element analysis showed that there are many cis-elements in the promoter of HuTZFs, such as plant growth and development, hormone, and abiotic stress-related cis-elements (Supplementary Figure S1B). According to the phylogenetic tree, conserved motif, and conserved domain, the 16 pitaya TZF proteins were divided into two groups: the RR-TZF group, including HuTZF1-10, and the ANK-RR-TZF group, including HuTZF11-16, similar to that of Arabidopsis (Supplementary Figure S2). All HuTZFs of the subfamily IX have the RR-TZF domain, that the RR (arginine-rich) domain localized in front of the TZF domain (Supplementary Figure S2A). HuTZF11-16 contains the ANK (Ankyrin repeat) domain and the RR-TZF domain belongs to the ANK-RR-TZF group (Supplementary Figure S2B).

Identification of HuCCCHs Response to Heat and Salt Stress
To screen which HuCCCHs respond to both heat and salt stress, we analyzed our previous transcriptomic data of pitaya seedlings under salt and heat treatment [5,6]. We found that sixteen of the HuCCCHs responded to heat treatment (Figure 2A), and 26 genes responded to salt treatment ( Figure 2B). Among them, nine genes were induced by both salt and heat stress, and they are HuTZFs3/4/12 and C3H31/32/39/40/54/67. To verify this result, the expression pattern of HuTZF3 was analyzed in different tissues and response to salt and heat stress by RT-qPCR ( Figure 2C-E). The results showed that expression of HuTZF3 was detected in different tissues. HuTZF3 had a high expression level in roots and stems, and a low expression level in petal and calyx ( Figure 2C). The expression of HuTZF3 gradually increased under salt stress ( Figure 2D), and its expression was rapidly induced by heat stress and reached a peak at 3 h ( Figure 2E), which confirmed the screening result above from the transcriptomic data [5,6].
to salt and heat stress by RT-qPCR ( Figure 2C-E). The results showed that expression of HuTZF3 was detected in different tissues. HuTZF3 had a high expression level in roots and stems, and a low expression level in petal and calyx ( Figure 2C). The expression of HuTZF3 gradually increased under salt stress ( Figure 2D), and its expression was rapidly induced by heat stress and reached a peak at 3 h ( Figure 2E), which confirmed the screening result above from the transcriptomic data [5,6].  [6]. The three-month-old seedlings grown in a greenhouse, were treated with 450 mM NaCl, or at 42 °C, for different times, then the samples were collected for RNAseq. (C-E) Expression pattern of HuTZF3 in different tissues of pitaya (C), and in pitaya seedlings under salt (D) and heat (E) treatment. Three-month-old pitaya seedlings were treated with 450 mM NaCl (D) at 42 °C (E). HuEF1-α was used as the internal reference gene. Values represent means ± SD (n = 3 technical repetitions). All RT-qPCR analyses for gene expression were performed in three biological replicates, with similar results.

Heterologous Expression of HuTZF3 Improved Salt and Heat Tolerance in Arabidopsis
To study whether HuTZF3 affects the salt and heat tolerance of plants, we heterologously expressed HuTZF3 in Arabidopsis with CaMV35S promoter (Figure 3). Three independent homozygous HuTZF3 overexpressing lines (OE-4/14/17), showed high expression of HuTZF3 ( Figure 3C), and were selected for further study. First, the salt tolerance of these transgenic Arabidopsis was tested under different concentrations of NaCl, using the seedlings. When the five-day-old seedlings were transferred to MS medium, containing 0, 100, 150, and 200 mM NaCl, and then cultured for 7 days, the root length of the OE lines was significantly longer than that of the wild type (WT); however,  [6]. The three-month-old seedlings grown in a greenhouse, were treated with 450 mM NaCl, or at 42 • C, for different times, then the samples were collected for RNA-seq. (C-E) Expression pattern of HuTZF3 in different tissues of pitaya (C), and in pitaya seedlings under salt (D) and heat (E) treatment. Three-month-old pitaya seedlings were treated with 450 mM NaCl (D) at 42 • C (E). HuEF1-α was used as the internal reference gene. Values represent means ± SD (n = 3 technical repetitions). All RT-qPCR analyses for gene expression were performed in three biological replicates, with similar results.

Heterologous Expression of HuTZF3 Improved Salt and Heat Tolerance in Arabidopsis
To study whether HuTZF3 affects the salt and heat tolerance of plants, we heterologously expressed HuTZF3 in Arabidopsis with CaMV35S promoter (Figure 3). Three independent homozygous HuTZF3 overexpressing lines (OE-4/14/17), showed high expression of HuTZF3 ( Figure 3C), and were selected for further study. First, the salt tolerance of these transgenic Arabidopsis was tested under different concentrations of NaCl, using the seedlings. When the five-day-old seedlings were transferred to MS medium, containing 0, 100, 150, and 200 mM NaCl, and then cultured for 7 days, the root length of the OE lines was significantly longer than that of the wild type (WT); however, there was no significant difference between the WT and OE lines when they were grown on MS without NaCl ( Figure 3A,B). When the four-week-old Arabidopsis of the WT and OE lines were grown in soil and then subjected to 300 mM NaCl, the OE lines also showed a higher survival rate to salt stress than the WT ( Figure 3D,E).
there was no significant difference between the WT and OE lines when they were grown on MS without NaCl ( Figure 3A,B). When the four-week-old Arabidopsis of the WT and OE lines were grown in soil and then subjected to 300 mM NaCl, the OE lines also showed a higher survival rate to salt stress than the WT ( Figure 3D,E). The heat tolerance of the transgenic Arabidopsis was also tested (Figure 4). When the two-week-old seedlings were exposed to heat (42 °C) and recovered under normal temperature (22 °C) ( Figure 4C), the survival rate of the seedlings in the OE lines was higher than that of the WT ( Figure 4A,B). The OE lines had a more than 90% survival rate, whereas WT was only about 44%. These results indicate that heterologous overexpression of HuTZF3 in Arabidopsis could improve its salt and heat tolerance. The heat tolerance of the transgenic Arabidopsis was also tested (Figure 4). When the two-week-old seedlings were exposed to heat (42 • C) and recovered under normal temperature (22 • C) ( Figure 4C), the survival rate of the seedlings in the OE lines was higher than that of the WT (Figure 4A,B). The OE lines had a more than 90% survival rate, whereas WT was only about 44%. These results indicate that heterologous overexpression of HuTZF3 in Arabidopsis could improve its salt and heat tolerance.
whereas WT was only about 44%. These results indicate that heterologous overexpression of HuTZF3 in Arabidopsis could improve its salt and heat tolerance.

Heterologous Expression of HuTZF3 Repressed Burst of Oxidative Stress in Arabidopsis
To analyze the accumulation of ROS in WT and the HuTZF3 overexpression lines, under salt and heat stress, the leaves were stained with DAB and NBT, to detect the production of H2O2 and O2 -. The results showed that the content of H2O2 and O2 -in the WT accumulated more than in the HuTZF3 overexpression lines ( Figure 5A,B); however, there was no significant difference between the transgenic lines and WT plants in the absence of stresses ( Figure 5A,B). The activities of CAT and POD in the HuTZF3 OE lines were higher than in the WT, under salt and heat stress ( Figure 5C). These results demonstrate that overexpression of HuTZF3 can improve the ROS scavenging ability of Arabidopsis, to reduce oxidative stress under salt and heat treatments.

Heterologous Expression of HuTZF3 Repressed Burst of Oxidative Stress in Arabidopsis
To analyze the accumulation of ROS in WT and the HuTZF3 overexpression lines, under salt and heat stress, the leaves were stained with DAB and NBT, to detect the production of H 2 O 2 and O 2 − . The results showed that the content of H 2 O 2 and O 2 − in the WT accumulated more than in the HuTZF3 overexpression lines ( Figure 5A,B); however, there was no significant difference between the transgenic lines and WT plants in the absence of stresses ( Figure 5A,B). The activities of CAT and POD in the HuTZF3 OE lines were higher than in the WT, under salt and heat stress ( Figure 5C). These results demonstrate that overexpression of HuTZF3 can improve the ROS scavenging ability of Arabidopsis, to reduce oxidative stress under salt and heat treatments.

HuTZF3 Is Co-Localized with PBs and SGs Markers in Arabidopsis
Since most TZFs are RNA-binding proteins involved in RNA regulation, and localized to PBs and SGs, the mRNA degradation factor DCP2 has decapping activity and is involved in PBs assembly, and the RNA-binding protein UBP1B, is required for SG formation. Therefore, DCP2 and UBP1B are known as PB marker and SG marker, respectively [25]. To explore whether HuTZF3 is localized to PBs and SGs, subcellular localization of HuTZF3 was performed. Protoplasts isolated from Arabidopsis leaves were co-transformed with HuTZF3-GFP and PBs marker RFP-DCP2 or SGs marker RFP-UBP1 ( Figure 6). The HuTZF3-GFP was diffusely localized in the cytoplasm under the control condition. However, after heat ( Figure 6A,B) and salt treatments ( Figure 6C,D), HuTZF3-GFP was redistributed to cytoplasmic foci, to co-localize with the PBs and SGs markers. Although SG cytoplasmic foci were not evident under normal conditions, SG foci appeared in the cytoplasm under heat and salt stress ( Figure 6B,D). These results showed that HuTZF3 is mainly localized in the cytoplasm under normal conditions and assembled into cytoplasmic foci to colocalize with PBs and SGs during the salt and heat treatment, indicating that HuTZF3 may participate in the RNA processing.

HuTZF3 Is Co-Localized with PBs and SGs Markers in Arabidopsis
Since most TZFs are RNA-binding proteins involved in RNA regulation, and localized to PBs and SGs, the mRNA degradation factor DCP2 has decapping activity and is involved in PBs assembly, and the RNA-binding protein UBP1B, is required for SG formation. Therefore, DCP2 and UBP1B are known as PB marker and SG marker, respectively [25]. To explore whether HuTZF3 is localized to PBs and SGs, subcellular localization of HuTZF3 was performed. Protoplasts isolated from Arabidopsis leaves were co-transformed with HuTZF3-GFP and PBs marker RFP-DCP2 or SGs marker RFP-UBP1 ( Figure 6). The HuTZF3-GFP was diffusely localized in the cytoplasm under the control condition. However, after heat ( Figure 6A,B) and salt treatments ( Figure 6C,D), HuTZF3-GFP was redistributed to cytoplasmic foci, to co-localize with the PBs and SGs markers. Although SG cytoplasmic foci were not evident under normal conditions, SG foci appeared in the cytoplasm under heat and salt stress ( Figure 6B,D). These results showed that HuTZF3 is mainly localized in the cytoplasm under normal conditions and assembled into cytoplasmic foci to co-localize with PBs and SGs during the salt and heat treatment, indicating that HuTZF3 may participate in the RNA processing.

Discussion
The CCCH-type zinc finger proteins have been confirmed to play important roles in plant growth, development, and stress adaption. Here, we identified a total of 81 CCCH genes from pitaya, which were divided into 11 subfamilies based on the classification in Arabidopsis [21], and nine of them responded to both heat and salt stress (Figures 1 and 2),

Discussion
The CCCH-type zinc finger proteins have been confirmed to play important roles in plant growth, development, and stress adaption. Here, we identified a total of 81 CCCH genes from pitaya, which were divided into 11 subfamilies based on the classification in Arabidopsis [21], and nine of them responded to both heat and salt stress (Figures 1 and 2), indicating that the nine CCCH genes may play roles in the tolerance of pitaya to both salt and heat stress. HuTZF3 may affect RNA processing to play a role in salt and heat tolerance of pitaya, since it is localized in the PBs and SGs (Figure 6). The IX subfamily of the HuCCCH family, contained 16 HuTZFs, which are characterized by two CCCH zinc finger motifs arranged in tandem ( Figure S2). Based on the presence of the Ankyrin repeat (ANK) domain, the 16 HuTZFs were further divided into two groups: RR-TZF group and ANK-RR-TZF group, which is consistent with phylogenetic analysis of the CCCH genes in Arabidopsis, rice, and maize [21,33]. Promoter cis-element analysis showed that HuTZFs are involved in plant growth and development, and respond to multiple hormones and environmental stress ( Figure S1), possibly like other plants [34][35][36]. Analysis of the transcriptomic data showed that the expression of nine genes could be regulated by both salt and heat stress (Figure 2A), implying that they may play roles in pitaya's tolerance to heat and salt stress.
Heterologous expression of HuTZF3, confirmed that some pitaya TZFs play a role in tolerance to salt and heat stress (Figures 3 and 4). HuTZF3 is expressed in different organs of pitaya, with a high level in the roots and stems, and its expression in pitaya seedlings is induced by both salt and heat stress ( Figure 2). This result implies that HuTZF3 may contribute to pitaya tolerance to salt and heat stress, like AtTZF1-3 [27,29] and rice OsTZF1, 5,8 [30,37,38]. Salinity and heat stress induced excessive ROS production and ultimately lead to oxidative stress. DAB and DBT staining revealed more ROS accumulation in the WT Arabidopsis than the HuTZF3 OE lines under salt and heat stress ( Figure 5A,B), the activities of CAT and POD in the HuTZF3 OE lines were higher than in WT Arabidopsis, under salt and heat stress ( Figure 5C). These results indicate that HuTZF3 could improve tolerance to oxidative stress, to confer salt and heat stress, like rice OsTZF1 [30].
In eukaryotes, cellular mRNAs are coated with proteins forming messenger ribonucleoprotein (mRNP) complexes [39]. The mRNP complex involves post-transcriptional regulation via controlling mRNA's whole life cycle, from pre-mRNA processing to mRNA transport, localization, stability, and translation [40]. Many inactive translation mRNAs often assemble with proteins to format cytoplasmic mRNP granules. Processing bodies (PBs) and stress granules (SGs) are two well-characterized mRNP granules, that are widely appear during various stresses [41,42]. PBs have a major involvement in mRNA decay and translation repression, SGs also contain many stalled preinitiation complexes [43]. Our results indicated that HuTZF3 can localize to PBs and SGs under salt and heat stress ( Figure 6). It is known that PB-and SG-localized OsTZF1 and OsTZF7 affect RNA stability, via binding mRNA, to delay senescence and improve plant stress tolerance [30,31]. The salt and heat stress could promote accumulation of HuTZF3 in PBs and SGs (Figure 6), this suggests that HuTZF3 might also be involved in the regulation of targeting RNA metabolism in post-transcriptional and translational levels, to confer salt and heat stress in pitaya.

Plant and Growth Conditions
Pitaya (Hylocereus polyrhizus) and Arabidopsis were used as the plant materials in this experiment. The pitaya seeds were germinated and cultivated in the plant growth room (25 • C, 16 h/8 h light/dark photoperiod). Arabidopsis was cultivated in the plant growth room (22 • C, 16 h/8 h light/dark photoperiod).

Abiotic Stress Treatment
For pitaya stress treatment, pitaya seeds were germinated in the soil and the seedlings were grown in plates filled with nutrient soil at 25 • C, the pitaya plants were subjected to salt or heat stress. For salt on primary root growth assay, Arabidopsis seeds were germinated vertically on MS medium for 5 days, then seedlings of similar root length were chosen to transfer to MS medium containing 0, 100, 150, and 200 mM NaCl, and vertically cultured for 7 days. For Arabidopsis stress treatment, Arabidopsis seedlings were transferred and grown in nutrient soil, after germination on MS medium, for 7 days, then four-week-old plants were watered with a solution with 300 mM NaCl for salt stress, or exposed to 42 • C in an incubator for heat stress.

RNA Isolation and RT-qPCR Analysis
Total RNA was extracted from pitaya and Arabidopsis using the Eastep Super Total RNA Extraction Kit (Promega, Beijing, China). First-strand cDNA was synthesized using GoScript TM Reverse Transcription Mix (Promega, Beijing, China). RT-qPCR reactions were performed with MonAmp TM ChemoHS qPCR Mix (Monad, Wuhan, China), by Roche Light Cycler 480 Real-time PCR System (Roche, Basel, Switzerland). Arabidopsis Actin2 and pitaya HuEF1-α were used as internal reference genes [45]. The primers of RT-qPCR are listed in Supplementary Table S2.

Vector Construction and Genetic Transformation
The full-length HuTZF3 cDNA was cloned into the pCAMBIA1302 vector driven by CaMV35S, to construct the HuTZF3 overexpression vector. Arabidopsis transgenic plants were produced by the Agrobacterium-mediated floral dipping method [46]. Positive transgenic plants were screened on MS medium by kanamycin and confirmed by PCR, until transgenic homozygous lines were obtained.

Histochemical and Physiological Analysis of Oxidative Stress
After the heat and salt treatment for 4 h, leaves were submerged in 1 mg/mL DAB or 1 mg/mL NBT solution within 10 h, for in situ detection of the superoxide (O 2 − ) anion and hydrogen peroxide (H 2 O 2 ), and washing in 95% ethanol [47]. Peroxidase (POD) and catalase (CAT) activities were measured using POD and CAT Assay Kits (Nanjing Jiancheng, Nanjing, China), according to the manufacturer's instructions.

Subcellular Localization of HuTZF3
The coding sequences of HuTZF3 were inserted into the pUC/GFP vector, to construct a fusion plasmid (HuTZF3-GFP). The RFP fusion plasmids were constructed by adding the coding sequence of DCP2 and UBP1 to pBI221/RFP, which were as described in [48]. Arabidopsis mesophyll protoplasts were isolated from four-week-old Arabidopsis leaves and transformed by PEG-CaCl 2 -mediated transfection. After incubating the transformed protoplasts at room temperature for 12-16 h in darkness, the protoplasts were then subjected to 39 • C for heat stress, or 150 mM NaCl for salt stress. Protoplasts were observed by using confocal laser microscopy Leica SP8 STED 3X (Leica Microsystems, Mannheim, Germany).

Conclusions
To comprehensively examine the CCCH gene family in pitaya, a genome-wide investigation identified 81 HuCCCHs, which were classified into 11 subfamilies. Analysis of the transcriptomic data found that 9 HuCCCHs, including HuTZF3, responded to both salt and heat stress. HuTZF3 is constitutively expressed in different organs of pitaya, with a high level in the roots and stems. RT-qPCR confirmed that expression of HuTZF3 is induced by both salt and heat stress. Overexpression of HuTZF3 improved Arabidopsis tolerance to salt and heat stress. Co-localization of HuTZF3 with PBs and SGs was observed under salt and heat stress. Our findings suggest that HuTZF3 may participate in RNA metabolism to cope with the salt and heat stress of pitaya.