The Arabidopsis Gene zinc finger protein 3(ZFP3) Is Involved in Salt Stress and Osmotic Stress Response

Plants are continuously challenged by various abiotic and biotic stresses. To tide over these adversities, plants evolved intricate regulatory networks to adapt these unfavorable environments. So far, many researchers have clarified the molecular and genetic pathways involved in regulation of stress responses. However, the mechanism through which these regulatory networks operate is largely unknown. In this study, we cloned a C2H2-type zinc finger protein gene ZFP3 from Arabidopsis thaliana and investigated its function in salt and osmotic stress response. Our results showed that the expression level of ZFP3 was highly suppressed by NaCl, mannitol and sucrose. Constitutive expression of ZFP3 enhanced tolerance of plants to salt and osmotic stress while the zfp3 mutant plants displays reduced tolerance in Arabidopsis. Gain- and Loss-of-function studies of ZFP3 showed that ZFP3 significantly changes proline accumulation and chlorophyll content. Furthermore, over-expression of ZFP3 induced the expressions of stress-related gene KIN1, RD22, RD29B and AtP5CS1. These results suggest that ZFP3 is involved in salt and osmotic stress response.


Introduction
Plants are constantly challenged by a variety of stresses such as high salinity, extreme temperature and drought [1,2]. Environmental stress has been one of the major factors affecting crops yield. These stresses cause huge losses in some cases is as high as 50% of the total yield of some major crops [1,3,4]. To cope with these stressful conditions, plants have evolved an intricate regulatory mechanism [5], which is mostly regulated through activation or inhibition of the expression of a series of transcription factors [5][6][7]. In the past few decades, a number of transcription factors have been found to participate in physiological responses of plants to abiotic stresses. These different transcription factors involved in abiotic stress generally belong to a large family which include C-repeat binding factors (CBFs), dehydration responsive element binding factors (DREBs), ethylene-responsive element binding factor (ERF), AP2/EREB, basic domain-leucine zipper (bZIP), NAC, MYB, MYC, WRKY and zinc finger proteins [6][7][8][9][10][11][12]. stress-related genes (KIN1, RD22, RD29B and AtP5CS1) in regulating salt and osmotic stress tolerance in Arabidopsis.

Plant materials and growth conditions
Arabidopsis thaliana ecotype Columbia Col-0 was used as the control in all experiments. The plants were grown in an environmentally controlled growth chamber programmed for cycles of 16 hours of light (95 μmol m -2 second -1 , 22˚C), 8 hours dark (18˚C) and relative humidity of 70-80% as we previously described [20,21]. Arabidopsis seeds were surface-sterilized with 5% NaClO: water (v/v) for 8 min and washed five times with distilled water. Seeds were plated on Murashige and Skoog (MS) medium and vernalize at 4˚C for 3 days in dark before being transferred to the growth chamber with growth condition described previously [23].

Isolation of a zfp3 loss-of-function mutant
A zfp3 mutant was identified from the Nottingham Arabidopsis Stock Centre (NASC; catalogue number N379591). Homozygous mutant was confirmed by screening on MS medium containing 5.25 mg/L sulfadiazine.

Construct cloning
For overexpression construct, sequences were first inserted into the pENTR-1A vector (Invitrogen) and then recombined into the destination vector using the Gateway LR reaction (Invitrogen) [20]. The destination vector was obtained from VIB (Flanders Interuniversity Institute for Biotechnology) as described before [18,20,21]. For cloning the overexpression construct, the gene-specific fragment was amplified from cDNA with primers containing SalI and NotI restriction sites. Then, the fragment was purified with a gel extraction kit (Axygen) before restriction digestion and cloning. The primers, which used for ZFP3 overexpression, were as follows: FP (5'-GCGCGTCGACAATCATCTCCTGAAAAAATCTATC-3'), RP (5'-TGGCG GCCGCACTAACAATCACATGAAAAAACAG-3'). The construct in binary vector was transferred into Agrobacterium tumefaciens Stain GV3101, which was then used to transform Arabidopsis thaliana ecotype Columbia Col-0 by the floral-dip method as described by Clough and Bent (1998) [32]. Transgenic seeds were first selected on Murashige and Skoog medium containing Kanamycin (50mg/L) and then confirmed by PCR with corresponding primers.

Stress treatment
For phenotypical analyses, seeds were sown on the 1/2 MS medium containing different concentrations of NaCl, mannitol and sucrose. Emergence of radicles from the seed coat was used as the standard of seed germination. The germination rate was calculated as the percentage of number of germinated seeds/number of total seeds, which was recorded daily for seven days.
For the root length measurement, seeds were cultured on 1/2 MS medium and grown on vertically placed plates for 3 days under normal condition and uniform seedlings were then transferred to 1/2 MS mediums with or without different concentrations of NaCl, mannitol and sucrose. After 7 days treatment, photos were taken and the root lengths of different plants were measured by Image-Pro plus 6.0.
For gene expression analysis, 10 days seedlings were transferred onto filter paper saturated with 0, 200mM NaCl, 250mM sucrose and 300mM mannitol in MS liquid media. The samples were harvested at different time points.

RNA extraction and real-time PCR
Total RNA was extracted from Arabidopsis plants using Trizol (Takara). First-strand cDNA was synthesized from 2 μg of total RNA by M-MLV transcriptase (Promega) and oligo (dT18) primer according to the manufacturer's instructions [21,23]. For real-time PCR analysis, reaction was performed on 2μl 4 times diluted cDNA templates, 10μl SYBR Green PCR mix (Takara) and 0.4μl of each primers (200 nM final concentration) in 20μl reactions. qRT-PCR was initiated with denaturation at 95˚C for 30 s, followed by 40 cycles each consisting of denaturation at 95˚C for 5 s and annealing/extension at 60˚C for 30 s. To determine the specificity of the reaction, a melting curve analysis of the product was performed immediately after the final PCR cycle by increasing the temperature from 60 to 90˚C at 0.2 to 1 s. UBQ10 (At4g05320) and TUB2 (At5g62690) were used as the endogenous control gene for qRT-PCR analyses. Relative expression of target genes was calculated as previously described [23,33]. The primers used are listed in Table 1.

Determination of chlorophyll content
The chlorophyll was extracted in 95% ethyl alcohol from the fresh leaves at darkness. Absorbance of solution was measured at wavelength of 470, 649 and 665 nm with spectrophotometer (UV-2450, Shimadzu, Japan) to analysis the content of chlorophyll(a) and chlorophyll(b), respectively.

Determination of proline content
Proline content of 10 days Arabidopsis seedlings was determined according to the procedure of Bates et al. (1973) [35].

Statistical analyses
All data displayed in figures were analyzed by means of ANOVA for significance using SPSS, version 14.0 software (SPSS Inc., Chicago, USA). Student's t-test was calculated to evaluate the difference at the probability of either 5% ( Ã , P < 0.05 with significant level) or 1% ( ÃÃ , P < 0.01 with significant level) [33]. Table 1. Primers used for quantitative real-time PCR, Arabidopsis thaliana ecotype Columbia (Col-0) was used for this study.

Gene
Sequence Reference

ZFP3 affects the response to salt and osmotic stress
To determine the effects of ZFP3 in abiotic stress responses, a zfp3 mutant (catalog no. N379591) was ordered and screened from the Nottingham Arabidopsis Stock Center. We also constructed 35S:ZFP3 transgenic lines and selected two representative lines, which showed higher level of ZFP3 expression in comparison to the wild type (Fig 1). Seeds of zfp3, 35S: ZFP3-6, 35S:ZFP3-11 and wild type were geminated on 1/2 MS medium with 0, 100, 150 and 200 mM NaCl. No significant difference among wild type, mutant and overexpression line was observed when seeds were grown on 1/2 MS medium (Fig 2a). When treated with 100 mM NaCl, zfp3 mutant exhibited significantly lower germination rates than wild type, while ZFP3 overexpression lines displayed significantly higher germination rates. However, as the concentration increases, the germination rates were decreased dramatically. The decrease in germination rates was more significant upon exposure to 150 and 200 mM NaCl. At the 3rd day and 4th day post germination on 150 mM NaCl, the rates of germination in zfp3 were significantly delayed compared to wild type. But 35S:ZFP3-6 and 35S:ZFP3-11 lines germinated significantly earlier than wild type (Fig 2c). On 200 mM NaCl medium, the differences of germination rate of wild type, zfp3, 35S:ZFP3-6 and 35S:ZFP3-11 were significant from the 3rd to7th day post germination (Fig 2d). To comprehend the role of ZFP3 in osmotic stress, we also examined the germination condition on 1/2 MS medium supplemented with different concentrations of mannitol and sucrose. As shown in Fig 3a to 3d, germination rates among the zfp3 mutant, ZFP3 overexpression lines and wild type were not significantly different on 1/2 MS medium. However, the zfp3 mutant displayed a significant decrease in germination rate compared to the wild type on 300, 400 and 500 mM mannitol. Contrary to zfp3 mutant, the overexpression lines showed significantly increased germination rates in these treatments. A similar trend in germination rates was observed on mediums containing different concentrations of sucrose.
The germination rates were significantly lower in zfp3 mutant and significantly higher in ZFP3 overexpression lines in comparison to wild type when treated with 200 mM sucrose. Upon exposure to 250 and 300 mM sucrose, the zfp3 mutant exhibited significant decrease in germination rates during most of exposure period (Fig 4a-4d). Meanwhile, when treated with 300    mM sucrose, the overexpression lines showed significantly higher germination rates than wild type from the 2nd to 7th day post germination (Fig 4d). The results indicate that loss of function of ZFP3 reduces the tolerance to salt and osmotic stress while overexpression of ZFP3 exhibited an increased tolerance to salt and osmotic stress in Arabidopsis thaliana.
To further confirm this conclusion, root length of the mutant, overexpression line and wild type were measured on different treatments. No significant difference in root length was recorded among wild type, zfp3, 35S:ZFP3-6 and 35S:ZFP3-11 seedlings on 1/2 MS mediums (Figs 2e and 2f, 3e and 3f, 4e and 4f). When treated with 100 and 150 mM NaCl, significant inhibition in root lengths was observed. The average root length of zfp3 seedlings was significantly shorter than wild type seedlings on 100 mM and 150 mM NaCl. Nevertheless, the average root lengths of 35S:ZFP3 seedlings were significantly longer in comparison to wild type seedlings (Fig 2e and 2f). When exposed to mannitol and sucrose, the zfp3 mutant was also more sensitive than wild type on root length. When treated with 200 mM mannitol, the root length of zfp3 seedlings was decreased significantly in comparison to wild type. However, 35S: ZFP3-6 and 35S:ZFP3-11 lines were about 16% longer than wild type (Fig 3e and 3f). On 300 mM mannitol medium, the root length of zfp3 seedlings was 16% shorter than wild type seedlings and that of ZFP3 overexpression lines seedlings were significantly longer than wild type seedlings (Fig 3e and 3f). The data displayed that root length of zfp3 mutant was significantly shorter than wild type under different sucrose treatments. Root length of zfp3 seedlings was shorter in comparison to wild type on both 150 mM and 250 mM sucrose medium (Fig 4e and  4f). Oppositely, root length of 35S:ZFP3-6 line was 16.9% and 17.5% longer than that of wild type (Fig 4e and 4f). Taken together, these data suggest that ZFP3 is a key regulator controlling salt and osmotic stress response in Arabidopsis.

ZFP3 expression is suppressed by salt and osmotic stress
In order to further confirm the function of ZFP3 in abiotic stress, real-time PCR analyses were done to detect the expression of ZFP3 gene under different concentrations of NaCl, mannitol and sucrose in wild type seedlings. As shown in Fig 5a, the expression level of ZFP3 was dramatically reduced to 0.12 fold after 10 hours of NaCl treatment. Similarly, the expression levels of ZFP3 were also rapidly induced by mannitol (Fig 5b) and sucrose (Fig 5c), which displayed a gradual decrease and only reached 0.30 and 0.46 of initial level after 10 hours. Taken together, these results confirm that ZFP3 expression is suppressed by abiotic stress response.

ZFP3 affects proline and chlorophyll contents under salt stress and osmotic stress
Proline, which works as a kind of compatible osmolyte, plays an important role in protecting plants from abrasive environmental stresses especially osmotic and salt stresses [36][37][38][39][40]. The content of proline was determined in wild type, zfp3 mutant and ZFP3 overexpression line (35S:ZFP3-6) with 0 mM, 100 mM NaCl, 200 mM mannitol and 150 mM sucrose treatments. As shown in Fig 6a-6c, the contents of proline were not significantly different among these three lines under normal environmental conditions. However, the overexpression line accumulated more proline than wild type, while the proline content of zfp3 mutant was significantly less than wild type when treated with NaCl, mannitol and sucrose (Fig 6a-6c).
Then, we further determined the chlorophyll contents of wild type, zfp3 and 35S:ZFP3 Arabidopsis plants under different conditions. Under normal condition, there were no significant differences among wild type, zfp3 and 35S:ZFP3 (Fig 6d and 6e). Chlorophyll a, chlorophyll b and chlorophyll a+b contents of zfp3 were decreased significantly compared to wild type under high concentration of NaCl, mannitol and sucrose conditions. However, higher chlorophyll content was found in overexpression line in comparison to wild type (Fig 6d and 6e).

ZFP3 regulates the expressions of stress-related genes
To further investigate the functions of ZFP3 in abiotic stresses, we analyzed the expressions of some stress-related marker genes such as KIN1 (At5g15960), RD22 (At5g25610), RD29B (At5g52300) and osmotic substance synthesis gene AtP5CS1 (At2g39800) that encodes pyrroline-5-carboxylate synthetase [15,38]. The plants of wild type, zfp3 mutant and ZFP3 overexpression line (35S:ZFP3-6) were treated with 200 mM NaCl, 300 mM mannitol and 250 mM sucrose for 5h and 10h respectively. As shown in Fig 7, for the normal 1/2 MS medium, there were no significant differences in all the genotypes in the expression pattern of four stressrelated genes except RD29B. However, the expressions of these genes in wild type, zfp3 mutant and overexpression line were all significantly induced when treated by NaCl, mannitol and  sucrose. The expressions of these genes in overexpression line were significantly higher than wild type while the expressions of these genes in zfp3 mutant were significantly lower than wild type (Fig 7). These results suggest that ZFP3 acts upstream of these genes and further research on these genes interact with ZFP3 to regulate network in salt stress and osmotic stress response is needed.

Discussion
In Arabidopsis C 2 H 2 -type ZFPs consists of 176 members and is one of largest transcription factor families. The cDNA of ZFP3 is 959 bp and encodes a zinc finger protein containing only a single zinc finger. C 2 H 2 -type ZFPs play a major role in growth and development of plants [30]. It was reported that they participate in various biological processes including signal transduction, DNA/RNA binding, morphogenesis, transcription regulation, abiotic and biotic response [4,[16][17][18][19]41]. Our previous work also have demonstrated that C2H2-type ZFPs GIS, GIS2, GIS3, ZFP8, ZFP5 and ZFP6 are all C 2 H 2 -type ZFPs control the trichome initiation by integrating GA and Cytokinin signals [18][19][20][21]23,42,43]. AtZFP1 was reported to plays a role in downstream of photomorphogenic activation [44]. Overexpression of ZFP2 was shown to delay flower abscission and ZFP2 may influence organ shed directly or indirectly in Arabidopsis [45]. An important feature of ZFP3 is the presence of a zinc finger motif. This motif is very conservative in both monocots and dicots, which indicate that they may have the similar biological function [46]. It was shown that ZFP3 protein is localized in nucleus, which suggests its potential functional diversity. ZFP3 was previously shown to regulate light and ABA signaling in seeds germination and plant development in Arabidopsis [31]. To further examine the function of ZFP3, we studied the phenotype of wild type, zfp3 mutant and ZFP3 overexpression line under stress condition. Our results demonstrated that ZFP3 is a key regulator controlling plants in response to salt stress and osmotic stress (Figs 2-4).
Chlorophyll content is an important parameter to determine the capacity of plant photosynthesis. Analysis of chlorophyll content revealed that the content of ZFP3 overexpression line is significantly higher compare to wild type while the mutant displayed an opposite result under stress conditions. Proline accumulation is a widespread phenomenon when plants suffer from a variety of hostile environmental stress [47]. Furthermore, proline is also known as a compatible osmolyte which can scavenge free radicals, keep the balance of intracellular redox, stabilize subcellular structures during stress, especially salt and osmotic stress [38,39]. In this study, the proline accumulation is elevated significantly in all of three lines. However, the elevated level of zfp3 mutant is much lower than that of wild type, while overexpression line is relatively higher in salt and osmotic stress (Fig 6a-6c). Proline is synthesized from glutamate or ornithine. In glutamate pathway, there are two steps in which glutamate is converted to glutamyl-5-semialdehyde (G5SA) by Δ 1 -pyrroline-5-carboxylate synthase (P5CS) and G5SA is converted to proline by the Δ 1 -pyrroline-carboxylate reductase (P5CR). Among them, the γ-glutamyl kinase activity of P5CS is the key rate-limiting factor [36][37][38]48,49]. Therefore we investigated the expression level of AtP5CS1. Our results show that the expression level of AtP5CS1 was significantly higher in overexpression line while the expression of this gene was significantly lower in the zfp3 mutant in comparison to wild type (Fig 7b, 7f and 7j). To further test the change of expression level in stress pathway genes, we analyzed several stress marker genes including KIN1, RD22 and RD29B (Fig 7). The results also demonstrated ZFP3 affects plants' tolerance to salt and osmotic stress by regulating the expressions of these genes. However, the mechanism of ZFP3 interacts with these genes controls salt and osmotic stress should be further investigated.