Silencing of the SlZF-31 gene decreases the salt stress tolerance and drought tolerance of tomato

The SlZF-31 gene is a member of the tomato C2H2 transcription factor family. Previous studies have shown that SlZF-31 gene expression is upregulated under drought stress and salt stress, but the specific function of this gene in tomato plants in response to these two kinds of stress is still unclear. To further explore the function of the SlZF-31 gene in tomato under drought stress and salt stress, we employed the virus-induced gene silencing method to reduce the expression of the SlZF-31 gene in tomato. The results showed that TRV2-SlZF-31 plants had higher levels of wilt and stem bending than CK and CK-TRV2 plants under drought and salt stress. The ABA content of TRV2-SlZF-31 plants were lower than those of CK and CK-TRV2 plants. The analysis of physiological indexes showed that the SOD and POD activity and the proline content of TRV2-SlZF-31 plants were lower than those of CK and CK-TRV2 plants, while the MDA content of TRV2-SLlZF-31 plants was higher than those of CK and CK-TRV2 plants. The accumulation of H2O2 and O2− in TRV2-SlZF-31 plants was greater than those in CK and CK-TRV2 plants. The values of the chlorophyll fluorescence parameters (ΦII and qL) of TRV2-SlZF-31 plants were significantly lower than those of CK and CK-TRV2 plants. These results showed that the silencing of the SlZF-31 gene reduces the drought resistance and salt tolerance of tomato. The finding of this study are expected to provide theoretical support for the study of abiotic stress in tomato.


Introduction
Tomatoes are one of the important vegetables in the world, and they inevitably encounter adverse environmental factors during their growth and development, including both biological factors and nonbiological factors. Among these factors, drought causes the greatest economic losses in plants among all abiotic stresses (Placide et al. 2014) and can have a significant effect on water metabolism in tomato plants.
For example, drought stress can reduce the stomatal opening rate to cause morphological, physiological and biochemical changes in tomato plants, thus hindering the normal growth and development of tomatoes (Torres-Ruiz et al. 2015). Tomatoes are a plant that is sensitive to salt. Salt treatment at an appropriate concentration can modulate the flavor, color and soluble substance content of tomato, which is beneficial for improving the sugar-acid ratio of tomato fruit and, thus, the quality of tomato. However, high salt concentrations often lead to slow growth and decrease the yield and quality of tomato . Therefore, salt stress disrupts the ion balance and cell structure in tomato, limits the absorption and utilization of water and nutrients in tomato roots, and thereby affects the growth and development of tomato. When plants are subjected to drought and salt stress, the content of abscisic acid (ABA) in plants increases, which activates the ABA hormone signaling pathway and induces the expression of downstream transcription factors (TFs). TFs activate gene expression through cis-acting elements to improve the tolerance of plants to the stress response  (Wang et al. 2011). There are many TFs related to abiotic stress in the whole tomato genetic system, among which the C2H2 zinc finger protein family is composed of TFs associated with abiotic stress. The C2H2 zinc finger protein forms a zinc finger structure composed of approximately 25-30 amino acids. The conserved amino acid sequence is C-X2 ~ 4-C-X3-P-X5-L-X2-H-X3 ~ 5-H (C: cysteine, H: histidine, X: amino acid, P: phenylalanine, L: leucine) (Shimeid 2008). Two cysteines and two histidines combine with zinc ions to form a coordination bond. These amino acid sequences can form a compact finger-like tetrahedral structure in the presence of coordination bonds and remain stable (Pabo et al. 2001). Within the zinc finger structure, there is a highly conserved sequence (QALGGH) located at the junction with DNA, and this conserved amino acid sequence is unique to plant zinc finger proteins, which indicates that C2H2 zinc finger proteins may play a regulatory role in the unique growth and development of plants (Huang et al. 2005).
In 1992, the first EPF1 gene of the C2H2 zinc finger was cloned from the petunia, and in recent years, the study of plant-type C2H2 zinc finger proteins in tobacco, Arabidopsis and tomato has been reported in plants; for example, C2H2type zinc finger proteins have been found to regulate plant growth and development and to play an important role in abiotic stress (Amarjeet et al. 2013;Englbrecht et al. 2004). The overexpression of the TaZFP1B gene of C2H2 type zinc indicating protein in wheat, can stimulate oxidative reactions and hormone signal transduction pathways in plants and increase the drought tolerance of wheat (Cheuk et al. 2020). We previously performed a genome-wide analysis of the C2H2-ZFP TF family and found that 7 and 5 genes were specifically expressed during drought stress and salt stress, respectively (Zhao et al. 2020).
Virus-induced gene silencing (VIGS) is a genetic technique that inhibits the expression of endogenous genes in plants by inserting recombinant viruses into target gene fragments and is used mainly for the functional analysis of genes (Benedito et al. 2004). VIGS technology has been widely used in the study of plant gene function in fields such as adversity stress, disease defense and plant growth research during the twenty-first century (Benedito et al. 2004;Anand et al. 2007;Senthil-Kumar et al. 2007). According to a survey of the literature, this technology has been applied in more than 30 species of plants, such as Arabidopsis, rice, tobacco, tomato, cucumber, cabbage and other species (Becker and Lange 2009). In recent years, the use of VIGS technology in tomato has also been increasing. In the present study, VIGS technology was used to reduce the expression level of the SlZF-31 gene to analyze and study the function of this gene under abiotic stress (drought stress and salt stress). This study will provide a theoretical basis and is of practical significance for future research on the function of this gene and C2H2 zinc finger proteins in general.

Plant materials
Seeds of the tomato variety "Moneymaker" were provided by the Tomato Research Institute of Northeast Agricultural University (Harbin, China) and were seeded in a multihole tray and incubated in the dark at 25 °C with 75% humidity for 5 d until seedling emergence. The seedlings were cultured in incubators at 30 °C with 75% humidity under a 16 h light, 8 h dark cycle until the first real leaf emerged, and the tomato seedlings were used for the VIGS experiment.

VIGS vector construction for the SlZF-31 gene and preparation of the Agrobacterium suspension
Total RNA was extracted from tomato leaves using the Trizol method described previously (Zhao et al. 2018a, b). The specific steps were modified according to the instructions provided for the reagent by Invitrogen. cDNA extraction was performed in accordance with the cDNA Synthesis Kit of Vazyme Biotech Co., Ltd. The primers for the gene SlZF-31 were designed according to the mRNA sequence of the SlZF-31 gene (Solyc08g063040.2.1) in the SGN tomato database. All primers employed in this study were designed using primer 5.0 software ( Table 1). The target fragment of the SlZF-31 gene was subjected to homologous recombination with the tobacco rattlesnake virus (pTRV2) vector, in which the specific steps were performed according to the Clon-ExpressII One Step Cloning Kit of Vazyme Biotech Co., Table 1 Primers used for target fragment amplification and qRT-PCR analysis Ltd. The ligated vector was transferred to an Escherichia coli DH5a culture in accordance with the instructions of the Escherichia coli transformation kit, and single colonies were selected for growth in liquid LB medium (50 µg/ mL kanamycin) and then sequenced. The sequenced vector pTRV2-SlZF-31 was transferred to Agrobacterium GV3101 for the preparation of the immersion solution according to the method of Huang et al. (Huang et al. 2014). Single colonies were transferred to liquid LB medium (50 µg/mL kanamycin, 50 µg/mL rifampicin). Then, the bacteria were transferred to IM medium (50 µg/mL kanamycin, 50 µg/mL rifampicin, 200 µM acetyleugenone) for 24 h, after which they were collected and transferred to IM medium (200 µM acetyleugenone) containing MgCl 2 to obtain an infection solution with an OD 600 of 0.2-0.3.

Infection of tomato plants to reduce the SlZF-31 gene expression level
Agrobacterium infection was carried out on tomato seedlings via vacuum infiltration with a syringe; 30 seedlings were infected at a time, among which 10 were used as the control plant (CK), 10 were used at the empty vector control plants (CK-TRV2), 10 were used as the SlZF-31 plants (SlZF-31-TRV 2 ). The experiment was repeated 3 times. The experimental steps were performed in accordance with the method described by Velasquez et al. (Velasquez et al. 2009).

Drought and salt stress treatment and sample collection
SlZF-31-silenced tomato plants at 25 d after infection were subjected to drought stress and salt stress (Zhang et al. 2014). After silenced SLZF-31 gene, tomato plant samples (CK, CK-TRV2 and SLZF-31-TRV2) were taken for 0 h, then the roots of these tomato plants were washed clean and the roots were inserted into 15% PEG 6000 solution for drought stress, and tomato leaves were collected at 1.5 h, 3 h, 6 h and 12 h after treatment. Meanwhile, tomato plants were inserted into 100 mM NaCl solution in the same way as above for salt stress, and tomato leaves were collected at the same 4 time points after treatment. The collected tomato leaves were used to detect various indicators in the VIGS experiment.

SlZF-31 gene expression pattern analysis
The silencing efficiency of the SlZF-31 gene was detected in tomato leaves collected at 25 d after infection. In this study, we analyzed SlZF-31 gene expression patterns in tomato leaves at different time points (0 h, 1.5 h, 3 h, 6 h and 12 h) after the silencing efficiency and expression levels of the SlZF-31 gene in TRV2-SlZF-31 tomato leaves were also analyzed by qRT-PCR at different time points (0 h, 1.5 h, 3 h, 6 h and 12 h) under the initiation of drought stress and salt stress. The same methods described above were employed for RNA extraction and cDNA synthesis from tomato leaves. The qRT-PCR method specifically followed that described by Zhao et al. (Zhao et al. 2015;Zhao et al. 2016). The primers used in the qRT-PCR analysis were designed by using the primer design software Primer 5.0 (Table 1). According to the CT values obtained with the qTOWER3G system (Analytik Jena, Germany), the relative expression of genes was calculated according to the 2 -ΔΔCT method with Actin as the internal reference gene (Livak 2001;Rotenberg et al. 2006). Bar charts were generated with Office 2016 software.

Determination of ABA content
The content of ABA hormone in CK, CK-TRV2 and TRV2-SlZF-31 tomato plants under drought and salt stress (0 h, 3 h and 12 h) was determined by high performance liquid chromatography (HPLC). For specific ABA hormone detection methods, refer to the 2018 article by Li et al. (2018). Weighed 0.1 g tomato samples, ground them and added 70-80% methanol solution (pH 3.5); Centrifuge overnight after extraction; The supernatant was vaporized at 40 °C under reduced pressure, and petroleum ether was added for static stratification; After extraction and decolorization for 2-5 times, triethylamine, PVPP and other solutions were added for shock incubation, and the supernatant was extracted after centrifugation; Adjust pH to 3.0 and extract with ethyl acetate; The product was evaporated at 40 °C under reduced pressure and dissolved by adding mobile phase solution in shock. After filtration, the product was placed under ultraviolet wave of 254 nm for detection.

Determination of physiological indexes (SOD, POD, proline and MDA)
The physiological indexes of superoxide dismutase (SOD) and peroxidase (POD) activity and proline and malondialdehyde (MDA) content were determined in tomato leaves (CK, CK-TRV2, TRV2-SlZF-31) after drought stress and salt stress. The SOD activity in tomato leaves was determined according to Giannopolitis and Ries (1977). The POD activity in tomato leaves was determined according to Chance and Maehly (1955). The proline content in tomato leaves was determined according to Cakmak and Horst (1991). The MDA content was determined with the thiobarbital acid method (Ali et al. 2017). These kits were purchased from Suzhou Keming Biotechnology Co., Ltd. A. 0.2 g of tomato samples were weighed for the determination of SOD, POD, proline and MDA indexes, add 1 ml of extraction solution to tomato samples and grind the samples to homogenateand; The supernatant was extracted after centrifugation at 8000 × g at 4 °C for 10 min; Add different reagents to the liquid and place at room temperature for rest, water bath, centrifugation; The colorimetric results were quantified at OD 560 nm for SOD, 470 nm for POD, 520 nm for proline and 532 nm and 600 nm for MDA.

NBT and DAB staining
Tomato leaves (CK, CK-TRV2, TRV2-SlZF-31) under drought stress and salt stress were assessed by NBT and DAB staining (Kumar et al. 2014). NBT (0.1 g) was dissolved in 50 ml of phosphate buffer, and 50 mg DAB was dissolved in 50 ml of distilled water, after which the pH was brought to 7.5. The leaves were placed in the NBT and DAB solutions overnight, and an appropriate amount of ethanol was added, followed by boiling in a water bath at 100 °C for 15 min. Finally, the results were observed on glass slides. A blue precipitate was observed in the tomato leaves after NBT staining, where a greater intensity of the blue precipitate indicated a higher O 2− content. A brown precipitate was observed in the tomato leaves after DAB staining, where a greater intensity of the brown precipitate indicated a higher H 2 O 2 content.

Chlorophyll fluorescence detection
This evaluation was conducted under drought stress and salt stress at different time points (0 h, 1.5 h, 3 h, 6 h and 12 h) in CK, CK-TRV2 and TRV2-SlZF-31 plants with a PhotosynQ MultispeQ multifunction plant measurement instrument made in the United States to measure the chlorophyll fluorescence parameters (PSII quantum yield (ΦII), non-photochemical quenching (Φ(NPQ)), photochemical quenching (qL) and electron transfer efficiency (ETR)).

Results of SlZF-31 gene silencing efficiency analysis
The expression level of the SlZF-31 gene in TRV2-SlZF-31 plants was detected by qRT-PCR. Figure 1 shows the gene expression levels of partially silenced plants, and the results showed that the expression level of the SlZF-31 gene in 10 TRV2-SlZF-31 plants was lower than that in CK and CK-TRV2 plants. SlZF-31 gene expression in TRV2-SlZF-31 plants was decreased by 42.6-85%, where the greatest decrease was 85%, and the average decrease was 79.4%. Tomato plants in which gene expression was decreased by more than 50% were selected for subsequent drought stress and salt stress treatment.

Expression pattern analysis of the SlZF-31 gene after drought stress and salt stress
The expression pattern of the SlZF-31 gene at different time points (0 h, 1.5 h, 3 h, 6 h, 12 h and 24 h) after drought stress and salt stress was detected by qRT-PCR. The results showed that the expression of the SlZF-31 gene in CK, CK-TRV2 and TRV2-SlZF-31 plants first increased and then decreased under drought stress and salt stress treatment, and that its expression was always lower in TRV2-SlZF-31 plants than in CK and CK-TRV2 plants. These results indicated that VIGS exerted a significant silencing effect on tomato SlZF-31 (Fig. 2).

Phenotypic observations of tomato plants under drought stress and salt stress
The phenotypes of CK, CK-TRV2 and TRV2-SlZF-31 plants were observed after PEG 6000 drought stress treatment, as shown in Fig. 3. CK and CK-TRV2 plants showed leaf wilting after 6 h of drought stress, which was aggravated after 12 h and was more serious after 24 h. The leaves of TRV2-SlZF-31 plants were wilted after 1.5 h of drought treatment, and the leaves were wilted and dried from 3 to 12 h. After 24 h of drought stress, stem bending degree increased gradually, and leaf wilting degree reached the highest level. Some of the leaves have dried up and some of the stalks have wilted and fallen. CK, CK-TRV2 and TRV2-SlZF-31 plants were observed after NaCl stress treatment. The CK plants showed leaf wilting after 12 h of treatment, and leaf wilting worsened after 24 h of treatment. CK-TRV2 plants showed leaf wilting after 3 h of treatment, and leaf wilting increased from 6 to 24 h, with no obvious change in the stem. The leaves of TRV-SlZF-31 plants began to wilt, and the stems were slightly bent after 3 h of salt stress. The degree of leaf wilting gradually increased from 6 to 12 h, and after 24 h of treatment, the leaves had dried out, and the stems were severely bent. Thus, the SlZF-31 gene plays a role in both drought and salt tolerance in tomato, and reducing the expression of the SlZF-31 gene decreases the drought and salt tolerance of tomato.

Determination of ABA content in tomatos
As shown in Fig. 4, the ABA content of TRV2-SLZF-31 for 0 h under drought and salt stress was slightly lower than that of CK and CK-TRV2, which indicates that TRV2-SlZF-31 gene may be involved in the synthesis and accumulation of plant ABA hormone. After drought stress and salt stress, the ABA content in tomato plants (TRV2-SlZF-31, CK and CK-TRV2) showed a trend of first increasing and then decreasing, and the ABA content of TRV2-SlZF-31 tomato plants was compared with CK and CK -TRV2 plants are significantly reduced.

Results of SOD, POD, proline and MDA quantification
As shown in Fig. 5, the SOD and POD activities and the contents of proline and MDA in CK, CK-TRV2 and TRV2-SlZF-31 plants increased after drought and salt treatment. At all time points (0 h, 1.5 h, 3 h, 6 h and 12 h) during drought treatment, the SOD and POD activities and the content of proline in TRV2-SlZF-31 plants were lower than those in CK and CK-TRV2 plants, while the MDA content in TRV2-SlZF-31 plants was higher than those in CK and CK-TRV2 plants. The results for the SOD activity, POD activity, proline content and MDA content in plants (CK, CK-TRV2 and TRV2-SlZF-31) subjected to salt stress were similar to those obtained under drought stress. Therefore, the degree of cell damage was more severe in TRV2-SLZF-31 plants than in CK and CK-TRV2 plants.

Results of NBT and DAB staining
As shown in Fig. 6, DAB and NBT staining was performed on CK, CK-TRV2 and TRV2-SlZF-31 at different time points (0 h, 1.5 h, 3 h, 6 h, 12 h and 24 h) during drought stress and salt stress, respectively. The results showed that leaf color gradually deepened with the extension of the treatment time.
The brown precipitate and the blue precipitate appeared in the leaves of CK, CK-TRV2 and TRV2-SlZF-31 plants stained with DAB and NBT, respectively, after drought stress. The area and color of the precipitates in the leaves of TRV2-SlZF-31 plants were larger and darker than those in

Chlorophyll fluorescence parameter detection under drought stress and salt stress
As shown in Fig. 7, under drought stress, the chlorophyll fluorescence parameters (ΦII, qL and ETR) of CK, CK-TRV2 and TRV2-SlZF-31 plants were reduced, among which the ΦII and qL values of TRV2-SlZF-31 plants were significantly lower than those of CK and CK-TRV2 plants, and the ETR of TRV2-SlZF-31 plants decreased the most. The Φ (NPQ) values of CK, CK-TRV2 and TRV2-SlZF-31 plants were increased. The changes in the chlorophyll fluorescence parameters of CK, CK-TRV2 and TRV2-SlZF-31 plants under salt stress were similar to those under drought stress.

Discussion
In the abiotic stress response, the regulatory network generated by the signal transduction pathways of different hormones is complex and diverse (Verma et al. 2016). ABA is one of the key hormones regulating plant growth, development and physiological activities. It not only regulates stomatal opening and controls organ shedding but also plays an important role in the response to adverse conditions and various types of stress (Yamaguchi-Shinozaki and Shinozaki 2006). When plants encounter drought, salt damage and other stresses, ABA levels increase, activating the ABA signal transduction pathway and inducing the expression of many genes related to stress resistance to reduce the damage to plants caused by stress and protect their normal growth (Wang et al. 2011). In this study, the ABA content of TRV2-SLZF-31 plants was lower than that of CK and CK-TRV2 plants under drought and salt stress. Therefore, we speculate that the SlZF-31 gene may be involved in the ABA hormone synthesis or metabolism pathway, so that this gene affects the drought and salt tolerance of tomato plants by affecting the signal transduction pathway of ABA hormone. Tian also found that STZFP1, a C2H2 type zinc finger protein gene in potato, may respond to plant salt and drought stress through ABA signaling pathway (Tian et al. 2010). TRV2-SlZF-31 plants showed no significant change in phenotype before stress treatment, indicating that the SlZF-31 gene had no significant effect on the plant phenotype after silencing. We observed the phenotypes of CK, CK-TRV2 and TRV2-SlZF-31 plants under drought and salt stress. It was found that the degrees of leaf wilting, drying and stem bending in TRV2-SlZF-31 plants were more serious than those in CK and CK-TRV2 plants under drought stress and salt stress. Therefore, we speculate that the SlZF-31 gene may play a role in both drought and salt tolerance in tomato, where reducing the expression of the gene SlZF-31 will decrease the drought and salt tolerance of tomato.  ROS production is one of the earliest responses of plants to drought stress and salt stress. ROS damage the cell membrane, thus affecting cell metabolism. To remove ROS from plant cells and protect macromolecules in plant cells, a series of antioxidant systems involving reactive oxygen scavenging enzymes, such as SOD and POD, are activated in plants (Apel and Hirt 2004). SOD can convert superoxide in plants into H 2 O 2 and O 2− to resist oxidative damage to plant cells (Ajithkumar and Panneerselvam 2014). POD can protect the cell structure and reduce damage to the membrane system and plays a highly significant role in the maintenance of normal physiological activities under adverse conditions (Apel and Hirt 2004). Proline is an osmotic regulator that is accumulated by plants in the process of drought resistance and salt resistance and reflects the resistance abilities of plants to some extent. MDA is the final product of membrane peroxidation in plants, and the accumulation of MDA damages the structure and function of the cell membrane. The MDA content represents the degree of membrane lipid peroxidation and the degree of damage to the membrane system and is therefore an important indicator of stress resistance in the plant (Kishor and Sreenivasulu 2014). The results of this experiment showed that under drought stress and salt stress, the SOD and POD activities and the proline content of TRV2-SlZF-31 plants were higher than those of CK and CK-TRV2 plants. The MDA content of TRV2-SlZF-31 plants was also higher than that of CK and CK-TRV2 plants, indicating that tomato plant cells suffered more damage after the expression of the SlZF-31 gene was reduced. Similar results were found in a study by Zhao et al., who reported that the SOD and POD activity and the proline content were lower in TRV2-SL-ZH13 plants than in CK plants after SL-ZH13 gene expression was reduced, while the content of MDA was higher than that of CK plants (2018a, b; 2019).
The accumulation of superoxide radicals (O 2− ) in tomato leaves was examined by NBT staining. The higher the O 2− accumulation is, the darker the blue precipitate will be in leaves stained with NBT (Ivan et al. 2009). DAB staining was used to detect the accumulation of H 2 O 2 . The higher the accumulation of H 2 O 2 is, the more intense the brown precipitate will be in leaves stained with DAB (Fryer et al. 2002). The experimental results showed that after drought stress and salt stress, the blue precipitate in the leaves of TRV2-SlZF-31 plants stained with NBT was darker than that in the leaves of CK and CK-TRV2 plants, and the brown precipitate in the leaves of TRV2-SlZF-31 plants stained with DAB was darker than that in the leaves of CK and CK-TRV2 plants. Therefore, the accumulation of O 2− and H 2 O 2 in TRV2-SlZF-31 plants was higher than those in CK and CK-TRV2 plants. This phenomenon indicated that SlZF-31 gene silencing did not directly affect the accumulation of reactive oxygen species in leaves, but after drought and salt stress, SlZF-31 gene silencing led to more intense accumulation of reactive oxygen species in leaves.
Drought, salt and other abiotic stresses can degrade chlorophyll in plants, thus reducing the absorption and transmission of light energy by chloroplasts (Kuang et al. 1980). Chlorophyll fluorescence parameters can reflect the PSII plant photosynthetic system of light absorption and utilization (Demmig-Adams and Adams 1996). ФII represents the actual photosynthetic efficiency of PSII (the PSII quantum efficiency of energy conversion). QL reflects the proportion of open PSII reaction centers (i.e., the degree of openness of the PSII reaction center). Ф (NPQ) represents the PSII regulatory energy dissipation quantum yield. ETR is the relative linear electron flow rate through the PSII optical system. In this study, the values of the chlorophyll fluorescence parameters (ΦII and qL) of TRV2-SlZF-31 plants were found to be significantly lower than those of CK and CK-TRV2 plants under drought stress and salt stress, and the ETR of TRV2-SlZF-31 plants showed the greatest decrease. We speculated that this might have occurred because of the decreased drought resistance and salt resistance of TRV2-SlZF-31 plants after the SlZF-31 gene was downregulated. Therefore, the chloroplast photosynthetic mechanism of TRV2-SlZF-31 plants under abiotic stress was more severely damaged than those of CK and CK-TRV2 plants, and the photosynthetic capacity of TRV2-SlZF-31 plants was significantly decreased. Lu and others show that the plant chloroplast photosynthetic mechanism is damaged under abiotic stress, the PSII original light energy conversion efficiency (ФII) is reduced, the photosynthetic ETR is reduced, the qL light energy available for photosynthesis is reduced, and the excess excitation energy (Ф (NPQ)) is increased, leading to a decrease in PSII function and photosynthetic capacity (Lu and Zhang 1999).
Prior to the abiotic stress treatment (drought stress and salt stress), there were no significant differences in the phenotype, physiological indexes (SOD, POD, proline and MDA), NBT staining or DAB staining between control plants and TRV2-SlZF-31 plants. After drought stress and salt stress, the phenotypes and related indexes of the control plants and TRV2-SlZF-31-silenced plants were significantly different, indicating that SlZF-31 gene silencing had no significant influence on the growth and development of tomato plants, while this gene did play a role in plant resilience to stress.

Conclusion
In conclusion, the degrees of withering and stem bending in TRV2-SlZF-31 plants were greater than those in CK and CK-TRV2 plants under drought stress and salt stress. The ABA content of TRV2-SlZF-31 plants were lower than those of CK and CK-TRV2 plants. The analysis of physiological indexes showed that the SOD and POD activity and the proline content of TRV2-SlZF-31 plants were lower than those of CK and CK-TRV2 plants, while the MDA content of TRV2-SlZF-31 plants was higher than those in CK and CK-TRV2 plants. The accumulation of H 2 O 2 and O 2− in TRV2-SlZF-31 plants was higher than those in CK and CK-TRV2 plants. The values of the chlorophyll fluorescence parameters (ΦII and qL) of TRV2-SlZF-31 plants were significantly lower than those of CK and CK-TRV2 plants, and the ETR of TRV2-SlZF-31 plants showed the greatest decrease. These results indicated that the downregulation of the SlZF-31 gene affected the response of tomato under drought and salt stress and reduced the drought and salt resistance of tomato plants.