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Saturday, 9 July 2016
An A20/AN1-zinc-finger domain containing protein gene in tea is differentially expressed during winter dormancy and in response to abiotic stress and plant growth regulators
Published Date
March 2015,Vol.1:1–7, doi:10.1016/j.plgene.2014.12.003
Open Access, Creative Commons license
An A20/AN1-zinc-finger domain containing proteingene in tea is differentially expressed during winter dormancy and in response to abiotic stress and plant growth regulators
Biotechnology Division, CSIR-Institute of Himalayan Bioresource Technology, Palampur, Himachal Pradesh 176061, India
Received 15 November 2014. Revised 31 December 2014. Accepted 31 December 2014. Available online 9 January 2015. Highlights
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A20/AN1-zinc-finger domain containing protein was cloned from tea.
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The gene belonged to a multi-gene family and was intron-less.
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The gene was expressed maximally in root and fruit compared to leaf, floral bud and stem.
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The gene responded to various cues in a temperature dependent manner.
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The gene exhibited up-regulation during winter dormancy.
Abstract
The present manuscript describes cloning and expression characterization of A20/AN1-zinc-finger domain containing protein (CsZfp) gene in an evergreen tree tea [Camellia sinensis (L.) O. Kuntze] in response to winter dormancy (WD), abiotic stresses (polyethylene glycol, hydrogen peroxide, and sodium chloride) and plant growth regulators [abscisic acid (ABA), and gibberellic acid (GA3)]. CsZfp encoded a putative protein of 173 amino acids with a calculated molecular weight of 18.44 kDa, an isoelectric point (pI) of 6.50 and grand average of hydropathicity (GRAVY) value of − 0.334. The gene did not have an intron, and belonged to a multi-gene family. During the period of active growth (PAG), CsZfp showed maximum expression in root and fruit as compared to leaf, floral bud and stem. Interaction studies between temperature and plant growth regulators on the expression of CsZfp showed that ABA upregulated CsZfp expression at growth temperature (GT; 25 °C) but had no effect at low temperature (LT; 4 °C). In response to GA3, upregulation was observed at LT but not at GT. Further, the expression was not modulated by LT either in the tissue harvested during PAG or during WD. It was interesting to record that the expression of CsZfp was upregulated by hydrogen peroxide and sodium chloride, whereas it was non-responsive to polyethylene glycol. The possible role of CsZfp in playing key but differential roles in tea to various abiotic stresses is discussed.
Abbreviations
ABA, abscisic acid
ANOVA, analysis of variance
CsZfp, Camellia sinensis A20/AN1-zinc-finger domain containing protein
cDNA, complementary DNA
DR, dormancy release
EST, expressed sequences tag
FB, flower bud
GA3, gibberellic acid
GT, growth temperature
LT, low temperature
ML, mature leaf
PAG, period of active growth
PCR, polymerase chain reaction
RACE, Rapid amplification of cDNA ends
REST, Relative Expression Software Tool
ROS, reactive oxygen species
TAB, two and a bud
WD, winter dormancy
Keywords
A20/AN1 zinc-finger protein
CsZfp
Camellia sinensis
Winter dormancy
Gene expression
1 Introduction
Tea [Camellia sinensis (L.) O. Kuntze] is a perennial evergreen tree grown in different agro-climatic zones across the world. Tender young shoots, consisting of an apical bud and associated two leaves, known as “two and a bud (TAB)”, are harvested at intervals of one to three weeks for the production of commercial tea. Growth of TAB is relatively uniform throughout the year near the equator. However, growth of TAB is restricted during winters in the plants at latitudes beyond 16° north and south of equator and the plants are said to be dormant, a phenomenon known as winter dormancy (WD). Duration of WD varies from 2 to 6 months, placing the areas experiencing WD at a disadvantage for crop yield compared to those areas where it does not occur (Kumar et al., 2012).
There is an interest to understand the phenomenon of WD and the evergreen nature of tea during winters. A subtracted cDNA library-based approach (Paul and Kumar, 2011) and RNA-sequencing on next generation sequencing (NGS) platform (Paul et al., 2014) were used to decipher the phenomenon. Analyses of unigenes obtained showed the operation of mechanisms of winter tolerance, and downregulation of genes involved in growth, development, protein synthesis and cell division (Paul and Kumar, 2011 and Paul et al., 2014). Also, the data explained the evergreen nature of the tea tree wherein inhibition of leaf abscission due to modulation of senescence related processes during WD in tea played a decisive role (Paul et al., 2014). Though subtracted cDNA library and RNA-sequencing on NGS platform produced a huge amount of biological information, limited information is available on the derived gene sequences needed for proper annotation and functional analysis because of the partial sequence data.
We previously identified an expressed sequence tag (EST; GH454325) that was upregulated in winter-dormant tissue of tea (Paul and Kumar, 2011). RNA-sequencing also identified expression of this gene (scaffold24632_86.0) in the transcriptome of winter-dormant tissue of tea (Paul et al., 2014). Preliminary analysis of the EST suggested the presence of A20 and AN1 zinc-finger domains. Realizing the importance of A20 and AN1 zinc-finger domain containing proteins in plant stresses response (Mukhopadhyay et al., 2004 and Vij and Tyagi, 2008) and that not much has been reported on this gene in its relationship with WD, the full-length cDNA of this gene was cloned through Rapid Amplification of cDNA Ends (RACE) followed by detailed expression analysis. BLAST analysis of the full-length cDNA suggested this to be encoding for an A20/AN1-zinc-finger protein (A20/AN1-ZFP), therefore the gene was called Camellia sinensis A20/AN1-zinc-finger domain containing protein (CsZfp). Proteins of these families have an A20 zinc-finger domain present at the N-terminus and the AN1 zinc-finger domain present at the C-terminus (Mukhopadhyay et al., 2004 and Vij and Tyagi, 2008). Evidence suggests that A20/AN1-ZFPs plays a crucial role in plant stress response. A20/AN1-Zfps are responsive to multiple environmental stresses like cold, salt, drought, submergence, wounding and heavy metals. Further, its overexpression confers tolerance to abiotic stresses in transgenic plants (Huang et al., 2008, Kang et al., 2011, Kanneganti and Gupta, 2008 and Mukhopadhyay et al., 2004). A few reports also suggested their role in plant development (Li et al., 2011). The present work describes the characterization of CsZfp and discusses its role in relation to abiotic stresses response.
2 Materials and methods
2.1 Plant materials and stress treatments
TEENALI, an Assamica clone of tea was used in the present work (Paul and Kumar, 2011). The tea bushes were well maintained at the tea experimental farm of our Institute (32°6′N latitude; 76°8′E longitude; 1289 ± 15 m above sea level) and subjected to usual cultural practices. TAB was collected during the period of active growth (PAG, July; maximum temperature, 25 ± 2 °C; minimum temperature, 20 ± 2 °C), WD (December; maximum temperature, 15 ± 2 °C; minimum temperature, 4 ± 2 °C), and dormancy release (DR, April; maximum temperature, 26 ± 3 °C; minimum temperature, 15 ± 2 °C), frozen immediately in liquid nitrogen and stored at − 80 °C until use. In addition to the above periods, TAB, mature leaf (ML), flower bud (FB), fruit, stem and root were also collected from a clonal bush from the field during the month of September, when both flower and fruit are available. Temperature data was collected from a weather observatory situated in the neighboring agriculture university, which is within 3 km of the periphery of the experimental tea farm.
In a separate experiment, shoot cuttings containing apical bud and associated five leaves were collected from clonal bushes of TEENALI from the field during PAG and WD and stabilized in deionized water overnight before the start of the experiment. Thereafter, shoot cuttings were transferred to deionized water (control), 100 μM 2-cis, 4-trans-abscisic acid (ABA; Sigma, USA) and 100 μM gibberellic acid (GA3; Sigma, USA), separately as described by Paul and Kumar (2011). These were housed in a plant growth chamber set at 25 ± 3 °C (growth temperature, GT) and 4 ± 2 °C (low temperature, LT) (light intensity, 200 μE m− 2 s− 1; RH, 70–80%; Saveer Biotech, India). For osmotic, oxidative and salinity stress, cuttings collected during PAG were transferred to 10% polyethylene glycol-8000 (Sigma, USA) and 0.25% hydrogen peroxide (Merck, Germany) and 100 mM sodium chloride (Sigma, USA) and housed in a plant growth chamber set at 25 ± 3 °C as described previously (Paul et al., 2012). Each experiment was carried out over a period of 48 h and the gene expression was analyzed in TAB as described in the relevant figures.
2.2 Rapid amplification of cDNA ends (RACE) and sequence analysis
As a part of our ongoing program on understanding the molecular basis of WD of tea, we deposited ESTs at Genbank at NCBI (accession numbers FF682697 to FF682833, GH454303 to GH454326, FE942774 to FE943102, and JG017532 to JG017536). For the present work, EST (accession number GH454325) was used to design primers for RACE (SMART RACE cDNA amplification Kit, BD Biosciences, USA) to clone full-length cDNA. Since the 3′ end of the gene was complete as cloned through screening of a subtracted cDNA library, 5′-RACE (Supplementary Fig. 1) was performed as per manufacturer's instructions using gene specific primers (GSPs): GSP1: 5′-TCCATCAAACCAGCACAAACAAACAAGT-3′ and GSP2: 5′-CCACTGAAATATCCATCAAACCAGCACA-3′. Total RNA isolated from winter dormant tissues as described by Muoki et al. (2011) was used for RACE-ready cDNA synthesis. The amplified fragment (Supplementary Fig. 1) was cloned in pGEM-T easy vector (Promega, USA). The nucleotide sequence was determined using BigDye terminator v 3.1 cycle sequencing mix (Applied Biosystems, USA) on an automated DNA sequencer (ABI 3130xl Genetic Analyzer, Applied Biosystems, USA) following the manufacturer's instructions. A search for homologous nucleic acid and protein sequences was performed using the BLAST algorithm (http://www.ncbi.nlm.nih.gov/). A multiple sequence alignment was generated by ClustalW multiple sequence alignment programs (http://www.ebi.ac.uk/clustalw/). The deduced amino acid sequence was analyzed by ProtParam Programs at ExPASy proteomics server (http://ca.expasy.org/) to calculate pI/Mw. The phylogenic tree was constructed using MEGA 6.06 software (Biodesign Institute, A240, Arizona State University, Tempe, AZ).
As a part of our ongoing program on understanding the molecular basis of WD of tea, we deposited ESTs at Genbank at NCBI (accession numbers FF682697 to FF682833, GH454303 to GH454326, FE942774 to FE943102, and JG017532 to JG017536). For the present work, EST (accession number GH454325) was used to design primers for RACE (SMART RACE cDNA amplification Kit, BD Biosciences, USA) to clone full-length cDNA. Since the 3′ end of the gene was complete as cloned through screening of a subtracted cDNA library, 5′-RACE (Supplementary Fig. 1) was performed as per manufacturer's instructions using gene specific primers (GSPs): GSP1: 5′-TCCATCAAACCAGCACAAACAAACAAGT-3′ and GSP2: 5′-CCACTGAAATATCCATCAAACCAGCACA-3′. Total RNA isolated from winter dormant tissues as described by Muoki et al. (2011) was used for RACE-ready cDNA synthesis. The amplified fragment (Supplementary Fig. 1) was cloned in pGEM-T easy vector (Promega, USA). The nucleotide sequence was determined using BigDye terminator v 3.1 cycle sequencing mix (Applied Biosystems, USA) on an automated DNA sequencer (ABI 3130xl Genetic Analyzer, Applied Biosystems, USA) following the manufacturer's instructions. A search for homologous nucleic acid and protein sequences was performed using the BLAST algorithm (http://www.ncbi.nlm.nih.gov/). A multiple sequence alignment was generated by ClustalW multiple sequence alignment programs (http://www.ebi.ac.uk/clustalw/). The deduced amino acid sequence was analyzed by ProtParam Programs at ExPASy proteomics server (http://ca.expasy.org/) to calculate pI/Mw. The phylogenetic tree was constructed using MEGA 6.06 software (Biodesign Institute, A240, Arizona State University, Tempe, AZ).
2.3 Intron and southern blot analysis
Genomic DNA was isolated from TAB using plant DNAzol (Invitrogen, USA). To check for the presence of intron within the coding region, gene-specific forward (5′-ATGGAGCAAAATGAGACAGGATGCC-3′; start codon underlined) and reverse (5′-CTAGAGCTTATCAAGCTTTTCAGC-3′; stop codon underlined) primers designed from the start and stop codon were used for polymerase chain reaction (PCR) with 3 μl of RACE-ready cDNA or 10 ng of genomic DNA. The PCR amplification involved an initial denaturation step at 94 °C for 1 min, followed by 28 cycles of denaturation at 94 °C for 10 s, annealing at 58 °C for 30 s, extension at 72 °C for 1 min, and final extension at 72 °C for 7 min. Cloning and sequencing were performed as described in the previous section.
Southern blotting was performed essentially as described by Sambrook et al. (1989). Genomic DNA (10 μg) was digested to completion with DraI, EcoRI, EcoRV and SpeI (no cut site within the probe) in separate reactions. Digested genomic DNA was separated by electrophoresis on a 0.7% agarose gel, denatured, and blotted onto a nylon membrane (Amersham Pharmacia, UK). The membrane was hybridized with full-length of CsZfp cDNA probe labeled with [α-32P] dATP. Hybridization was performed overnight at 55 °C in ExpressHyb™ solution (Clontech, USA). After hybridization, the blot was washed twice with 2 × SSC and 0.01% SDS for 20 min each at room temperature and twice with 0.1 × SSC and 0.1% SDS for 10 min each at 55 °C. The blot was then exposed to X-ray film and stored at − 80 °C until developed as described by Bhardwaj et al. (2010).
2.4 Expression analysis
Gene expression was performed as described by Paul and Kumar (2011). Total RNA was treated with DNase I, Ampl Grade (Invitrogen, USA) to remove contaminating genomic DNA. First-strand cDNA was Synthesized using 2 μg of DNA free RNA, SuperScript III First-strand synthesis Kit (Invitrogen, USA) and an oligo (dT)12–18primer (Invitrogen, USA). Gene expression was performed using forward: 5′-CAACAACAGACTGAACTTGC-3′ and reverse: 5′-GCAGTCATGTTTGTCAGAGT-3′ gene-specific primers on a Stratagene Mx3000P system (Agilent Technologies, Germany) using 2 × Brilliant III SYBR® Green qPCR Master Mix (Agilent Technologies, Germany). All qPCRs were run in triplicate with a no-template control to check for contamination. PCR was conducted under the following conditions: 10 min at 95 °C (enzyme activation), 40 cycles each of 30 s at 95 °C, 30 s at 55 °C and 72 °C for 30 s and a final melting curve analysis was performed (55° to 95 °C) to verify the specificity of amplicons. The raw threshold cycle (Ct) values were normalized against a housekeeping gene actin (forward: 5′-GCCATATTTGATTGGAATGG-3′ and reverse: 5′-GGTGCCACAACCTTGATCTT-3′ primers) using the Relative Expression Software Tool (REST) (Pfaffl et al., 2002).
Each experiment was repeated three times with separate biological materials. One way factorial analysis of variance (ANOVA) was used to test the effects of treatments on gene expression; Duncan's multiple range test (P < 0.05) was used to compare means post hoc using the software Statistica version 7.0 (StatSoft Inc., USA).
3 Results and discussion
3.1 CsZFP is a member of the AN1/A20-zinc-finger proteins family
The complete cDNA of CsZfp was 0.997 kb long (GenBank accession no. DQ869863). A single open reading frame starts with nucleotide 96 and ends at nucleotide 617 including a 5′ untranslated region (95 bp) and a long 3′ untranslated region (380 bp) (Fig. 1a). The open reading frame encoded for a protein of 173 amino acids with a predicted molecular weight of 18.44 kDa and pI of 6.50. The ProtParam analysis suggested that the protein is hydrophilic with GRAVY value of − 0.334. The database search using the amino acid sequence as query showed homology to several characterized AN1/A20-ZFPs from plants including the Solanum lycopersicumStress Associated Protein 5 (SlSAP5) (Solanke et al., 2009), Oryza sativa Stress Associated Protein 8 (OsSAP8) (Kanneganti and Gupta, 2008), Solanum pennelliiStress Associated Protein 5 (SpSAP5) (Solanke et al., 2009) and Saccharum officinarum Stress Associated Protein 1 (SoSAP1) (Li et al., 2011) (Fig. 1b). All these proteins showed homology to CsZFP in the AN1-type and A20-like zinc-finger region (Fig. 1b). An AN1-type zinc-finger region is present at the C-terminus of the protein stretching between amino acids 113 and 151. It has a consensus sequence of Cx2-4Cx9-12Cx2Cx4Cx2Hx5HxC, where x represents any amino acid. The conserved cysteine and histidine residues might help to form a zinc finger (indicated in bold type in Fig. 1b). There are four conserved cysteine residues at amino acids 18, 22, 34, and 37 toward the N-terminus of the protein (Fig. 1b). This region is similar to the A20 (an inhibitor of cell death)-like zinc finger protein (Dixit et al., 1990). Phylogenetic analysis of CsZFP with other plant A20/AN1-ZFPs revealed that CsZFP showed close similarity to Malus domestica Stress-Associated Protein 8-like (MdSAP8), Prunus mume Stress-Associated Protein 8-like (PmSAP8) and Prunus persica Zinc Finger Protein (PpZFP) (Fig. 1c). Although MdSap8 and PmSap8 are not characterized yet, an expressed sequence tag (EST) of PpZfp was reported to be enriched in dormant buds of P. persica (Leida et al., 2010).
Fig. 1. Isolation and characterization of CsZfp of tea: nucleotide and deduced amino acid sequence (a), multiple sequence alignment (b), phylogenetic tree for deduced amino acid sequences of CsZfp and other A20/AN1-zinc-finger protein (c) and southern analysis of CsZfp (d). Start and stop codons are indicated by asterisks and double asterisks, respectively. Nucleotides in small letters represent 5′ and 3′ untranslated regions. The A20 and AN1 zinc-finger domains are also indicated by a line over the domains. Conserved cysteine and histidine residues are indicated by bold type. Residues varying between sequences are shown with gray background. The neighbor-joining tree was constructed using MEGA 6.06 with 1000 bootstrap replications. The GenBank accession numbers mentioned in parentheses are as follows: PpZFP (Prunus persica, XP_007218502.1), PmSAP8 (Prunus mume, XP_008231312.1), MdSAP8 (Malus domestica, XP_008375158.1), PdSAP8 (Phoenix dactylifera, XP_008782899.1), VvSAP8 (Vitis vinifera, XP_002283046.1), SlSAP5 (Solanum lycopersicum, ACM68442.1), SpSAP5 (Solanum pennellii, ACM68455.1), FaZnF (Festuca arundinacea, AEZ53300.1), OsSAP8 (Oryza sativa, A2YEZ6.2), SoSAP1 (Saccharum officinarum, ADM33793.1), AtSAP2 (Arabidopsis thaliana, NP_001185194.1), AtSAP5 (A. thaliana, NP_566429.1), AtSAP3 (A. thaliana, NP_001189620.1), AtSAP4 (A. thaliana, NP_565844.1), AtSAP6 (A. thaliana, NP_190848.1), OsZFP177 (O. sativa, AAP37480.1), AtSAP1 (A. thaliana, NP_172706.1), AtSAP7 (A. thaliana, NP_192941.1), AtSAP9 (A. thaliana, NP_194013.1), AtSAP8 (A. thaliana, NP_680686.1) and AtSAP10 (A. thaliana, NP_194268.1). Values at the node denote percentage bootstrap value. The genetic distances are indicated by the horizontal bar.
Comparison of full-length cDNA of CsZfp with a genomic fragment generated by PCR amplification and sequencing revealed the coding region of the genomic clone of CsZfp to be continuous, without an intron (Supplementary Fig. 2). The indica rice, O. sativa Stress associated protein 1 (OsSap1), identified as the first plant protein having A20 and AN1 zinc-fingers present at the N- and C-terminus, respectively, was also an intron-less gene (Mukhopadhyay et al., 2004). Southern blotting analysis of tea genomic DNA digested with DraI, EcoRI, EcoRV and SpeI with a probe encompassing the full-length cDNA of CsZfp resulted in multiple bands (Fig. 1d). This finding indicates the presence of the CsZfp gene family in the genome of tea. This was in agreement with the result of Vij and Tyagi (2008), who reported the presence of 14, 18, 10, 19, 10 and 18 A20/AN1-Zfp in Arabidopsis thaliana, O. sativa, Physcomitrella patens, Populus trichocarpa, Vitis vinifera, and Sorghum bicolor, respectively.
Comparison of full-length cDNA of CsZfp with a genomic fragment generated by PCR amplification and sequencing revealed the coding region of the genomic clone of CsZfp to be continuous, without an intron (Supplementary Fig. 2). The indica rice, O. sativa Stress associated protein 1 (OsSap1), identified as the first plant protein having A20 and AN1 zinc-fingers present at the N- and C-terminus, respectively, was also an intron-less gene (Mukhopadhyay et al., 2004). Southern blotting analysis of tea genomic DNA digested with DraI, EcoRI, EcoRV and SpeI with a probe encompassing the full-length cDNA of CsZfp resulted in multiple bands (Fig. 1d). This finding indicates the presence of the CsZfp gene family in the genome of tea. This was in agreement with the result of Vij and Tyagi (2008), who reported the presence of 14, 18, 10, 19, 10 and 18 A20/AN1-Zfp in Arabidopsis thaliana, O. sativa, Physcomitrella patens, Populus trichocarpa, Vitis vinifera, and Sorghum bicolor, respectively.
3.2 CsZfp is ubiquitously expressed in various organs with highest expression in root
Expression of CsZfp was ubiquitous in all the tissues, with the highest expression in root followed by fruit, FB, ML, TAB and stem (Fig. 2) suggesting that CsZFP might play an essential role in root development. In rice, most of the A20/AN1-Zfp genes, Zfp175, Zfp176, Zfp177, Zfp184 and Zfp185, exhibited high constitutive expression in roots, leaves, culms and spikes; whereas the expression of Zfp161 and Zfp181 was higher in roots and in leaves, respectively. Tissue specificity of A20/AN1-Zfp was also observed, as Zfp157 was specifically expressed in leaves. However the expression of Zfp156, Zfp168 and Zfp229 was below the detection level in all the tissues (Huang et al., 2008). The indica rice OsSap1 transcript was also detected at higher levels in root and pre-pollination stage panicle (Mukhopadhyay et al., 2004). SoSap1, a S. officinarum A20/AN1-Zfp, exhibited higher expression in mature stalk as compared to immature stems (Li et al., 2011). SoSap1 was suggested to function in sugarcane maturation processes.
Fig. 2. Expression of CsZfp in apical bud and the associated two leaves (TAB, two and a bud), mature leaf (ML; leaf at node position 5th with reference to the apical bud), flower bud (FB), fruit, stem and root of a field grown tea bush. Transcriptional change values, as calculated by relative expression software tool (REST), are shown in Supplementary Table 1a. Data represent the average of three biological replicates. Statistical significance of differences was determined by one way ANOVA. Vertical bars ± SD; *P < 0.05, significant difference.
3.3 CsZfp is responsive to ABA and GA3 at GT (25±3°C) and LT (4±2°C), respectively
The expression kinetics of CsZfp was investigated in response to abscisic acid and gibberellic acid in PAG and WD tissues placed at LT and GT. These cues were selected based upon their possible involvement in the process of WD and gene modulation (Paul and Kumar, 2011). Expression studies revealed that CsZfp was significantly upregulated in response to ABA in PAG and WD tissues kept at GT; whereas at LT, the expression was the same as the untreated controls (Fig. 3a,c). Interestingly, significant upregulation was also observed in response to GA3 in both PAG and WD tissues kept at LT, though at GT the expression was not affected (Fig. 3b,d). The expression was not modulated by LT either in the tissue harvested during PAG or during WD (Fig. 3).
Fig. 3. Effect of abscisic acid (ABA, 100 μM) and gibberellic acid (GA3, 100 μM) on the expression of CsZfp in apical bud and the associated two leaves (two and a bud, TAB) harvested during the period of active growth (PAG) and winter dormancy (WD) and placed at two temperatures (growth temperature; GT, 25 ± 3 °C and low temperature; LT, 4 ± 2 °C). Various experiments are shown in panels a to d. Each experiment was carried out over a period of 48 h and the analysis was performed at different time points. Stem cuttings kept in deionized water at the two temperatures served as the control. Transcriptional change values are shown in Supplementary Table 1b. Data represent the average of three biological replicates. Statistical significance of differences between the control and treatment group was determined by one way ANOVA. Vertical bars ± SD; *P < 0.05, significant difference.
Study of the interactive effects of temperature, osmotic stress, and ABA in the regulation of expression of a transgene (RD29A-Luc) consisting of the firefly luciferase coding sequence (Luc) driven by the stress-responsive RD29A promoter in A. thaliana seedlings indicated that both positive and negative interactions existed among the studied stress factors in regulating gene expression (Xiong et al., 1999). At normal growth temperature (22 °C), osmotic stress and ABA acted synergistically to induce the transgene expression. LT inhibited the response to osmotic stress or to combined treatment of osmotic stress and ABA, whereas LT and ABA treatments were additive in inducing transgene expression. Although high temperature alone did not activate the transgene, it significantly amplified the effects of ABA and osmotic stress. Previously, Paul and Kumar (2011) also reported shared gene networks in response to WD, temperature and plant hormones in tea.
In O. sativa, a GA3 inducible A20/AN1-Zfp (OsDog: O. sativa dwarf rice with overexpression of gibberellin-induced gene) was suggested to function in regulating gibberellins (GA) homeostasis and in negative maintenance of plant cell elongation (Liu et al., 2011). Transgenic O. sativa overexpressing the OsDog showed dwarf phenotypes, deficient in cell elongation. Further, the overexpressing line showed decreased levels of GA1 concomitantly with reduced expression of GA3ox2 (encodes GA 3-oxidase, which converts GA20 to GA1) and enhanced expression of GA2ox1and GA2ox3 (GA2ox1 and GA2ox3 encode GA 2-oxidases, which transfers GA1 and its immediate precursors into inactive forms). Several studies suggested an involvement of GA in different kinds of abiotic stresses (Achard et al., 2008 and Magome et al., 2008). LT reduces GA content through enhanced expression of GA-inactivating GA 2-oxidase genes (Achard et al., 2008). Low levels of GA in turn increase stability of DELLA proteins (Castillon et al., 2007). DELLA proteins are a class of nuclear proteins that belong to the GRAS (GAI, RGA, and SCARECROW) family of plant transcriptional regulators (Magome et al., 2008). DELLA proteins play an important role in regulation of sensitivity to GA, given that they were involved in negative GA signaling. DELLA proteins also conferred tolerance to salt stress by reducing reactive oxygen species (ROS) accumulation through the upregulation of genes encoding for ROS detoxification (Achard et al., 2008). Similarly in tea, regulating GA homeostasis at LT might be a desirable feature to mitigate LT mediated cell injury, where CsZfp might play key role.
3.4 CsZfp is upregulated by oxidative and salinity stress, whereas it is non-responsive to osmotic stress
A20/AN1-Zfps were modulated by various abiotic stresses (Huang et al., 2008, Kang et al., 2011, Kanneganti and Gupta, 2008 and Mukhopadhyay et al., 2004), and hence the expression of CsZfp was studied in response to various cues. CsZfp was upregulated by sodium chloride and hydrogen peroxide stress, whereas it is non-responsive to polyethylene glycol-8000 treatment (Fig. 4). A significant upregulation was observed at 1 and 24 h of sodium chloride and hydrogen peroxide treatments, respectively.
Fig. 4. Expression of CsZfp in response to polyethylene glycol-8000 (10%), sodium chloride (100 mM) and hydrogen peroxide (0.25%) in the apical bud and the associated two leaves (TAB) harvested during the period of active growth (PAG) as measured by quantitative real-time PCR. Each experiment was carried out over a period of 48 h and the analysis was performed at different time points. Stem cuttings kept in deionized water served as the control. Transcriptional change values are shown in Supplementary Table 1c. Data represent the average of three biological replicates. Statistical significance of differences between the control and treatment groups was determined by one way ANOVA. Vertical bars ± SD; *P < 0.05, significant difference.
Similar differential expression was observed with O. sativa Zfp177 (Huang et al., 2008). OsZfp177 was significantly induced by cold and heat stresses, but downregulated by salt stress. For polyethylene glycol-6000 stimulated drought stress, OsZfp177 was upregulated at 2 h followed by downregulation at 6 h of polyethylene glycol-6000 treatment. Further, overexpression of OsZfp177 in tobacco plants increased tolerance to both high and LT and hydrogen peroxide stress but led to an increased sensitivity to dehydration and salt stresses (Huang et al., 2008). On the contrary, a Festuca arundinacea A20/AN1-Zfp gene FaZnf was induced by sodium chloride, heat, wilting, wounding, polyethylene glycol-6000, but not by LT or ultraviolet light (Martin et al., 2012). FaZnf was shown to influence transcription of oxidative stress pathway genes in F. arundinacea calli overexpressing FaZnf.
3.5 CsZFP exhibits upregulation during winter dormancy
Tea plant experiences abiotic stresses during winter months (Kumar et al., 2012, Paul et al., 2014 and Vyas et al., 2007). The pattern of transcript accumulation in response to seasonal variation was examined in field-grown tea bushes (Fig. 5). CsZfpexpression was the lowest during PAG while the highest expression was observed during WD. This coincided with the growth cessation of TAB. The expression declined during dormancy release (DR), when TAB resumed growth (Fig. 5). The phylogenetic tree (Fig. 1c) suggested a close similarity of CsZFP to PpZFP. Leida et al. (2010)reported upregulation of PpZfp in the dormant bud of P. persica. Further, expression of PpZfp showed an initial gradual decrease by about a month in ‘Zincal 5’, a cultivar with a shorter period of dormancy, as compared to ‘Springlady’, a cultivar with longer dormancy period. Though CsZfp was upregulated during WD, the expression of the gene was not modulated by LT and polyethylene glycol-8000, but hydrogen peroxide treatment did modulate its expression (Fig. 4), suggesting that oxidative stress might regulate the expression of this gene. In fact tea does experience oxidative stress during winters and a positive association of WD with oxidative stress does exist in this species (Vyas et al., 2007).
Fig. 5. Expression of CsZfp in apical bud and associated two leaves (two and a bud, TAB) during the period of active growth (PAG), winter dormancy (WD), and dormancy release (DR) in field-grown tea bush. Transcriptional change values are shown in Supplementary Table 1d. Data represent the average of three biological replicates. Statistical significance of differences was determined by one way ANOVA. Vertical bars ± SD; *P < 0.05, significant difference.
For survival in adverse conditions, plants activate a stress response which involves several signaling and regulatory proteins. Genes of the A20/AN1-Zfp family have been shown to be involved in animal immune responses and plant stress responses (Heyninck and Beyaert, 2005, Huang et al., 2008, Kang et al., 2011, Kanneganti and Gupta, 2008 and Mukhopadhyay et al., 2004). It has been suggested that A20/AN1-ZFP might act as regulatory protein by DNA or protein binding through A20 and/or AN1 zinc-fingers and regulates the activity of other key stress-related genes such as receptor-like cytoplasmic kinases (RLCKs; RLCKs are variants of receptor-like kinases without transmembrane domain) during stress and helps in imparting subsequent tolerance (Giri et al., 2011). The A20 domain was shown to mediate the interaction of an O. sativa A20/AN1-ZFP (OsSAP1) with itself and its close homologs OsSAP11 and OsRLCK253. The interactions between OsSAP1⁄11 and with OsRLCK253 occur at the nuclear membrane, plasma membrane and in the nucleus (Giri et al., 2011).
Environmental stresses change the cellular ionic balance and this change in cellular redox state acts as a signal, activating the cellular defense response (Farah and Amberg, 2007). A. thaliana A20/AN1-ZFP (AtSAP12) has been proposed to act as redox sensor and undergoes conformational changes from oxidized to reduced state in a reversible manner during stress (Ströher et al., 2009). It was proposed that AtSAP12 can interact with other proteins and modulates stress signaling during conformation changes (Ströher et al., 2009).
Ubiquitination is a posttranslational modification system that is involved in protein quality control and protein stability through the covalent attachment of the 76 amino acid ubiquitin polypeptide to a target protein (Ben-Neriah, 2002). It was suggested that A. thaliana A20/AN1-ZFPs might have ubiquitination activity and might be involved in downregulating the pathway associated with abiotic stress associated injuries, such as cell death, by ubiquitinating the key proteins and targeting them for degradation (Vij and Tyagi, 2008). AtSAP5, a member of A. thaliana A20/AN1-ZFP was shown to have E3 ubiquitin ligase activity in vitro (Kang et al., 2011). The E3 ubiquitin ligase activity was mediated primarily through the AN1 zinc-finger motif. Transgenic A. thaliana plants, overexpressing AtSAP5 exhibited increased tolerance to abiotic stresses such as salt stress, osmotic stress and water deficit, suggesting that AtSAP5 acts as a positive regulator of stress responses mediated through ubiquitination in A. thaliana.
CsZFP might act through ubiquitin related mechanisms, through interactions with protein kinases, or through interactions with other A20/AN1-ZFP, non-A20/AN1-ZFP proteins or transcription factors. It is quite possible that CsZFP might have many functions as an abiotic stress factor as do many stress related proteins.
The following are the supplementary data related to this article.
Supplementary Table 1. (a) Expression of CsZfp in apical bud and the associated two leaves (two and a bud, TAB), mature leaf (ML, leaf at node position 5th with reference to the apical bud), flower bud (FB), fruit, stem and root of a field-grown tea bush.
(b) Effect of abscisic acid (ABA, 100 μM) and gibberellic acid (GA3, 100 μM) on the expression of CsZfp in apical bud and the associated two leaves (two and a bud, TAB) harvested during the period of active growth (PAG) and winter dormancy (WD) and placed at two temperatures (GT, 25 ± 3 °C and LT, 4 ± 2 °C). Each experiment was carried out over a period of 48 h and the analysis was performed at different time points. Stem cuttings kept in deionized water for the two temperatures served as the control.
(c) Expression of CsZfp in response to polyethylene glycol-8000 (10%), hydrogen peroxide (0.25%) and sodium chloride (100 mM) in apical bud and the associated two leaves (TAB) as measured by quantitative real-time PCR. Each experiment was carried out over a period of 48 h and the analysis was performed at different time points. Stem cuttings kept in deionized water served as the control.
(d) Expression of CsZfp in apical bud and associated two leaves (TAB) during the period of active growth (PAG), winter dormancy (WD), and dormancy release (DR) in field-grown tea bush for two consecutive years. Transcriptional change values were calculated by relative expression software tool (REST). “Materials and Methods” section details normalization of data using housekeeping gene.
Supplementary Fig. 2. Intron analysis. Amplification of CsZfp open reading frame using genomic DNA (L1) and cDNA (L2) as template. The polymerase chain reaction (PCR) was performed with 10 ng of genomic DNA or with 3 μl of RACE-ready cDNA synthesized from total RNA isolated from winter dormant tissues (described in detailed in Materials and Methods Section) and 0.5 μM each of forward: 5′-ATGGAGCAAAATGAGACAGGATG-3′ and reverse: 5′-CTAGAGCTTATCAAGCTTTTCAGC-3′ gene-specific primers in a final volume of 25 μl. The PCR products were separated on a 1.2% agarose gel stained with ethidium bromide; visualized using a gel documentation system (Alpha DigiDocTM, Alpha Innotech, USA). M1 and M2 represent 100 and 500 bp molecular weight marker of DNA, respectively.
The authors are thankful to the Director, CSIR-IHBT for providing necessary facilities and Council of Scientific and Industrial Research (CSIR) for providing financial assistance through network projects BSC-0107, and BSC-0109. AP is grateful to CSIR for awarding Junior and Senior Research Fellowships. MS represents CSIR-IHBT publication number 3758.
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