C2H2-type Zinc Finger Protein Research Articles

Characterization of functional, noncovalently assembled zinc finger nucleases

Zinc finger nuclease (ZFN) is a chimeric restriction enzyme made of a C2H2-type zinc finger protein (ZFP) and the FokI nuclease domain (FN). ZFN technology has been considered as a powerful tool for genome editing. Here, we report a new type of ZFN system based on the coiled-coil interaction used as a noncovalent assembler. Like conventional ZFNs, noncovalently assembled ZFNs (ncZFNs) structurally have two domains, a ZFP and a FN. Each domain carries one of antiparallel heterodimeric leucine zippers, respectively, to form an ncZFN through leucine zipper assembly. The characterization of ncZFNs revealed that they behave as fully functional sequence-specific endonucleases, comparable to those of conventional ZFNs. Interestingly, some ncZFNs displayed augmented off-target cleavage, possibly by degenerate DNA binding of the ZFP domain of ncZFNs based on our data. We postulate that DNA cleavage of ncZFN(s) seems to be more sensitive to the ZFP binding to lesser-optimal sites. Facile design of ncZFNs through the mix-and-assemble approach could be applicable to other DNA binding proteins for evaluation of sequence-specificity. In addition, our work establishes that the coiled-coil interaction could be used as the peptide-based noncovalent assembler for the formation of a noncovalently-linked functional multidomain protein.

ZAT11, a zinc finger transcription factor, is a negative regulator of nickel ion tolerance in Arabidopsis.

ZAT11, a Zinc Finger of Arabidopsis Thaliana 11, is a dual-function transcriptional regulator that positively regulates primary root growth but negatively regulates Ni (2+) tolerance. Zinc Finger of Arabidopsis Thaliana 11 (ZAT11) is a C2H2-type zinc finger protein that has been reported to function as an active transcriptional repressor. However, the biological function of ZAT11 remains unknown. Here we show that GFP-tagged ZAT11 is targeted to the nucleus. Analysis of plants expressing ZAT11 promoter-GUS showed that ZAT11 is highly expressed in roots and particularly in root tips. To identify the biological function of ZAT11, we constructed three independent lines of ZAT11 overexpressing transgenic plant (ZAT11 OE). ZAT11 OE enhanced the elongation of primary root but reduced the metal tolerance against nickel ion (Ni(2+)). The reduced Ni(2+) tolerance of ZAT11 OE was correlated with decreased accumulation of Ni(2+) in plants. The decreased accumulation of Ni(2+) in ZAT11 OE was caused by the reduced transcription of a vacuolar Ni(2+) transporter gene. Taken together, our results suggest that ZAT11 is a dual function transcriptional regulator that positively regulates primary root growth but negatively regulates Ni(2+) tolerance.

A novel rice C2H2-type zinc finger protein, ZFP36, is a key player involved in abscisic acid-induced antioxidant defence and oxidative stress tolerance in rice.

C2H2-type zinc finger proteins (ZFPs) have been shown to play important roles in the responses of plants to oxidative and abiotic stresses, and different members of this family might have different roles during stresses. Here a novel abscisic acid (ABA)- and hydrogen peroxide (H₂O₂)-responsive C2H2-type ZFP gene, ZFP36, is identified in rice. The analyses of ZFP36-overexpressing and silenced transgenic rice plants showed that ZFP36 is involved in ABA-induced up-regulation of the expression and the activities of superoxide dismutase (SOD) and ascorbate peroxidase (APX). Overexpression of ZFP36 in rice plants was found to elevate the activities of antioxidant enzymes and to enhance the tolerance of rice plants to water stress and oxidative stress. In contrast, an RNA interference (RNAi) mutant of ZFP36 had lower activities of antioxidant enzymes and was more sensitive to water stress and oxidative stress. ABA-induced H₂O₂ production and ABA-activated mitogen-activated protein kinases (MAPKs) were shown to regulate the expression of ZFP36 in ABA signalling. On the other hand, ZFP36 also regulated the expression of NADPH oxidase genes, the production of H₂O₂, and the expression of OsMPK genes in ABA signalling. These results indicate that ZFP36 is required for ABA-induced antioxidant defence, for the tolerance of rice plants to water stress and oxidative stress, and for the regulation of the cross-talk between NADPH oxidase, H₂O₂, and MAPK in ABA signalling.

Involvement of C2H2 zinc finger proteins in the regulation of epidermal cell fate determination in Arabidopsis.

Cell fate determination is a basic developmental process during the growth of multicellular organisms. Trichomes and root hairs of Arabidopsis are both readily accessible structures originating from the epidermal cells of the aerial tissues and roots respectively, and they serve as excellent models for understanding the molecular mechanisms controlling cell fate determination and cell morphogenesis. The regulation of trichome and root hair formation is a complex program that consists of the integration of hormonal signals with a large number of transcriptional factors, including MYB and bHLH transcriptional factors. Studies during recent years have uncovered an important role of C2H2 type zinc finger proteins in the regulation of epidermal cell fate determination. Here in this minireview we briefly summarize the involvement of C2H2 zinc finger proteins in the control of trichome and root hair formation in Arabidopsis.

An EAR-Dependent Regulatory Module Promotes Male Germ Cell Division and Sperm Fertility in Arabidopsis.

The production of the sperm cells in angiosperms requires coordination of cell division and cell differentiation. In Arabidopsis thaliana, the germline-specific MYB protein DUO1 integrates these processes, but the regulatory hierarchy in which DUO1 functions is unknown. Here, we identify an essential role for two germline-specific DUO1 target genes, DAZ1 and DAZ2, which encode EAR motif-containing C2H2-type zinc finger proteins. We show that DAZ1/DAZ2 are required for germ cell division and for the proper accumulation of mitotic cyclins. Importantly, DAZ1/DAZ2 are sufficient to promote G2- to M-phase transition and germ cell division in the absence of DUO1. DAZ1/DAZ2 are also required for DUO1-dependent cell differentiation and are essential for gamete fusion at fertilization. We demonstrate that the two EAR motifs in DAZ1/DAZ2 mediate their function in the male germline and are required for transcriptional repression and for physical interaction with the corepressor TOPLESS. Our findings uncover an essential module in a regulatory hierarchy that drives mitotic transition in male germ cells and implicates gene repression pathways in sperm cell formation and fertility.

The Reaction of Arsenite with Proteins Relies on Solution Conditions

Arsenic is a biologically interesting element with both antitumor and carcinogenic effects. Zinc finger proteins (ZFPs) have been confirmed to be the cellular targets of arsenite; however, arsenite inhibits ZFPs much less efficiently in vitro than in vivo. The molecular mechanism of this difference is unknown. In this work, we found that the reaction of arsenite with ZFPs relies on the presence of small biomolecules such as glutathione (GSH), histidine, and cysteine (Cys). The weak acidity also enhances the reaction. Further study shows that the coordination of zinc is much more susceptible than that of arsenic to these solution conditions, which enhance the competition of arsenic. Notably, different from C3H-type ZFPs, the C2H2-type ZFPs are more significantly influenced by the presence of thiol-containing molecules in the reaction. GSH and Cys can facilitate the reaction by participation of the coordination to As(III) together with C2H2-type ZFPs. Consequently, the reactions are promoted both thermodynamically and kinetically via the formation of ternary complexes GSH-As-ZFP or Cys-As-ZFP. These results indicate that the reactions between arsenite and proteins are considerably modulated by environments such as the small biomolecules and the acidity of the solution. This finding clarifies the discrepancy observed in the reactions of arsenite in vitro versus in cells, and provides an insight into the molecular mechanism of arsenite.

Identification of a C2H2-type zinc finger gene family from Eucalyptus grandis and its response to various abiotic stresses

C2H2-type zinc finger proteins belong to a major family of transcription factors that play a crucial role in plant stress responses. In this study, seven C2H2-type zinc finger genes (EgrZFP1-7) in Eucalyptus grandis were cloned using the RACE PCR method. EgrZFP1-7 proteins included a QALGGH motif, two zinc finger domains, and an EAR motif. They belong to a Q-type C2H2 zinc finger protein family and are classified into the subset C1. EgrZFP4 and EgrZFP6 had a higher transcription in roots than in leaves and stems, whereas the expression of the other genes did not differ in various plant tissues. The expression of EgrZFP genes in seedlings was induced by low temperatures. Time course experiments at temperatures lower than 4 °C revealed oscillating expression of EgrZFP1-6 during the cold treatment. However, EgrZFP7 showed a phasic expression pattern at the same conditions. The expression of EgrZFP1-6 was found to be enhanced by 200 mM NaCl, whereas the expression of EgrZFP7 was inhibited.

Activation of C2H2-type zinc finger genes induces dwarfism in Arabidopsis thaliana

Zinc finger proteins compose one of the largest transcription factor families in plants, but only a handful have been functionally specified in plant growth. In this study, a semidominant mutant, Arabidopsis thaliana dwarf1 (Atdwa1), was discovered from activation tagging screening using the SKI015 TDNA vector. The Atdwa1 mutant displayed severe dwarfism and loss of apical dominance, as well as other pleiotropic defects, such as earlier flowering, fewer leaves, and shorter sliliques than those of the wild-type plant. Thermal asymmetric interlaced-polymerase chain reaction analysis revealed that activated T-DNA was inserted into the At5g54330 gene, disrupting its coding sequence. We also demonstrated that At5g54330 expression was nullified by the insertion, whereas expression of its downstream genes, At5g54340, At5g54350, and At5g54360, were activated. A database search revealed that all three downstream genes encode proteins related to C2H2-type zinc finger proteins. Overexpression of each gene resulted in dwarfism similar to that of Atdwa1. These findings indicate that Atdwa1 mutant was caused by synergistic manifestation of concomitant activation of At5g54340 to At5g54360, and that the zinc finger proteins may play a role in regulation of plant growth and development.

Cucumber SUPERMAN Has Conserved Function in Stamen and Fruit Development and a Distinct Role in Floral Patterning

The Arabidopsis SUPERMAN (SUP) gene encodes a C2H2 type zinc finger protein that is required for maintaining the boundaries between stamens and carpels, and for regulating development of ovule outer integument. Orthologs of SUP have been characterized in bisexual flowers as well as dioecious species, but it remains elusive in monoecious plants with unisexual flowers on the same individual. Here we isolate the SUP ortholog in Cucumis sativus L (CsSUP), a monoecious vegetable. CsSUP is predominantly expressed in female specific organs: the female flower buds and ovules. Ectopic expression of CsSUP in Arabidopsis can partially complement the fruit development in sup-5 mutant, and its over-expression in wide-type leads to reduced silique length, suppressed stamen development and distorted petal patterning. Our data suggest that CsSUP plays conserved as well as distinct roles during flower and fruit development, and it may function in the boundaries and ovules to balance petal patterning, stamen and ovule development in Arabidopsis.

Cloning and Characterization of Brassica juncea Zinc Finger Protein Transcription Factor Gene Bj26

Zinc-finger proteins, forming an important transcriptional factor family, are involved in gene transcriptional regulation, development and stress-responses in plants. The previous studies revealed that BjC-P is a fungus-inducible promoter from Brassica juncea and W-box-like-4 is the core element responsive to fungal infection. Here, we report the molecular cloning and characterization of a zinc finger protein-encoding gene(designated Bj26). Bj26 was screened out from the Brassica juncea cDNA library by the Yeast One-Hybrid System. Bioinformatic analysis showed that Bj26 contains open reading frame of 735 bp that encodes a new C2H2-type zinc finger protein with an isoelectric point(pI) of 9.2 and a molecular weight of 26.6 kD. The protein consists of two typical zinc-finger domains and contains two conserved QALGGH amino acid sequences. Subcellular localization showed that Bj26 is located in the nuclear. The histochemical and quantitative GUS assays, through transient gene expression in Nicotiana benthamiana leaf, showed that Bj26 can bind to the core element W-box-like-4 and activate the function of BjC-P. qRT-PCR analysis showed that the gene Bj26 expression obviously increased under the induction of the fungal elicitor(Hexa-N-Acetyl-Chitohexaose). CDS alignments and phylogenetic analysis of Bj26 and other C2H2-type proteins from Arabidopsis thaliana(At) and Oryza sativa(Os)(MEGA 6) showed Bj26 shares high similarity with that from Arabidopsis thaliana(At). All of the above results suggested that Bj26 protein mediates the process of plant response to fungal pathogen.

Drosophila Hindsight and mammalian RREB-1 are evolutionarily conserved DNA-binding transcriptional attenuators

The Drosophila Hindsight (hnt) gene encodes a C2H2-type Zinc-finger protein, HNT, that plays multiple developmental roles including control of embryonic germ band retraction and regulation of retinal cell fate and morphogenesis. While the developmental functions of the human HNT homolog, RREB-1, are unknown, it has been shown to function as a transcriptional modulator of several tumor suppressor genes. Here we investigate HNT's functional motifs, target genes and its regulatory abilities. We show that the C-terminal region of HNT, containing the last five of its 14 Zinc fingers, binds in vitro to DNA elements very similar to those identified for RREB-1. We map HNT's in vivo binding sites on salivary gland polytene chromosomes and define, at high resolution, where HNT is bound to two target genes, hnt itself and nervy (nvy). Data from both loss-of-function and over-expression experiments show that HNT attenuates the transcription of these two targets in a tissue-specific manner. RREB-1, when expressed in Drosophila, binds to the same polytene chromosome sites as HNT, attenuates expression of the hnt and nvy genes, and rescues the germ band retraction phenotype. HNT's ninth Zinc finger has degenerated or been lost in the vertebrate lineage. We show that a HNT protein mutant for this finger can also attenuate target gene expression and rescue germ band retraction. Thus HNT and RREB-1 are functional homologs at the level of DNA binding, transcriptional regulation and developmental control.

Regulation of cell proliferation and migration in gallbladder cancer by zinc finger X-chromosomal protein

Gallbladder carcinoma (GBC) is one of the mostly aggressive and fatal malignancies. However, little is known about the oncogenic genes that contributed to the development of GBC. Zinc finger X-chromosomal protein (ZFX) was a novel member of the Krueppel C2H2-type zinc-finger protein family and its down-regulation led to impaired cell growth in human laryngeal squamous cell carcinoma. Here, we aim to investigate the function of ZFX in GBC cell proliferation and migration. Loss of function analysis was performed on GBC cell line (GBC-SD) using lentivirus-mediated siRNA against ZFX. The proliferation, in vitro tumorigenesis (colony-formation) ability as well as cell migration was significantly suppressed after GBC-SD cells which were infected with ZFX-siRNA-expressing lentivirus (Lv-shZFX). Our finding suggested that ZFX promoted the growth and migration of GBC cells and could present a potential molecular target for gene therapy of GBC.

Phosphorylation of the zinc finger transcriptional regulator ZAT6 by MPK6 regulates Arabidopsis seed germination under salt and osmotic stress

C2H2-type zinc finger proteins (ZFPs) play diverse roles in plant response to abiotic stresses. ZAT6, an Arabidopsis C2H2-type ZFP, has been reported to regulate root development and nutrient stress responses. However, its roles in regulation of abiotic stress response are incompletely known. Here, we demonstrate that salt or osmotic stress triggers a strong increase in ZAT6 expression in leaves. Transgenic plants overexpressing ZAT6 showed improved seed germination under salt and osmotic stress. Intriguingly, ZAT6 interacts with a stress-responsive mitogen-activated protein kinase MPK6 in vitro and in planta. ZAT6 is phosphorylated by both recombinant and plant endogenous MPK6. Serine 8 and serine 223 in ZAT6 were identified as the sites phosphorylated by MPK6. In contrast to wild-type form of ZAT6, overexpression of phosphorylation mutant form did not display significantly enhanced salt and osmotic stress tolerance. Altogether, our results suggest that phosphorylation by MPK6 is required for the functional role of ZAT6 in seed germination under salt and osmotic stress.

Overexpression of a C2H2-type zinc finger protein gene, ZAT11, leads to enhanced primary root growth and increased nickel ion sensitivity in Arabidopsis

The C2H2-type zinc finger proteins play crucial roles in various developmental processes and environmental stress tolerance in plants. Zinc Finger of Arabidopsis Thaliana11 (ZAT11), has been previously reported as an active repressor. We show here that GFP-tagged ZAT11 is targeted to the nucleus. Transgenic plants overexpressing ZAT11 show enhanced primary root growth. Analysis of β-glucuronidase (GUS) expression in transgenic plants expressing a ZAT11 promoter–GUS reporter chimeric gene confirmed that ZAT11 was expressed in roots and its expression was particularly high in root tips. ZAT11-overexpressing transgenic plants were specifically sensitive to nickel ions (Ni2+). Transcript levels of a tonoplast-localized Ni2+ transporter gene, IREG2, were approximately 2-fold decreased in transgenic plants overexpressing ZAT11 and this correlated with a reduced capacity for Ni2+ accumulation in their roots. Altogether, our results reveal that ZAT11 functions as a positive regulator of primary root growth but a negative regulator of Ni2+ tolerance, suggesting a novel biological function of Arabidopsis C2H2-type zinc finger protein in metal detoxification.

The conserved Bud20 zinc finger protein is a new component of the ribosomal 60S subunit export machinery.

The nuclear export of the preribosomal 60S (pre-60S) subunit is coordinated with late steps in ribosome assembly. Here, we show that Bud20, a conserved C(2)H(2)-type zinc finger protein, is an unrecognized shuttling factor required for the efficient export of pre-60S subunits. Bud20 associates with late pre-60S particles in the nucleoplasm and accompanies them into the cytoplasm, where it is released through the action of the Drg1 AAA-ATPase. Cytoplasmic Bud20 is then reimported via a Kap123-dependent pathway. The deletion of Bud20 induces a strong pre-60S export defect and causes synthetic lethality when combined with mutant alleles of known pre-60S subunit export factors. The function of Bud20 in ribosome export depends on a short conserved N-terminal sequence, as we observed that mutations or the deletion of this motif impaired 60S subunit export and generated the genetic link to other pre-60S export factors. We suggest that the shuttling Bud20 is recruited to the nascent 60S subunit via its central zinc finger rRNA binding domain to facilitate the subsequent nuclear export of the preribosome employing its N-terminal extension.

Cloning and functional analysis of <I>SCTF-1</I> encoding a C<SUB>2</SUB>H<SUB>2</SUB>-type zinc finger protein from soybean

The zinc finger protein is one of the proteins with finger-like domain. Some of them are transcription factors which play important role in plant growth and plant resistance to abiotic stresses. In this paper, a novel C2H2-type zinc finger protein gene SCTF-1 (GenBank accession number JQ692081) was isolated from soybean (Glycine max (L.) Merr.) This gene has a 699 bp ORF (open reading frame) with no intron and encodes a 24.9 kDa protein with 233 amino acids. Its isoelectric point (pI) is 8.33. The SCTF-1 protein contains two typical C2H2-type zinc finger domains. Both of them have highly conserved amino acid sequence-QALGGH which is a particular characteristic of plant. Transient expression of the GFP-SCTF-1 protein in onion epidermal cell showed that SCTF-1 was localized in cell nuclei. RT-PCR results showed that SCTF-1 gene was expressed with high levels in flowers and leaves in soybean, but low in roots and stems. The expression of SCTF-1 gene was strongly induced by low temperature in the soybean seedlings. Overexpression of SCTF-1 enhanced cold tolerance of transgenic tobacco (Nicotiana tabacum L.) compared to the control.

Over-expression of GsZFP1, an ABA-responsive C2H2-type zinc finger protein lacking a QALGGH motif, reduces ABA sensitivity and decreases stomata size

A cDNA of the gene GsZFP1 was cloned from Glycine soja. GsZFP1 encodes a protein with one C2H2-type zinc finger motif. The QALGGH motif, which exists in most plant C2H2-type zinc finger proteins (ZFPs), does not exist in GsZFP1. Real-time RT-PCR revealed that GsZFP1 expression was significantly up-regulated by exogenous ABA, both in leaves and roots. Over-expression of this gene, in Arabidopsis thaliana, resulted in a reduced sensitivity to ABA during seed germination and seedling growth. Transcript levels of some stress and ABA marker genes, including RD29A, RD22, NCED3, COR47, COR15A and KIN1 were increased, in the GsZFP1 over-expression lines, when plants were treated with exogenous ABA. We further studied the effects of GsZFP1 over-expression on the regulation of genes involved in ABA signaling. Negative ABA signaling regulators, such as ABI1 and ABI2, were up-regulated in over-expression lines, while positive ABA signaling regulators, such as ABF4, ABI5, GTG1, GTG2, PYR1/RCAR11, PYL2/RCAR13, SnRK2.2 and SnRK2.3, were down-regulated, in comparison to wild type plants. GsZFP1 over-expression lines also exhibited small stomata, impairment of ABA-induced stomata closure. The data presented, herein, suggests that GsZFP1 plays a crucial role in ABA signaling in A. thaliana, GsZFP1 may be a promising gene for negative regulating ABA signaling. Our findings broaden our understanding of this C2H2 ZFP subtype's function, and add to the body of evidence that has been developed in earlier studies.

Differential expression of ZFX gene in gastric cancer

Gastric cancer accounts for 8% of the total cancer cases and 10% of total cancer deaths worldwide. In Iran, gastric cancer is the leading cause of national cancer-related mortality. Most human cancers show substantial heterogeneity. The cancer stem cell (CSC) hypothesis has been proposed to reconcile this heterogeneity. ZFX encodes a member of the krueppel C2H2-type zinc-finger protein family that is required as a transcriptional regulator for self-renewal of stem cells. A total of 30 paired tissue gastric samples were examined for ZFX gene expression by quantitative real-time RT-PCR. Although the relative expression of the gene was significantly high in 47% of the examined tumour tissues, its expression was low in the others (53%). There was a statistically significant association between the ZFX gene expression and different tumour types and grades. This is the first report that shows ZFX was differentially expressed in gastric cancer. Of note, it was overexpressed in diffused-type and grade III gastric tumoural tissues. Due to this, ZFX may have the potential to be used as a target for therapeutic interventions.

GsZFP1, a new Cys2/His2-type zinc-finger protein, is a positive regulator of plant tolerance to cold and drought stress

Plant acclimation to environmental stress is controlled by a complex network of regulatory genes that compose distinct stress-response regulons. The C2H2-type zinc-finger proteins (ZFPs) have been implicated in different cellular processes involved in plant development and stress responses. Through microarray analysis, an alkaline (NaHCO(3))-responsive ZFP gene GsZFP1 was identified and subsequently cloned from Glyycine soja. GsZFP1 encodes a 35.14kDa protein with one C2H2-type zinc-finger motif. The QALGGH domain, conserved in most plant C2H2-type ZFPs, is absent in the GsZFP1 protein sequence. A subcellular localization study using a GFP fusion protein indicated that GsZFP1 is localized to the nucleus. Real-time RT-PCR analysis showed that GsZFP1 was induced in the leaf by ABA (100μM), salt (200mM NaCl), and cold (4°C), and in the root by ABA (100μM), cold (4°C), and drought (30% PEG 6000). Over-expression of GsZFP1 in transgenic Arabidopsis resulted in a greater tolerance to cold and drought stress, a decreased water loss rate, and an increase in proline irrespective of environmental conditions. The over-expression of GsZFP1 also increased the expression of a number of stress-response marker genes, including CBF1, CBF2, CBF3, NCED3, COR47, and RD29A in response to cold stress and RAB18, NCED3, P5CS, RD22, and RD29A in response to drought stress, especially early during stress treatments. Our studies suggest that GsZFP1 plays a crucial role in the plant response to cold and drought stress.

Expression of ripening-related genes in cold-stored tomato fruit

The effects of chilling on fruit ripening and the expression of ripening-related genes have been investigated in M82IL2-2, a wild species introgression breeding tomato line ( Solanum lycopersicum × Solanum pennellii). Fruit harvested at the breaker stage of ripening were stored at 3 °C for 0, 1, 2 and 4 weeks, followed by 20 °C for 0–14 d. Fruit stored for 1 week ripened normally, as assessed by red color development and softening at 20 °C, but those stored for 2 or 4 weeks showed delayed or inhibited ripening. The concentrations of the carotenoids, phytoene, phytofluene, zeta (ζ)-carotene, gamma (γ)-carotene and lycopene, but not lutein and β-carotene, were reduced in chilled fruit. Microarray analysis showed that after storage at 3 °C for 4 weeks, 352 genes were up-regulated by chilling, whereas 321 genes were down-regulated, while after 7 d at 20 °C, 180 and 126 genes, respectively, were up- and down-regulated in chilled fruit. Chilling-induced changes included expression of transcriptional repressors such as a C2H2-type zinc finger protein. Expression of genes involved in color development, including phytoene synthase1 ( PSY1), carotenoid isomerase ( CRTISO), geranylgeranyl diphosphate synthase 2 ( GGPPS2), and 1-deoxy- d-xylulose-5-phosphate synthase ( DXS), showed reduced expression during and after chilling, as did genes encoding the cell wall modifying proteins polygalacturonase (PG), pectin esterase1 (PE1), β-galactosidase ( TBG4), expansin1 ( LeExp1), and xyloglucan endotransglucosylase-hydrolase 5 ( XTH5). Alcohol dehydrogenase 2 ( ADH2) and alcohol acyltransferase ( AAT) gene expression was also reduced by chilling. Alteration of ethylene production correlated with the altered ACC synthases ( ACS2, ACS4), and ACC oxidase ( ACO1) expression. The expression of genes involved in the ethylene signal transduction pathway, such as LeETR1, NR, LeETR4, LeCTR1, LeEIL3, LeEIL4, and LeERF3, was altered by chilling, suggesting that ethylene perception and sensitivity were affected. Chilling also reduced gene expression of a ripening-regulated transcription factor, LeMADS-RIN. The effect of chilling on ethylene biosynthesis, ethylene perception, the expression of a transcription factor necessary for ripening, and transcriptional repressors may contribute to the alteration of fruit ripening in tomato.