Silencing of the SL-ZH13 Transcription Factor Gene Decreases the Salt Stress Tolerance of Tomato

in Journal of the American Society for Horticultural Science
View More View Less
  • 1 College of Horticulture and Landscape Architecture, Northeast Agricultural University, Harbin, China 150030
  • | 2 College of Agronomy, Northeast Agricultural University, Harbin, China 150030
  • | 3 College of Horticulture and Landscape Architecture, Northeast Agricultural University, Harbin, China 150030

Zinc finger-homeodomains (ZF-HDs) are considered transcription factors that are involved in a variety of life activities in plants, but their function in regulating plant salt stress tolerance is unclear. The SL-ZH13 gene is significantly upregulated under salt stress treatment in tomato (Solanum lycopersicum) leaves, per our previous study. In this study, to further understand the role that the SL-ZH13 gene played in the response process of tomato plants under salt stress, the virus-induced gene silencing (VIGS) method was applied to down-regulate SL-ZH13 expression in tomato plants, and these plants were treated with salt stress to analyze the changes in salt tolerance. The silencing efficiency of SL-ZH13 was confirmed by quantitative real-time PCR analysis. SL-ZH13-silenced plants wilted faster and sooner than control plants under the same salt stress treatment condition, and the main stem bending angle of SL-ZH13-silenced plants was smaller than that of control plants. Physiological analysis showed that the activities of superoxide dismutase, peroxidase, and proline content in SL-ZH13-silenced plants were lower than those in control plants at 1.5 and 3 hours after salt stress treatment. The malondialdehyde content of SL-ZH13-silenced plants was higher than that in control plants at 1.5 and 3 hours after salt stress treatment; H2O2 and O2- accumulated much more in leaves of SL-ZH13-silenced plants than in leaves of control plants. These results suggested that silencing of the SL-ZH13 gene affected the response of tomato plants to salt stress and decreased the salt stress tolerance of tomato plants.

Abstract

Zinc finger-homeodomains (ZF-HDs) are considered transcription factors that are involved in a variety of life activities in plants, but their function in regulating plant salt stress tolerance is unclear. The SL-ZH13 gene is significantly upregulated under salt stress treatment in tomato (Solanum lycopersicum) leaves, per our previous study. In this study, to further understand the role that the SL-ZH13 gene played in the response process of tomato plants under salt stress, the virus-induced gene silencing (VIGS) method was applied to down-regulate SL-ZH13 expression in tomato plants, and these plants were treated with salt stress to analyze the changes in salt tolerance. The silencing efficiency of SL-ZH13 was confirmed by quantitative real-time PCR analysis. SL-ZH13-silenced plants wilted faster and sooner than control plants under the same salt stress treatment condition, and the main stem bending angle of SL-ZH13-silenced plants was smaller than that of control plants. Physiological analysis showed that the activities of superoxide dismutase, peroxidase, and proline content in SL-ZH13-silenced plants were lower than those in control plants at 1.5 and 3 hours after salt stress treatment. The malondialdehyde content of SL-ZH13-silenced plants was higher than that in control plants at 1.5 and 3 hours after salt stress treatment; H2O2 and O2- accumulated much more in leaves of SL-ZH13-silenced plants than in leaves of control plants. These results suggested that silencing of the SL-ZH13 gene affected the response of tomato plants to salt stress and decreased the salt stress tolerance of tomato plants.

Salt stress is a problem for plant growth and agricultural productivity. During different developmental stages, cultivated plants are also exposed to changes in the environment and respond by activating gene expression (Yanez et al., 2009). In studies of stress tolerance in plants, it is found that the transcription factor activates or inhibits the expression of related genes through interactions with other related proteins or itself and then plays an important role in regulating the adaptability of plants to adverse conditions.

The homeobox (HB) gene encodes a highly conserved 60–61 amino acid homeodomain (HD), which confers sequence-specific DNA binding function by folding into a characteristic three alpha helix structure (Ariel et al., 2007; Hanes and Brent, 1989; Zhao et al., 2011). Plants have evolved the specific HD-Zip transcriptional factor family (Ariel et al., 2007), the members of which bear a unique leucine zipper domain at the C-terminus. Zinc fingers, which consist of two pairs of conserved cysteine and/or histidine residues binding a single zinc ion to form a finger-shaped loop (Klug and Schwabe, 1995), are necessary motifs that are found widely in regulatory proteins (Hu et al., 2018; Krishna et al., 2003; Takatsuji, 1999). According to the nature, number, and spacing pattern of zinc-binding residues, zinc fingers could be classified into different types (Englbrecht et al., 2004; Kosarev et al., 2002; Li et al., 2001; Wang et al., 2016; Yanagisawa, 2004). ZF-HD proteins are one of the HD-containing protein families.

The ZF-HD subfamily of homeobox genes has been researched in some model plants. They are considered transcription factors that regulate biotic and abiotic stress, and plant development processes. Functional studies of ZF-HD in abiotic stress responses have been reported in different plant species. AtZHD1 is a transcriptional regulator that binds to the promoter region of ERD1 (early response to dehydration stress 1), and its expression is induced by drought, salinity, and abscisic acid [ABA (Tran et al., 2007)]. The overexpression of NAC and AtZHD1 increases drought tolerance in arabidopsis [Arabidopsis thaliana (Wang et al., 2014)]. Thirty-one ZF-HD genes were identified in chinese cabbage (Brassica rapa ssp. pekinensis), and most of these genes are significantly induced under abiotic stresses (Wang et al., 2016).

Salt stress mainly causes oxidative damage in plants due to the overproduction of reactive oxygen species [ROS (Ali et al., 2017a; Ashraf, 2009)]. Superoxide anions (O2-) and hydrogen peroxide (H2O2) are important ROS that can initiate a series of destructive processes, leading to gradual lipid peroxidation and the inactivation of antioxidant enzymes (Tanou et al., 2009). Plants developed an antioxidative defense mechanism for the detoxification of excessively produced ROS. Among various antioxidants, superoxide dismutase (SOD) and peroxidase (POD) are enzymatic ones, while proline (Pro) is a non-enzymatic metabolite. These antioxidants all serve as ROS quenchers, thus protecting cells from oxidative damage (Yadav et al., 2016). The overproduction of ROS results in the accumulation of malondialdehyde (MDA) due to lipid peroxidation. MDA content estimation provides an effective means for assessing the extent of membrane damage (Ali et al., 2017b).

In our previous study, 22 ZF-HD genes (SL-ZH1SL-ZH22) were identified from tomato (Hu et al., 2018), and most of these genes were responsive to abiotic stresses of cold, drought, and salinity. SL-ZH13 is one of the salinity-responsive genes. This gene is upregulated significantly under salt stress treatment in tomato leaves. In this study, to understand the function of the SL-ZH13 gene and determine the relationship between SL-ZH13 expression and salt tolerance ability of tomato plants, we applied virus-induced gene silencing technology to decrease the expression level of SL-ZH13 in tomato plants and analyzed changes in the salt tolerance ability in these plants.

Materials and Methods

Plant material.

Tomato ‘Moneymaker’ was provided by Tomato Research Institute of Northeast Agricultural University (Harbin, China). All plants were grown at the Horticultural Experimental Station of Northeast Agricultural University. At the two- to three-leaf stage, the seedlings were used in a VIGS study.

Target fragments amplification and vectors construction.

Total RNA was extracted from ‘Moneymaker’ seedling leaves using the Trizol method (Trizol; Invitrogen, Shanghai, China) according to the manufacturer’s instructions. First-strand cDNA was synthesized using an M-MLVRTase cDNA synthesis system (M-MLVRTase cDNA synthesis kit; Takara, Dalian, China) according to the manufacturer’s instructions. Primers (Table 1) of the SL-ZH13 fragment were designed using the primer design tool of the National Center for Biotechnology Information [NCBI (Ye et al., 2012)], based on the sequence of Solyc03g098060 from Sol Genomics Network [SGN (Fernandez-Pozo et al., 2014)]. The resulting PCR products were analyzed by agarose gel electrophoresis; bands with the correct size were excised from the gel and purified with a PCR purification system (PCR purification kit, Takara). The purified products were cloned into the pMD18-T vector (Takara) and sequenced (Sangon Biotech Co., Shanghai, China).

Table 1.

Primers used for SL-ZH13 fragment amplification and qRT-PCR analysis of SL-ZH13 expression pattern.

Table 1.

After the target fragment was identified, re-amplifications were applied using the primers with added restriction site sequences (EcoRI and BamHI). Target products were excised from the gel, purified and cloned into tobacco rattle virus RNA2 (TRV2), which was digested with restriction endonucleases EcoRI and BamHI (Takara). The cloned TRV2 vectors were transformed into competent cells of Escherichia coli DH5α and incubated at 37 °C overnight. The white clones grown on lysogeny broth (LB) medium containing kanamycin/X-gal/isopropyl β-D-thiogalactoside (IPTG) were picked and cultured in a liquid LB culture with 50 μg·mL−1 kanamycin, and the plasmids were extracted and verified by sequencing. The identified TRV2-SL-ZH13 strain was cultured in a liquid LB culture with 50 μg·mL−1 kanamycin and used for plasmid extraction. The TRV constructs were transformed into Agrobacterium tumefaciens GV3101 according to the method of Huang et al. (2008).

In this experiment, silencing of phytoene desaturase (PDS), which causes the plants to photobleach, was used as a silencing experimental efficiency control. This gene was silenced in a separate experimental group parallel to the experimental group with silencing of the target gene SL-ZH13. GV3101 carrying the TRV2-PDS vector we used in this experiment was constructed in our previous study (Zhao et al., 2017) and preserved at the Tomato Research Institute of Northeast Agricultural University.

Infiltration of tomato seedlings.

Seedlings at the two- to three-leaf stage were used for infiltration experiments. For each target fragment, 20 seedlings were prepared for one experimental repeat, with three experimental repeats in total. All steps were performed as described by Velásquez et al. (2009).

Confirmation of gene silencing efficency by quantitative real-time PCR.

The leaves were collected from the VIGS-treated seedlings and the control seedlings at 25 d after infiltration. RNA extraction and cDNA synthesis were carried out as mentioned above. Quantitative real-time PCR (qRT-PCR) was performed according to a previous study (Zhao et al., 2016). The primers used for target fragment amplification were used as qRT-PCR primers (Table 1). The data were analyzed using the 2–∆∆CT method (Livak and Schmittgen, 2001) with EFα1 as a reference gene for normalization (Rotenberg et al., 2006).

Salt stress treatment and phenotypic observation.

According to the method of our previous study (Hu et al., 2018), SL-ZH13 silenced seedlings (selected based on the qRT-PCR result of gene silencing efficiency confirmation) and control seedlings were grown in nutrient solution for 24 h and then irrigated with 250 mm NaCl for 24 h. Tomato leaf samples were gathered at 1.5, 3, 6, 12, and 24 h after treatment and stored at –80 °C for analysis. Three biological replicates were carried out. The angle between the part of the main stem inside the bottle and the part of the main stem outside the bottle was measured as the plant bending angle. Ten plants of each sample were measured, and the bending angle values were used to obtain the mean bending angle. Bending angle value data were analyzed with analysis of variance (ANOVA) (significance level 0.05) using SAS (version 9.1.3; SAS Institute, Cary, NC).

Assay of SOD, POD, Pro and MDA.

The SOD activity was determined following the method of Giannopolitis and Ries (1977), and the activity of POD was determined following Chance and Maehly (1955). The Pro content in leaf homogenates was estimated by the method of Bates et al. (1973) with the help of the standard curve. The MDA content was estimated following the method of Cakmak and Horst (1991) and determined following the method of Ali et al. (2017a). The activities of SOD and POD and the content of MDA were estimated using the same method of mean bending angle mentioned above.

Nitroblue tetrazolium (NBT) and 3,3′ diaminobenzidine (DAB) staining.

Leaves collected from control plants and SL-ZH13 silenced plants at 0, 1.5, and 3 h after treatment were used for superoxide radical staining. NBT staining was performed according to Rao and Davis (1999), and DAB staining was performed according to Fryer et al. (2002) and Ivan et al. (2009). O2.- was visualized as a blue color produced by NBT precipitation. H2O2 was visualized as a brown color due to DAB polymerization.

Gene expression pattern analysis.

To further investigate the expression level of SL-ZH13 in gene-silenced plants during the treatment process, leaves collected at 1.5, 3, 6, 12, and 24 h after treatment were used for qRT-PCR analysis. RNA extraction and cDNA synthesis were carried out as mentioned above. qRT-PCR was performed according to a previous study (Zhao et al., 2016). The primers used for target fragment amplification were used as qRT-PCR primers (Table 1). The data were analyzed using the 2–∆∆CT method (Livak and Schmittgen, 2001) with EFα1 as a reference gene for normalization (Rotenberg et al., 2006).

Results

Verification of PDS silencing efficiency.

Silencing of the PDS control gene caused photobleaching in tomato plants. Photobleaching was observed as soon as 12 d after infiltration. Of 45 plants in three experimental repeats in total, 41 (91%) showed the characteristic photobleaching phenotype 20 d after infiltration. Empty TRV2 vector-control plants and untreated-control plants grew normally. SL-ZH13-silenced plants did not show obvious abnormalities in tomato plant morphology.

The expression levels of target gene SL-ZH13-silenced plants were compared with the expression levels in wild-type control plants and empty vector control plants. The results showed that the expression level of the SL-ZH13 gene decreased significantly after SL-ZH13 silencing in most plants, but there were five plants showing normal expression levels of the SL-ZH13 gene. Fifteen successfully silenced plants that showed obviously down-regulated expression patterns were used for subsequent experiments (Fig. 1).

Fig. 1.
Fig. 1.

Changes in SL-ZH13 expression. Twenty SL-ZH13-silenced tomato plants were analyzed (VIGS-1 to VIGS-20); 15 SL-ZH13-silenced plants showed down-regulated expression pattern of the SL-ZH13 gene. CK = control plant; VIGS-TRV2 = empty vector control plants.

Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 143, 5; 10.21273/JASHS04477-18

Plant phenotype observation under salt stress treatment.

As shown in Fig. 2, all plants under salt stress treatment were wilted. This phenomenon was observed at 1.5 h after treatment and was even worse at 3 h after treatment. Plant wilt was very obvious on stems and petioles but not on leaves. Petioles and stems were curved. The mean bending angle of SL-ZH13-silenced plants during wilting is smaller than that of CK and CK-TRV2 plants.

Fig. 2.
Fig. 2.

Phenotype changes of SL-ZH13-silenced (VIGS) tomato plants under salt stress treatment. Plant wilt was very obvious on stems and petioles but not on leaves. Petioles and stems were curved. The mean bending angle of SL-ZH13-silenced plants during wilting is smaller than that of control (CK) and empty vector control plants (CK-TRV2) plants. (A) CK plants at 0 h after salt stress treatment; (B) CK plants at 1.5 h after salt stress treatment; (C) CK plants at 3 h after salt stress treatment; (D) mean bending angle of SL-ZH13-silenced plants; (E) CK-TRV2 plants at 0 h after salt stress treatment; (F) CK-TRV2 plants at 1.5 h after salt stress treatment; (G) CK-TRV2 plants at 3 h after salt stress treatment; (H) mean bending angle of CK-TRV2 plants; (I) SL-ZH13-silenced plants at 0 h after salt stress treatment; (J) SL-ZH13-silenced plants at 1.5 h after salt stress treatment; (K) SL-ZH13-silenced plants at 3 h after salt stress treatment; and (L) mean bending angle of SL-ZH13-silenced plants. Red lines show the bending angle of plants.

Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 143, 5; 10.21273/JASHS04477-18

Gene expression pattern analysis.

To further confirm the effect of gene silencing throughout the whole treatment process and to compare expression levels between control plants and SL-ZH13-silenced plants directly, qRT-PCR analysis was applied to leaves gathered from control plants and SL-ZH13-silenced plants at 0, 1.5, 3, 6, 12, and 24 h after salt stress treatment. The results (Fig. 3) showed that the SL-ZH13 gene expression level increased after salt stress treatment in control plants and peaked at 3 h after salt stress treatment. In SL-ZH13-silenced plants, the expression levels were very low and stable.

Fig. 3.
Fig. 3.

SL-ZH13 expression patterns of different tomato plants under salt stress treatment. The expression level of the SL-ZH13 gene was very low and stable in SL-ZH13-silenced plants (VIGS). CK = control plants; CK-TRV2 = empty vector control plants.

Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 143, 5; 10.21273/JASHS04477-18

Assay of SOD, POD, Pro, and MDA.

SOD and POD activities and Pro content were increased significantly in all studied plants due to the imposition of salt stress (Fig. 4). Salt-induced increases in SOD and POD activities and Pro content were larger in the control plants CK and CK-TRV2 compared with the SL-ZH13-silenced plants. Similar to the SOD and POD activities and Pro content, the leaf MDA content also increased significantly due to the imposition of salt stress, while a smaller increase was found in control plants compared with SL-ZH13-silenced plants.

Fig. 4.
Fig. 4.

Activities of superoxide dismutase (SOD) and peroxidase (POD) and contents of proline (Pro) and malondialdehyde (MDA) in tomato plants under salt stress treatment. The SOD and POD activities and Pro content in SL-ZH13-silenced (VIGS) plants were lower than those in the control (CK) and empty vector control (CK-TRV2) plants at 1.5 and 3 h after salt stress treatment. The MDA content in SL-ZH13-silenced plants was higher than that in the control plants CK and CK-TRV2 at 1.5 and 3 h after salt stress treatment.

Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 143, 5; 10.21273/JASHS04477-18

NBT and DAB staining.

Superoxide radical detection and quantification were performed using the NBT staining method. As shown in Fig. 5, the levels of superoxide radical staining before salt treatment were quite similar between control plants and SL-ZH13-silenced plants. A blue coloration appeared in all studied plants treated with salt. The blue coloration in SL-ZH13-silenced plants was stronger than that in the control plants CK and CK-TRV2 at the same treatment time. A brown precipitate in the presence of H2O2 was detected in all DAB strained leaves. Similar to the NBT staining result, the brown precipitate in SL-ZH13-silenced plants was stronger than that in the control plants CK and CK-TRV2.

Fig. 5.
Fig. 5.

Nitroblue tetrazolium (NBT) and 3,3′ diaminobenzidine (DAB) staining of tomato leaves. The blue coloration in SL-ZH13-silenced (VIGS) plants was stronger than that in the control (CK) and empty vector control (CK-TRV2) plants at the same treatment time; the brown precipitate in SL-ZH13-silenced plants was stronger than that in the control plants CK and CK-TRV2. (A) CK samples at 0 h after salt stress treatment; (B) CK samples at 1.5 h after salt stress treatment; (C) CK samples at 3 h after salt stress treatment; (D) CK-TRV2 samples at 0 h after salt stress treatment; (E) CK-TRV2 samples at 1.5 h after salt stress treatment; (F) CK-TRV2 samples at 3 h after salt stress treatment; (G) SL-ZH13-silenced plants at 0 h after salt stress treatment; (H) SL-ZH13-silenced plants at 1.5 h after salt stress treatment; and (I) SL-ZH13-silenced plants at 3 h after salt stress treatment.

Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 143, 5; 10.21273/JASHS04477-18

Discussion

In this study, VIGS technology was used to down-regulate the SL-ZH13 gene. qRT-PCR analysis results showed that the gene silencing success rate of twenty plants was 75% (plants with a SL-ZH13 gene expression level reduction of more than 50% were classified as successfully silenced plants); the average silencing efficiency of 15 successfully silenced plants was 54%. This silencing efficiency is relatively low compared with our previous study (Zhao et al., 2017). The phenotypic observation indicated that the down-regulation of SL-ZH13 gene expression in this study caused a change in the plant phenotype under salt stress treatment. At 1.5 and 3 h after salt stress treatment, SL-ZH13-silenced plants showed more serious wilting than control plants and stems drooped more obviously. Another analysis also showed changes caused by SL-ZH13 silencing. These results indicated that the influence of gene function analysis in this study caused by the relatively low silencing efficiency is limited.

Under stressful biotic and abiotic conditions, enhanced formation of ROS attacks nucleic acids, photosynthetic pigments, proteins and lipids, as well as induces both cellular damage and protective responses (Caverzan et al., 2016; Lin and Kao, 2000). Plant cells contain a wide range of antioxidant enzymes, such as SOD, POD, CAT, and glutathione-S-transferase (GST), and nonenzymatic metabolites (e.g., Pro), which serve as ROS quenchers, thus protecting cells from oxidative damage (Yadav et al., 2016). MDA is a by-product of lipid peroxidation and an indicator of abiotic stress-induced oxidative damage in plants. Damage to lipids further results in the generation of ROS and subsequent oxidative stress. In this study, SOD and POD activities as well as Pro and MDA contents were measured to evaluate plant antioxidant ability under salt stress. According to our results, SOD and POD activities as well as Pro and MDA contents all increased in plants treated with salt stress, while the activities of SOD and POD and the content of Pro in SL-ZH13-silenced plants were all lower than those in control plants at the same treated time. The MDA content of SL-ZH13-silenced plants was higher than that of control plants at the same treated time. This result indicated that silencing of the SL-ZH13 gene reduced the salt stress tolerance of tomato plants. Similar results were also found in plant salt stress tolerance ability studies in arabidopsis (Zhang et al., 2016). The study indicated that arabidopsis overexpressing the Tamarix hispida zinc finger protein ThZFP1 showed more tolerance to salt and osmotic stress. The POD and SOD activities, Pro content and expression levels of relative genes, such as delta-pyrroline-5-carboxylate synthetase (P5CS), all increased (Sun et al., 2007; Zang et al., 2015).

DAB and NBT staining methods have been used in many abiotic stress tolerance-related gene functional studies (Fryer et al., 2002). DAB reacts with H2O2 in the presence of peroxidases to produce a brown polymerization product, making the production of H2O2 visible in leaves infiltrated with DAB. O2- was visualized as a blue color produced by NBT precipitation (Fryer et al., 2002). These analyses could intuitively show the damage situation of the study plants. In this study, DAB and NBT staining results showed that H2O2 and O2- accumulated in tomato leaves treated with salt stress, and this accumulation increased with treatment time. At 1.5 and 3 h after salt stress treatment, brown and blue colors of leaves from SL-ZH13-silenced plants were all deeper than colors of control plant leaves. The results showed that salt stress caused more damage to plants with SL-ZH13 gene silencing and coincided with SOD and POD activity as well as Pro and MDA content changes.

In all analyses of this study, there were no remarkable differences between control plants and SL-ZH13-silenced plants before salt stress treatment. This finding may suggest that the down-regulation of the SL-ZH13 gene did not cause remarkable damage to the plants.

In conclusion, we obtained tomato plants with SL-ZH13 gene silencing using the VIGS method. The silencing efficiency was confirmed by the down-regulation of SL-ZH13 gene expression. SL-ZH13- silenced plants wilted faster and sooner than control plants under the same salt stress treatment conditions. Physiological analysis showed that the SOD and POD activities and Pro content in SL-ZH13- silenced plants were lower than those in control plants at 1.5 and 3 h after salt stress treatment. The MDA content of SL-ZH13-silenced plants was higher than that in control plants at 1.5 and 3 h after salt stress treatment. H2O2 and O2- accumulated much more in leaves from SL-ZH13-silenced plants than those from control plants. These results suggested that silencing of the SL-ZH13 gene affected the response of tomato plants under salt stress and decreased the salt stress tolerance of tomato plants.

1

These authors contributed equally to this work.

Literature Cited

  • Ali, Q., Javed, M.T., Noman, A., Haider, M.Z., Waseem, M., Iqbal, N., Waseem, M., Shah, M.S., Shahzad, F. & Perveen, R. 2017a Assessment of drought tolerance in mung bean cultivars/lines as depicted by the activities of germination enzymes, seedling’s antioxidative potential and nutrient acquisition Arch. Agron. Soil Sci. 64 84 102

    • Search Google Scholar
    • Export Citation
  • Ali, Q., Daud, M.K., Haider, M.Z., Alid, S., Rizwand, M., Aslame, N., Nomanf, A., Iqbala, N., Shahzada, F., Deebac, F., Alig, I. & Zhu, S.J. 2017b Seed priming by sodium nitroprusside improves salt tolerance in wheat (Triticum aestivum L.) by enhancing physiological and biochemical parameters Plant Physiol. Biochem. 119 50 58

    • Search Google Scholar
    • Export Citation
  • Ariel, F.D., Manavella, P.A., Dezar, C.A. & Chan, R.L. 2007 The true story of the HD-Zip family Trends Plant Sci. 12 419 426

  • Ashraf, M. 2009 Biotechnological approach of improving plant salt tolerance using antioxidants as markers Biotechnol. Adv. 27 84 93

  • Bates, L.S., Waldren, R.P. & Teare, I.D. 1973 Rapid determination of free proline for water stress studies Plant Soil 39 205 208

  • Cakmak, I. & Horst, W.J. 1991 Effect of aluminium on lipid peroxidation, superoxide dismutase, catalase, and peroxidase activities in root tips of soybean (Glycine max) Physiol. Plant. 83 463 468

    • Search Google Scholar
    • Export Citation
  • Caverzan, A., Casassola, A. & Brammer, S.P. 2016 Antioxidant responses of wheat plants under stress Genet. Mol. Biol. 39 1 6

  • Chance, B. & Maehly, A. 1955 Assay of catalases and peroxidases Methods Enzymol. 2 764 775

  • Englbrecht, C.C., Schoof, H. & Böhm, S. 2004 Conservation, diversification and expansion of C2H2 zinc finger proteins in the Arabidopsis thaliana genome BMC Genomics 5 39

    • Search Google Scholar
    • Export Citation
  • Fernandez-Pozo, N., Menda, N., Edwards, J.D., Saha, S., Tecle, I.Y., Strickler, S.R., Bombarely, A., Fisher-York, T., Pujar, A., Foerster, H., Yan, A. & Mueller. L.A. 2015 The Sol Genomics Network (SGN) - From genotype to phenotype to breeding Nucleic Acids Res. 43 1036 1041

    • Search Google Scholar
    • Export Citation
  • Fryer, M.J., Oxborough, K., Mullineaux, P.M. & Baker, N.R. 2002 Imaging of photo-oxidative stress responses in leaves J. Expt. Bot. 53 1249 1254

  • Giannopolitis, C.N. & Ries, S.K. 1977 Superoxide dismutase occurrence in higher plants Plant Physiol. 59 309 314

  • Hanes, S.D. & Brent, R. 1989 DNA specificity of the bicoid activator proteinis determined by homeodomain recognition helix residue 9 Cell 57 1275 1283

    • Search Google Scholar
    • Export Citation
  • Hu, J.k., Gao, Y.M., Zhao, T.T., Li, J.F., Yao, M.N. & Xu, X.Y. 2018 Genome-wide identification and expression pattern analysis of Zinc-finger homeodomain transcription factors in tomato under abiotic stress J. Amer. Soc. Hort. Sci. 143 14 22

    • Search Google Scholar
    • Export Citation
  • Huang, W.Z., Ma, X.R., Wang, Q.L., Gao, Y.F., Xue, Y., Niu, X.L., Yu, G.R. & Liu, Y.S. 2008 Significant improvement of stress tolerance in tobacco plants by overexpressing a stress-responsive aldehyde dehydrogenase gene from maize (Zea mays) Plant Mol. Biol. 68 451 463

    • Search Google Scholar
    • Export Citation
  • Ivan, C., Matthieu, B., Cécile, S., Fanny, R. & Gwenola, G. 2009 Differential patterns of reactive oxygen species and antioxidative mechanisms during atrazine injury and sucrose-induced tolerance in Arabidopsis thaliana plantlets BMC Plant Biol. 9 28

    • Search Google Scholar
    • Export Citation
  • Klug, A. & Schwabe, J.W. 1995 Protein motifs 5. Zinc fingers Federation Amer. Soc. Expt. Biol. 9 597 604

  • Kosarev, P., Mayer, K.F. & Hardtke, C.S. 2002 Evaluation and classification of RING-finger domains encoded by the Arabidopsis genome Genome Biol. 3 1 12

    • Search Google Scholar
    • Export Citation
  • Krishna, S.S., Majumdar, I. & Grishin, N.V. 2003 Structural classification of zinc fingers: Survey and summary Nucleic Acids Res. 31 532 550

  • Li, J.J., Jia, D.X. & Chen, X.M. 2001 HUA1, a regulator of stamen and carpel identities in Arabidopsis, codes for a nuclear RNA binding protein Plant Cell 13 2269 2281

    • Search Google Scholar
    • Export Citation
  • Lin, C.C. & Kao, C.H. 2000 Effect of NaCl stress on H2O2 metabolism in rice leaves Plant Growth Regulat. 30 1151 1155

  • Livak, K.J. & Schmittgen, T.D. 2001 Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method Methods 25 402 408

    • Search Google Scholar
    • Export Citation
  • Rao, M.V. & Davis, K.R. 1999 Ozone-induced cell death occurs via two distinct mechanisms in arabidopsis: The role of salicylic acid Plant J. 17 603 614

    • Search Google Scholar
    • Export Citation
  • Rotenberg, D., Thompson, T.S., German, T.L. & Willis, D.K. 2006 Methods for effective real-time RT-PCR analysis of virus-induced gene silencing J. Virol. Methods 138 49 59

    • Search Google Scholar
    • Export Citation
  • Sun, J.Q., Jiang, H.L., Xu, Y.X., Li, H.M., Wu, X.Y., Xie, Q. & Li, C.Y. 2007 The CCCH-Type zinc finger proteins AtSZF1 and AtSZF2 regulate salt stress responses in Arabidopsis Plant Cell Physiol. 8 1148 1158

    • Search Google Scholar
    • Export Citation
  • Takatsuji, H. 1999 Zinc-finger proteins: The classical zinc finger emerges in contemporary plant science Plant Mol. Biol. 39 1073 1078

  • Tanou, G., Molassiotis, A. & Diamantidis, G. 2009 Hydrogen peroxide- and nitric oxide-induced systemic antioxidant prime-like activity under NaCl-stress and stress-free conditions in citrus plants J. Plant Physiol. 166 1904 1913

    • Search Google Scholar
    • Export Citation
  • Tran, L.S., Nakashima, K., Sakuma, Y., Osakabe, Y., Qin, F., Simpson, S.D., Maruyama, K., Fujita, Y., Shinozaki, K. & Yamaguchi-Shinozaki, K. 2007 Co-expression of the stress-inducible zinc finger homeodomain ZFHD1 and NAC transcription factors enhances expression of the ERD1 gene in Arabidopsis Plant J. 49 46 63

    • Search Google Scholar
    • Export Citation
  • Velásquez, A.C., Chakravarthy, S. & Martin, G.B. 2009 Virus-induced gene silencing (VIGS) in Nicotiana benthamiana and tomato. JoVE. 28:1292

  • Wang, H., Yin, X., Li, X., Wang, L., Zheng, Y., Xu, X. & Wang, X. 2014 Genome-wide identification, evolution and expression analysis of the grape (Vitis Vinifera L.) zinc finger-homeodomain gene family Intl. J. Mol. Sci. 15 5730 5748

    • Search Google Scholar
    • Export Citation
  • Wang, W.L., Wu, P., Li, Y. & Hou, X.L. 2016 Genome-wide analysis and expression patterns of ZF-HD transcription factors under different developmental tissues and abiotic stresses in chinese cabbage Mol. Genet. Genomics 291 1451 1464

    • Search Google Scholar
    • Export Citation
  • Yadav, G., Srivastava, P.K., Parihar, P., Tiwari, S. & Prasad, S.M. 2016 Oxygen toxicity and antioxidative responses in arsenic stressed Helianthus annuus L. seedlings against UV-B J. Photochem. Photobiol. B 165 58 70

    • Search Google Scholar
    • Export Citation
  • Yanagisawa, S. 2004 Dof domain proteins: Plant-specific transcription factors associated with diverse phenomena unique to plants Plant Cell Physiol. 45 386 391

    • Search Google Scholar
    • Export Citation
  • Yanez, M., Caceres, S., Orellana, S., Bastias, A., Verdugo, I., Ruiz-Lara, S. & Casaretto, J.A. 2009 An abiotic stress-responsive bZIP transcription factor from wild and cultivated tomatoes regulates stress-related genes Plant Cell Rpt. 28 1497 1507

    • Search Google Scholar
    • Export Citation
  • Ye, J., Coulouris, G., Zaretskaya, I., Cutcutache, I., Rozen, S. & Madden, T. 2012 Primer-BLAST: A tool to design target-specific primers for polymerase chain reaction BMC Bioinformatics 13 134

    • Search Google Scholar
    • Export Citation
  • Zang, D., Wang, C., Ji, X. & Wang, Y. 2015 Tamarix hispida zinc finger protein ThZFP1 participates in salt and osmotic stress tolerance by increasing proline content and SOD and POD activities Plant Sci. 235 111 121

    • Search Google Scholar
    • Export Citation
  • Zhang, A.D., Liu, D.D., Hua, C.M., Yan, A., Liu, B.H., Wu, M.J., Liu, Y.H., Huang, L.L., Ali, I. & Gan, Y.B. 2016 The Arabidopsis gene zinc finger protein 3(ZFP3) is involved in salt stress and osmotic stress response PLoS One 11 e0168367

    • Search Google Scholar
    • Export Citation
  • Zhao, T.T., Jiang, J.B., Liu, G., He, S.S., Zhang, H., Chen, X.L., Li, J.F. & Xu, X.Y. 2016 Mapping and candidate gene screening of tomato Cladosporium fulvum-resistant gene Cf-19, based on high-throughput sequencing technology BMC Plant Biol. 16 51

    • Search Google Scholar
    • Export Citation
  • Zhao, T.T., Yang, H.H., Jiang, J.B., Liu, G., Zhang, H., Xiao, D., Chen, X.L., Li, J.F. & Xu, X.Y. 2017 Silencing of the SAMDC gene decreases resistance of tomato to Cladosporium fulvum Physiol. Mol. Plant. 102 1 7

    • Search Google Scholar
    • Export Citation
  • Zhao, Y., Zhou, Y.Q., Jiang, H.Y., Li, X.Y., Gan, D.F., Pen, X.J., Zhu, S.W. & Chen, B.J. 2011 Systematic analysis of aequences and expression patterns of drought-responsive members of the HD-Zip gene family in maize PLoS One 6 e28488

    • Search Google Scholar
    • Export Citation

Contributor Notes

This study was funded by the National Key R&D Progrem of China (2017YFD0101900), China Agriculture Research System (CARS-25-A-15), Breeding of New Staple Vegetable Varieties of Heilongjiang Province (GA15B103) Natural Science Foundation of Heilongjiang Province (C2017024) and Youth Talent Support Program of Northeast Agricultural University (17QC07).

Corresponding authors. E-mail: qshchen@126.com or xxy709@126.com.

  • View in gallery

    Changes in SL-ZH13 expression. Twenty SL-ZH13-silenced tomato plants were analyzed (VIGS-1 to VIGS-20); 15 SL-ZH13-silenced plants showed down-regulated expression pattern of the SL-ZH13 gene. CK = control plant; VIGS-TRV2 = empty vector control plants.

  • View in gallery

    Phenotype changes of SL-ZH13-silenced (VIGS) tomato plants under salt stress treatment. Plant wilt was very obvious on stems and petioles but not on leaves. Petioles and stems were curved. The mean bending angle of SL-ZH13-silenced plants during wilting is smaller than that of control (CK) and empty vector control plants (CK-TRV2) plants. (A) CK plants at 0 h after salt stress treatment; (B) CK plants at 1.5 h after salt stress treatment; (C) CK plants at 3 h after salt stress treatment; (D) mean bending angle of SL-ZH13-silenced plants; (E) CK-TRV2 plants at 0 h after salt stress treatment; (F) CK-TRV2 plants at 1.5 h after salt stress treatment; (G) CK-TRV2 plants at 3 h after salt stress treatment; (H) mean bending angle of CK-TRV2 plants; (I) SL-ZH13-silenced plants at 0 h after salt stress treatment; (J) SL-ZH13-silenced plants at 1.5 h after salt stress treatment; (K) SL-ZH13-silenced plants at 3 h after salt stress treatment; and (L) mean bending angle of SL-ZH13-silenced plants. Red lines show the bending angle of plants.

  • View in gallery

    SL-ZH13 expression patterns of different tomato plants under salt stress treatment. The expression level of the SL-ZH13 gene was very low and stable in SL-ZH13-silenced plants (VIGS). CK = control plants; CK-TRV2 = empty vector control plants.

  • View in gallery

    Activities of superoxide dismutase (SOD) and peroxidase (POD) and contents of proline (Pro) and malondialdehyde (MDA) in tomato plants under salt stress treatment. The SOD and POD activities and Pro content in SL-ZH13-silenced (VIGS) plants were lower than those in the control (CK) and empty vector control (CK-TRV2) plants at 1.5 and 3 h after salt stress treatment. The MDA content in SL-ZH13-silenced plants was higher than that in the control plants CK and CK-TRV2 at 1.5 and 3 h after salt stress treatment.

  • View in gallery

    Nitroblue tetrazolium (NBT) and 3,3′ diaminobenzidine (DAB) staining of tomato leaves. The blue coloration in SL-ZH13-silenced (VIGS) plants was stronger than that in the control (CK) and empty vector control (CK-TRV2) plants at the same treatment time; the brown precipitate in SL-ZH13-silenced plants was stronger than that in the control plants CK and CK-TRV2. (A) CK samples at 0 h after salt stress treatment; (B) CK samples at 1.5 h after salt stress treatment; (C) CK samples at 3 h after salt stress treatment; (D) CK-TRV2 samples at 0 h after salt stress treatment; (E) CK-TRV2 samples at 1.5 h after salt stress treatment; (F) CK-TRV2 samples at 3 h after salt stress treatment; (G) SL-ZH13-silenced plants at 0 h after salt stress treatment; (H) SL-ZH13-silenced plants at 1.5 h after salt stress treatment; and (I) SL-ZH13-silenced plants at 3 h after salt stress treatment.

  • Ali, Q., Javed, M.T., Noman, A., Haider, M.Z., Waseem, M., Iqbal, N., Waseem, M., Shah, M.S., Shahzad, F. & Perveen, R. 2017a Assessment of drought tolerance in mung bean cultivars/lines as depicted by the activities of germination enzymes, seedling’s antioxidative potential and nutrient acquisition Arch. Agron. Soil Sci. 64 84 102

    • Search Google Scholar
    • Export Citation
  • Ali, Q., Daud, M.K., Haider, M.Z., Alid, S., Rizwand, M., Aslame, N., Nomanf, A., Iqbala, N., Shahzada, F., Deebac, F., Alig, I. & Zhu, S.J. 2017b Seed priming by sodium nitroprusside improves salt tolerance in wheat (Triticum aestivum L.) by enhancing physiological and biochemical parameters Plant Physiol. Biochem. 119 50 58

    • Search Google Scholar
    • Export Citation
  • Ariel, F.D., Manavella, P.A., Dezar, C.A. & Chan, R.L. 2007 The true story of the HD-Zip family Trends Plant Sci. 12 419 426

  • Ashraf, M. 2009 Biotechnological approach of improving plant salt tolerance using antioxidants as markers Biotechnol. Adv. 27 84 93

  • Bates, L.S., Waldren, R.P. & Teare, I.D. 1973 Rapid determination of free proline for water stress studies Plant Soil 39 205 208

  • Cakmak, I. & Horst, W.J. 1991 Effect of aluminium on lipid peroxidation, superoxide dismutase, catalase, and peroxidase activities in root tips of soybean (Glycine max) Physiol. Plant. 83 463 468

    • Search Google Scholar
    • Export Citation
  • Caverzan, A., Casassola, A. & Brammer, S.P. 2016 Antioxidant responses of wheat plants under stress Genet. Mol. Biol. 39 1 6

  • Chance, B. & Maehly, A. 1955 Assay of catalases and peroxidases Methods Enzymol. 2 764 775

  • Englbrecht, C.C., Schoof, H. & Böhm, S. 2004 Conservation, diversification and expansion of C2H2 zinc finger proteins in the Arabidopsis thaliana genome BMC Genomics 5 39

    • Search Google Scholar
    • Export Citation
  • Fernandez-Pozo, N., Menda, N., Edwards, J.D., Saha, S., Tecle, I.Y., Strickler, S.R., Bombarely, A., Fisher-York, T., Pujar, A., Foerster, H., Yan, A. & Mueller. L.A. 2015 The Sol Genomics Network (SGN) - From genotype to phenotype to breeding Nucleic Acids Res. 43 1036 1041

    • Search Google Scholar
    • Export Citation
  • Fryer, M.J., Oxborough, K., Mullineaux, P.M. & Baker, N.R. 2002 Imaging of photo-oxidative stress responses in leaves J. Expt. Bot. 53 1249 1254

  • Giannopolitis, C.N. & Ries, S.K. 1977 Superoxide dismutase occurrence in higher plants Plant Physiol. 59 309 314

  • Hanes, S.D. & Brent, R. 1989 DNA specificity of the bicoid activator proteinis determined by homeodomain recognition helix residue 9 Cell 57 1275 1283

    • Search Google Scholar
    • Export Citation
  • Hu, J.k., Gao, Y.M., Zhao, T.T., Li, J.F., Yao, M.N. & Xu, X.Y. 2018 Genome-wide identification and expression pattern analysis of Zinc-finger homeodomain transcription factors in tomato under abiotic stress J. Amer. Soc. Hort. Sci. 143 14 22

    • Search Google Scholar
    • Export Citation
  • Huang, W.Z., Ma, X.R., Wang, Q.L., Gao, Y.F., Xue, Y., Niu, X.L., Yu, G.R. & Liu, Y.S. 2008 Significant improvement of stress tolerance in tobacco plants by overexpressing a stress-responsive aldehyde dehydrogenase gene from maize (Zea mays) Plant Mol. Biol. 68 451 463

    • Search Google Scholar
    • Export Citation
  • Ivan, C., Matthieu, B., Cécile, S., Fanny, R. & Gwenola, G. 2009 Differential patterns of reactive oxygen species and antioxidative mechanisms during atrazine injury and sucrose-induced tolerance in Arabidopsis thaliana plantlets BMC Plant Biol. 9 28

    • Search Google Scholar
    • Export Citation
  • Klug, A. & Schwabe, J.W. 1995 Protein motifs 5. Zinc fingers Federation Amer. Soc. Expt. Biol. 9 597 604

  • Kosarev, P., Mayer, K.F. & Hardtke, C.S. 2002 Evaluation and classification of RING-finger domains encoded by the Arabidopsis genome Genome Biol. 3 1 12

    • Search Google Scholar
    • Export Citation
  • Krishna, S.S., Majumdar, I. & Grishin, N.V. 2003 Structural classification of zinc fingers: Survey and summary Nucleic Acids Res. 31 532 550

  • Li, J.J., Jia, D.X. & Chen, X.M. 2001 HUA1, a regulator of stamen and carpel identities in Arabidopsis, codes for a nuclear RNA binding protein Plant Cell 13 2269 2281

    • Search Google Scholar
    • Export Citation
  • Lin, C.C. & Kao, C.H. 2000 Effect of NaCl stress on H2O2 metabolism in rice leaves Plant Growth Regulat. 30 1151 1155

  • Livak, K.J. & Schmittgen, T.D. 2001 Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method Methods 25 402 408

    • Search Google Scholar
    • Export Citation
  • Rao, M.V. & Davis, K.R. 1999 Ozone-induced cell death occurs via two distinct mechanisms in arabidopsis: The role of salicylic acid Plant J. 17 603 614

    • Search Google Scholar
    • Export Citation
  • Rotenberg, D., Thompson, T.S., German, T.L. & Willis, D.K. 2006 Methods for effective real-time RT-PCR analysis of virus-induced gene silencing J. Virol. Methods 138 49 59

    • Search Google Scholar
    • Export Citation
  • Sun, J.Q., Jiang, H.L., Xu, Y.X., Li, H.M., Wu, X.Y., Xie, Q. & Li, C.Y. 2007 The CCCH-Type zinc finger proteins AtSZF1 and AtSZF2 regulate salt stress responses in Arabidopsis Plant Cell Physiol. 8 1148 1158

    • Search Google Scholar
    • Export Citation
  • Takatsuji, H. 1999 Zinc-finger proteins: The classical zinc finger emerges in contemporary plant science Plant Mol. Biol. 39 1073 1078

  • Tanou, G., Molassiotis, A. & Diamantidis, G. 2009 Hydrogen peroxide- and nitric oxide-induced systemic antioxidant prime-like activity under NaCl-stress and stress-free conditions in citrus plants J. Plant Physiol. 166 1904 1913

    • Search Google Scholar
    • Export Citation
  • Tran, L.S., Nakashima, K., Sakuma, Y., Osakabe, Y., Qin, F., Simpson, S.D., Maruyama, K., Fujita, Y., Shinozaki, K. & Yamaguchi-Shinozaki, K. 2007 Co-expression of the stress-inducible zinc finger homeodomain ZFHD1 and NAC transcription factors enhances expression of the ERD1 gene in Arabidopsis Plant J. 49 46 63

    • Search Google Scholar
    • Export Citation
  • Velásquez, A.C., Chakravarthy, S. & Martin, G.B. 2009 Virus-induced gene silencing (VIGS) in Nicotiana benthamiana and tomato. JoVE. 28:1292

  • Wang, H., Yin, X., Li, X., Wang, L., Zheng, Y., Xu, X. & Wang, X. 2014 Genome-wide identification, evolution and expression analysis of the grape (Vitis Vinifera L.) zinc finger-homeodomain gene family Intl. J. Mol. Sci. 15 5730 5748

    • Search Google Scholar
    • Export Citation
  • Wang, W.L., Wu, P., Li, Y. & Hou, X.L. 2016 Genome-wide analysis and expression patterns of ZF-HD transcription factors under different developmental tissues and abiotic stresses in chinese cabbage Mol. Genet. Genomics 291 1451 1464

    • Search Google Scholar
    • Export Citation
  • Yadav, G., Srivastava, P.K., Parihar, P., Tiwari, S. & Prasad, S.M. 2016 Oxygen toxicity and antioxidative responses in arsenic stressed Helianthus annuus L. seedlings against UV-B J. Photochem. Photobiol. B 165 58 70

    • Search Google Scholar
    • Export Citation
  • Yanagisawa, S. 2004 Dof domain proteins: Plant-specific transcription factors associated with diverse phenomena unique to plants Plant Cell Physiol. 45 386 391

    • Search Google Scholar
    • Export Citation
  • Yanez, M., Caceres, S., Orellana, S., Bastias, A., Verdugo, I., Ruiz-Lara, S. & Casaretto, J.A. 2009 An abiotic stress-responsive bZIP transcription factor from wild and cultivated tomatoes regulates stress-related genes Plant Cell Rpt. 28 1497 1507

    • Search Google Scholar
    • Export Citation
  • Ye, J., Coulouris, G., Zaretskaya, I., Cutcutache, I., Rozen, S. & Madden, T. 2012 Primer-BLAST: A tool to design target-specific primers for polymerase chain reaction BMC Bioinformatics 13 134

    • Search Google Scholar
    • Export Citation
  • Zang, D., Wang, C., Ji, X. & Wang, Y. 2015 Tamarix hispida zinc finger protein ThZFP1 participates in salt and osmotic stress tolerance by increasing proline content and SOD and POD activities Plant Sci. 235 111 121

    • Search Google Scholar
    • Export Citation
  • Zhang, A.D., Liu, D.D., Hua, C.M., Yan, A., Liu, B.H., Wu, M.J., Liu, Y.H., Huang, L.L., Ali, I. & Gan, Y.B. 2016 The Arabidopsis gene zinc finger protein 3(ZFP3) is involved in salt stress and osmotic stress response PLoS One 11 e0168367

    • Search Google Scholar
    • Export Citation
  • Zhao, T.T., Jiang, J.B., Liu, G., He, S.S., Zhang, H., Chen, X.L., Li, J.F. & Xu, X.Y. 2016 Mapping and candidate gene screening of tomato Cladosporium fulvum-resistant gene Cf-19, based on high-throughput sequencing technology BMC Plant Biol. 16 51

    • Search Google Scholar
    • Export Citation
  • Zhao, T.T., Yang, H.H., Jiang, J.B., Liu, G., Zhang, H., Xiao, D., Chen, X.L., Li, J.F. & Xu, X.Y. 2017 Silencing of the SAMDC gene decreases resistance of tomato to Cladosporium fulvum Physiol. Mol. Plant. 102 1 7

    • Search Google Scholar
    • Export Citation
  • Zhao, Y., Zhou, Y.Q., Jiang, H.Y., Li, X.Y., Gan, D.F., Pen, X.J., Zhu, S.W. & Chen, B.J. 2011 Systematic analysis of aequences and expression patterns of drought-responsive members of the HD-Zip gene family in maize PLoS One 6 e28488

    • Search Google Scholar
    • Export Citation
All Time Past Year Past 30 Days
Abstract Views 215 0 0
Full Text Views 233 88 10
PDF Downloads 148 80 22