Abstract

   C[2]H[2]-type zinc finger proteins are classic and extensively studied
   members of the zinc finger family. C[2]H[2]-type zinc finger proteins
   participate in plant growth, development and stress responses. In this
   study, 99 C[2]H[2]-type zinc finger protein genes were identified and
   classified into four groups, and many functionally related cis-elements
   were identified. Differential C[2]H[2]-ZFP gene expression and specific
   responses were analyzed under drought, cold, salt, and pathogen
   stresses based on RNA-Seq data. Thirty-two C[2]H[2] genes were
   identified in response to multiple stresses. Seven, 3, 5, and 8 genes
   were specifically expressed under drought, cold, salt, and pathogenic
   stresses, respectively. Five glycometabolism and sphingolipid-related
   pathways and the endocytosis pathway were enriched by KEGG analysis.
   The results of this study represent a foundation for further study of
   the function of C[2]H[2]-type zinc finger proteins and will provide us
   with genetic resources for stress tolerance breeding.

   Keywords: C[2]H[2]-type zinc finger gene family, biotic stress, abiotic
   stress, transcription factor, tomato

Introduction

   Tomato (Solanum lycopersicum) is one of the most important vegetable
   crops of Solanaceae ([37]Mueller et al., 2005). However, the yield and
   quality of tomato are greatly affected by various biotic and abiotic
   stresses such as pathogen infection, low temperature, salt, and drought
   when the plants are exposed to complex environmental conditions. Upon
   stress perception, transcription factors (TFs) bind to their target
   genes to regulate their expression and orchestrate biochemical and
   physiological modifications critical for stress tolerance and the
   adaptation of plant growth ([38]Hichri et al., 2014).

   C[2]H[2]-type zinc finger proteins (C[2]H[2]-ZFPs), which are members
   of an important TF family, are also called TFIIIA-type ZFPs or
   classical ZFPs and are widely distributed in eukaryotic genomes
   ([39]Huang et al., 2005). There are 176, 189, 211, and 109
   C[2]H[2]-ZFPs in Arabidopsis, rice, maize, and poplar, respectively
   ([40]Englbrecht et al., 2004; [41]Agarwal et al., 2007; [42]Liu et al.,
   2015; [43]Wei et al., 2015). The C[2]H[2]-ZFPs of eukaryotes generally
   have a specific conserved sequence consisting of 25–30 amino acids:
   X-X-C-X(1-5)-C-X(12)-H-X(3-6)-H (X: any amino acid; number: the number
   of amino acids). The two C (Cys) and two H (His) residues in the
   sequence form a coordination bond with a zinc ion and then form a
   tetrahedral structure composed of a two-stranded antiparallel β-sheet
   and an α-helix ([44]Carl et al., 2001). EPF1 (later renamed ZPT2-1) was
   identified in Petunia as the first plant-specific ZFP. Takatsuji
   discovered that EPF1 interacts with the promoter region of the
   5-enolpyruvylshikimate-3-phosphate synthase gene (EPSPS) and that the
   expression of EPF1 parallels the expression of EPSPS ([45]Hiroshi et
   al., 1994). More zinc finger TFs have been subsequently identified in
   other plants and have been found to play crucial roles in the
   regulation of development and responses to biotic and abiotic stresses
   ([46]Sang et al., 2004; [47]Jiang and Pan, 2012).

   Many C[2]H[2]-ZFPs genes involved in biotic and abiotic stresses have
   been studied in detail. GmZF1 and GmZFP3, two soybean C[2]H[2]-ZFPs,
   positively regulate the cold response and negatively regulate the
   drought response, respectively. Both of these proteins might be
   involved in the ABA-dependent pathway during the stress response
   ([48]Zhang et al., 2016a). OsMSR15 contains two C[2]H[2]-ZFP motifs,
   and its expression is strongly upregulated by cold, drought, and heat
   stresses in different rice tissues at different developmental stages
   ([49]Zhang et al., 2016b). The expression of a novel ZFP gene, StZFP1,
   cloned from potato, is increased after salt stress as well as after
   infection by Phytophthora infestans and exogenous ABA ([50]Tian et al.,
   2010). Overexpression of the CAZFP1 gene, a zinc-finger protein gene
   isolated from pepper leaves, enhances resistance against infection by
   the pathogens Xanthomonas campestris and Colletotrichum coccodes in
   transgenic Arabidopsis plants ([51]Sang et al., 2004). In tobacco, ZFT1
   functions as a transcription repressor and binds to the EP1S sequence.
   Overexpression of ZFT1 renders tobacco plants more tolerant to TMV
   ([52]Uehara et al., 2005). In this study, C[2]H[2]-ZFP genes are
   identified from the tomato genome by a bioinformatic analysis method
   and are analyzed for many aspects, including their phylogenetic
   relationships, gene structures, conserved protein motifs, chromosomal
   locations and promoter cis-elements. The abiotic and biotic stress
   response genes of the C[2]H[2]-ZFP family are screened under drought,
   salt, cold, and pathogen infection stresses based on corresponding
   RNA-Seq data. The results of the present work will provide us with
   comprehensive genome-wide knowledge of the tomato C[2]H[2]-ZFP TF
   family and will also provide us with potential gene resources for
   biotic and abiotic stress tolerance breeding.

Materials and Methods

Identification and Phylogenetic Analysis of the C[2]H[2]-ZFP Gene Family

   Tomato genome sequence data were obtained from the Solanaceae Genomics
   Network (SGN)^[53]1 ([54]Tomato Genome, 2012). The tomato genome
   version was GCF_000188115.3_SL2.50. Arabidopsis genes were obtained
   from the Arabidopsis Information Resource (TAIR^[55]2). The
   C[2]H[2]-ZFPs of tomato (SlZFs) were predicted using the HLH hidden
   Markov model (HMM) profile obtained from Pfam^[56]3 (PF00096) and
   analyzed manually using the SMART^[57]4 database to confirm the
   presence of C[2]H[2]-ZFP domains (SM000355) [33,34]. Finally, the
   obtained genes were compared with members of the C[2]H[2]-ZFP gene
   family in PlnTFDB v3.0^[58]5 ([59]Riano-Pachon et al., 2007). The
   ExPaSy site^[60]6 ([61]Elisabeth et al., 1999) was used to calculate
   the molecular weights and isoelectric points (pI) of the deduced
   polypeptides. Multiple sequence alignment was performed using ClustalX
   1.83 ([62]Julie et al., 1997). Phylogenetic analyses were performed
   using the neighbor-joining method in MEGA 5.0 ([63]Tamura et al., 2011)
   and one unrooted neighbor-joining tree was constructed with 1,000
   bootstrap replications.

Exon/Intron Structure Analysis and Identification of Conserved Motifs

   The exon/intron arrangement of the ZFP genes was generated by the GSDS
   (Gene structure display server)^[64]7 using both DNA sequences and
   their corresponding coding sequences ([65]Hu et al., 2015).
   Additionally, online MEME (Multiple Expectation Maximization for Motif
   Elicitation)^[66]8 was performed to search for conserved motifs for
   each C[2]H[2]-ZFP gene with the following parameters: the maximum
   number of motifs was set to 15, and the optimum motif width was set to
   6 to 50 residues ([67]Bailey et al., 2009). Each structural motif
   annotation was performed using the Pfam and SMART tools.

Chromosomal Location

   The chromosomal location data of the C[2]H[2]-ZFP genes were obtained
   from SGN, and a map was generated via MapInspect software^[68]9.
   Different colors were used to represent different groups of
   C[2]H[2]-ZFP genes. Pink, green, blue, and orange represent the I, II,
   III, and IV groups, respectively. This approach allowed different
   groups of genes to be more easily distinguished in the distribution of
   the chromosome.

Promoter cis-Element Analysis

   The transcription start site was designated + 1. The promoter sequences
   (from −2 kb to + 1 bp) of all C[2]H[2]-ZFP genes were obtained from
   Phytozome and analyzed using the program PlantCARE online^[69]10. The
   cis-elements were predicted and located.

Plant Materials and Stress Treatments

   Tomato Micro-Tom and [70]CGN18423 (which carries the Cf-19 gene that
   confers resistance to Cladosporium fulvum) were from the Tomato
   Research Institute of Northeast Agricultural University and were grown
   in a growth chamber (Conviron, Canada) with a light intensity of 120 μM
   photons m^–2 s^–1 (photoperiod 16 h, day/night temperature 22/18°C).
   Next, 100 healthy Micro-Tom seedlings were selected for subsequent
   analyses (10 seedlings used for organ analysis and 90 for drought,
   salt, cold stress treatments). The tomato seedlings used for drought,
   salt, and cold stress treatments were transferred to hydroponics and
   grown for 48 h when they were in the raising period of the four-leaf
   stage. Then, 30 seedlings were treated with 15% PEG 6000 to simulate
   drought stress. Young leaves were collected and frozen in liquid
   nitrogen at 0, 3 and 6 h after drought treatment. Thirty seedlings were
   transferred to a 5°C growth chamber for cold treatment. Young leaves
   were collected and frozen in liquid nitrogen at different time points
   (0, 4, and 12 h) after treatment. Additionally, 30 tomato seedlings
   were exposed to 200 mM sodium chloride (NaCl). Young leaves were
   collected and frozen in liquid nitrogen at different time points (0, 2,
   and 8 h) after treatment. Three biological replicates were performed
   for each time point. The materials and methods for pathogen stress are
   shown in [71]Zhao et al. (2019).

RNA Isolation and cDNA Synthesis

   Total RNA was extracted from leaf samples using a plant RNA mini kit
   (Watson, China) according to the manufacturer’s handbook. The
   first-strand cDNA was transcribed with the TransScript^® One-Step gDNA
   Removal and cDNA Synthesis SuperMix Kit according to the manufacturer’s
   instructions.

Primer Design and qRT-PCR Verification for Different Tissues and Organs

   Because of the large number of genes in the gene family and to rule out
   absolute tissue-specific preferences for subsequent analysis using