Plant Gene 1 (2015) 1–7

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Plant Gene

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An A20/AN1-zinc-finger domain containing gene in tea is differentially expressed during winter dormancy and in response to abiotic stress and plant growth regulators

Asosii Paul 1,SanjayKumar⁎

Biotechnology Division, CSIR-Institute of Himalayan Bioresource Technology, Palampur, Himachal Pradesh 176061, India article info abstract

Article history: The present manuscript describes cloning and expression characterization of A20/AN1-zinc-finger domain Received 15 November 2014 containing protein (CsZfp) gene in an evergreen tree tea [Camellia sinensis (L.) O. Kuntze] in response to winter Received in revised form 31 December 2014 dormancy (WD), abiotic stresses (polyethylene glycol, hydrogen peroxide, and sodium chloride) and plant Accepted 31 December 2014 growth regulators [abscisic acid (ABA), and gibberellic acid (GA )]. CsZfp encoded a putative protein of 173 Available online 9 January 2015 3 amino acids with a calculated molecular weight of 18.44 kDa, an isoelectric point (pI) of 6.50 and grand average − Keywords: of hydropathicity (GRAVY) value of 0.334. The gene did not have an intron, and belonged to a multi-gene A20/AN1 zinc-finger protein family. During the period of active growth (PAG), CsZfp showed maximum expression in root and fruit as CsZfp compared to leaf, floral bud and stem. Interaction studies between temperature and plant growth regulators Camellia sinensis on the expression of CsZfp showed that ABA upregulated CsZfp expression at growth temperature (GT; 25 °C)

Winter dormancy 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. © 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction There is an interest to understand the phenomenon of WD and the evergreen nature of tea during winters. A subtracted cDNA library- Tea [Camellia sinensis (L.) O. Kuntze] is a perennial evergreen tree based approach (Paul and Kumar, 2011) and RNA-sequencing on next grown in different agro-climatic zones across the world. Tender young generation sequencing (NGS) platform (Paul et al., 2014)wereusedto shoots, consisting of an apical bud and associated two leaves, known as decipher the phenomenon. Analyses of unigenes obtained showed the “two and a bud (TAB)”, are harvested at intervals of one to three weeks operation of mechanisms of winter tolerance, and downregulation of for the production of commercial tea. Growth of TAB is relatively uniform genes involved in growth, development, protein synthesis and cell divi- throughout the year near the equator. However, growth of TAB is restrict- sion (Paul and Kumar, 2011; Paul et al., 2014). Also, the data explained ed during winters in the plants at latitudes beyond 16° north and south of the evergreen nature of the tea tree wherein inhibition of leaf abscission equator and the plants are said to be dormant, a phenomenon known as due to modulation of senescence related processes during WD in tea winter dormancy (WD). Duration of WD varies from 2 to 6 months, plac- played a decisive role (Paul et al., 2014). Though subtracted cDNA ing the areas experiencing WD at a disadvantage for crop yield compared library and RNA-sequencing on NGS platform produced a huge amount to those areas where it does not occur (Kumar et al., 2012). 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. Abbreviations: ABA, abscisic acid; ANOVA, analysis of variance; CsZfp, Camellia sinensis We previously identified an expressed sequence tag (EST; GH454325) A20/AN1-zinc-finger domain containing protein; cDNA, complementary DNA; DR, dormancy that was upregulated in winter-dormant tissue of tea (Paul and Kumar, release;EST,expressedsequencestag;FB,flowerbud;GA3,gibberellicacid;GT,growthtem- perature;LT,lowtemperature;ML,matureleaf;PAG,periodofactivegrowth;PCR,polymer- 2011). RNA-sequencing also identified expression of this gene ase chain reaction; RACE, Rapid amplification of cDNA ends; REST, Relative Expression (scaffold24632_86.0) in the transcriptome of winter-dormant tissue of Software Tool; ROS, reactive oxygen species; TAB, two and a bud; WD, winter dormancy. tea (Paul et al., 2014). Preliminary analysis of the EST suggested the ⁎ Corresponding author. Tel.: +91 1894 233339; fax: +91 1894 230411. presence of A20 and AN1 zinc-finger domains. Realizing the E-mail addresses: [email protected], [email protected] (S. Kumar). 1 Current address: National Institute of Plant Genome Research, Aruna Asaf Ali Marg, importance of A20 and AN1 zinc-finger domain containing in P.O. Box No. 10531, New Delhi 110067, India. plant stresses response (Mukhopadhyay et al., 2004; Vij and Tyagi, http://dx.doi.org/10.1016/j.plgene.2014.12.003 2352-4073/© 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 2 A. Paul, S. Kumar / Plant Gene 1 (2015) 1–7

2008) and that not much has been reported on this gene in its relation- through screening of a subtracted cDNA library, 5′-RACE (Supplementa- ship with WD, the full-length cDNA of this gene was cloned through ry Fig. 1) was performed as per manufacturer's instructions using gene Rapid Amplification of cDNA Ends (RACE) followed by detailed expression specific primers (GSPs): GSP1: 5′-TCCATCAAACCAGCACAAACAAACAA analysis. BLAST analysis of the full-length cDNA suggested this to be GT-3′ and GSP2: 5′-CCACTGAAATATCCATCAAACCAGCACA-3′.Total encoding for an A20/AN1-zinc-finger protein (A20/AN1-ZFP), therefore RNA isolated from winter dormant tissues as described by Muoki et al. the gene was called Camellia sinensis A20/AN1-zinc-finger domain (2011) was used for RACE-ready cDNA synthesis. The amplified containing protein (CsZfp). Proteins of these families have an A20 zinc- fragment (Supplementary Fig. 1) was cloned in pGEM-T easy vector finger domain present at the N-terminus and the AN1 zinc-finger domain (Promega, USA). The nucleotide sequence was determined using BigDye present at the C-terminus (Mukhopadhyay et al., 2004; Vij and Tyagi, terminator v 3.1 cycle sequencing mix (Applied Biosystems, USA) on an 2008). Evidence suggests that A20/AN1-ZFPs plays a crucial role in plant automated DNA sequencer (ABI 3130xl Genetic Analyzer, Applied stress response. A20/AN1-Zfps are responsive to multiple environmental Biosystems, USA) following the manufacturer's instructions. A search stresses like cold, salt, drought, submergence, wounding and heavy for homologous nucleic acid and protein sequences was performed metals. Further, its overexpression confers tolerance to abiotic stresses using the BLAST algorithm (http://www.ncbi.nlm.nih.gov/). A multiple in transgenic plants (Huang et al., 2008; Kang et al., 2011; Kanneganti sequence alignment was generated by ClustalW multiple sequence and Gupta, 2008; Mukhopadhyay et al., 2004). A few reports also sug- alignment programs (http://www.ebi.ac.uk/clustalw/). The deduced gested their role in plant development (Li et al., 2011). The present amino acid sequence was analyzed by ProtParam Programs at ExPASy work describes the characterization of CsZfp and discusses its role in rela- proteomics server (http://ca.expasy.org/) to calculate pI/Mw. The phy- tion to abiotic stresses response. logenetic tree was constructed using MEGA 6.06 software (Biodesign In- stitute, A240, Arizona State University, Tempe, AZ). 2. Materials and methods 2.3. Intron and southern blot analysis 2.1. Plant materials and stress treatments Genomic DNA was isolated from TAB using plant DNAzol (Invitrogen, TEENALI, an Assamica clone of tea was used in the present work USA). To check for the presence of intron within the coding region, gene- (Paul and Kumar, 2011). The tea bushes were well maintained at specificforward(5′-ATG GAGCAAAATGAGACAGGATGCC-3′;startcodon the tea experimental farm of our Institute (32°6′N latitude; 76°8′E underlined) and reverse (5′-CTAGAGCTTATCAAGCTTTTCAGC-3′; stop longitude; 1289 ± 15 m above sea level) and subjected to usual cultural codon underlined) primers designed from the start and stop codon practices. TAB was collected during the period of active growth (PAG, were used for polymerase chain reaction (PCR) with 3 μl of RACE-ready July; maximum temperature, 25 ± 2 °C; minimum temperature, 20 ± cDNA or 10 ng of genomic DNA. The PCR amplification involved an initial 2 °C), WD (December; maximum temperature, 15 ± 2 °C; minimum denaturation step at 94 °C for 1 min, followed by 28 cycles of denaturation temperature, 4 ± 2 °C), and dormancy release (DR, April; maximum at 94 °C for 10 s, annealing at 58 °C for 30 s, extension at 72 °C for 1 min, temperature, 26 ± 3 °C; minimum temperature, 15 ± 2 °C), frozen im- and final extension at 72 °C for 7 min. Cloning and sequencing were mediately in liquid nitrogen and stored at −80 °C until use. In addition performed as described in the previous section. to the above periods, TAB, mature leaf (ML), flower bud (FB), fruit, stem Southern blotting was performed essentially as described by and root were also collected from a clonal bush from the field during Sambrook et al. (1989).GenomicDNA(10μg) was digested to comple- the month of September, when both flower and fruit are available. tion with DraI, EcoRI, EcoRV and SpeI (no cut site within the probe) in Temperature data was collected from a weather observatory situated separate reactions. Digested genomic DNA was separated by electro- in the neighboring agriculture university, which is within 3 km of the phoresis on a 0.7% agarose gel, denatured, and blotted onto a nylon periphery of the experimental tea farm. membrane (Amersham Pharmacia, UK). The membrane was hybridized In a separate experiment, shoot cuttings containing apical bud and with full-length of CsZfp cDNA probe labeled with [α-32P] dATP. Hybrid- associated five leaves were collected from clonal bushes of TEENALI ization was performed overnight at 55 °C in ExpressHyb™ solution from the field during PAG and WD and stabilized in deionized water (Clontech, USA). After hybridization, the blot was washed twice with overnight before the start of the experiment. Thereafter, shoot cuttings 2× SSC and 0.01% SDS for 20 min each at room temperature and twice were transferred to deionized water (control), 100 μM 2-cis, 4-trans- with 0.1× SSC and 0.1% SDS for 10 min each at 55 °C. The blot was abscisic acid (ABA; Sigma, USA) and 100 μM gibberellic acid (GA ; 3 then exposed to X-ray film and stored at −80 °C until developed as Sigma, USA), separately as described by Paul and Kumar (2011).These described by Bhardwaj et al. (2010). were housed in a plant growth chamber set at 25 ± 3 °C (growth tem- perature, GT) and 4 ± 2 °C (low temperature, LT) (light intensity, 200 μEm−2 s−1;RH,70–80%; Saveer Biotech, India). For osmotic, 2.4. Expression analysis oxidative and salinity stress, cuttings collected during PAG were transferred to 10% polyethylene glycol-8000 (Sigma, USA) and 0.25% Gene expression was performed as described by Paul and Kumar hydrogen peroxide (Merck, Germany) and 100 mM sodium chloride (2011). Total RNA was treated with DNase I, Ampl Grade (Invitrogen, (Sigma, USA) and housed in a plant growth chamber set at 25 ± 3 °C USA) to remove contaminating genomic DNA. First-strand cDNA was Syn- as described previously (Paul et al., 2012). Each experiment was carried thesized using 2 μg of DNA free RNA, SuperScript III First-strand synthesis out over a period of 48 h and the gene expression was analyzed in TAB Kit (Invitrogen, USA) and an oligo (dT)12–18 primer (Invitrogen, USA). as described in the relevant figures. Gene expression was performed using forward: 5′-CAACAACAGACTGA ACTTGC-3′ and reverse: 5′-GCAGTCATGTTTGTCAGAGT-3′ gene-specific 2.2. Rapid amplification of cDNA ends (RACE) and sequence analysis primers on a Stratagene Mx3000P system (Agilent Technologies, Germany) using 2× Brilliant III SYBR® Green qPCR Master Mix (Agilent As a part of our ongoing program on understanding the molecular Technologies, Germany). All qPCRs were run in triplicate with a no- basis of WD of tea, we deposited ESTs at Genbank at NCBI (accession template control to check for contamination. PCR was conducted under numbers FF682697 to FF682833, GH454303 to GH454326, FE942774 the following conditions: 10 min at 95 °C (enzyme activation), 40 cycles to FE943102, and JG017532 to JG017536). For the present work, EST eachof30sat95°C,30sat55°Cand72°Cfor30sandafinal melting (accession number GH454325) was used to design primers for RACE curve analysis was performed (55° to 95 °C) to verify the specificity of (SMART RACE cDNA amplification Kit, BD Biosciences, USA) to clone amplicons. The raw threshold cycle (Ct) values were normalized against full-length cDNA. Since the 3′ end of the gene was complete as cloned a housekeeping gene actin (forward: 5′-GCCATATTTGATTGGAATGG-3′ A. Paul, S. Kumar / Plant Gene 1 (2015) 1–7 3 and reverse: 5′-GGTGCCACAACCTTGATCTT-3′ primers) using the Relative 3. Results and discussion Expression Software Tool (REST) (Pfaffl et al., 2002). Each experiment was repeated three times with separate biological 3.1. CsZFP is a member of the AN1/A20-zinc-finger proteins family materials. One way factorial analysis of variance (ANOVA) was used to test the effects of treatments on gene expression; Duncan's multiple The complete cDNA of CsZfp was 0.997 kb long (GenBank accession range test (P b 0.05) was used to compare means post hoc using the soft- no. DQ869863). A single open reading frame starts with nucleotide 96 ware Statistica version 7.0 (StatSoft Inc., USA). and ends at nucleotide 617 including a 5′ untranslated region a c

PpZFP 99 PmSAP8 98 MdSAP8 89 CsZFP 89 PdSAP8 VvSAP8 48 47 SlSAP5 91 100 SpSAP5 FaZnF

89 92 OsSAP8 98 SoSAP1 72 AtSAP2 AtSAP5 35 AtSAP3 AtSAP4 75 47 100 AtSAP6 OsZFP177 AtSAP1

97 AtSAP7 82 AtSAP9 AtSAP8 87 AtSAP10

0.1

b A20 domain d

AN1 domain

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 se- quences 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. Nucle- otides 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 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 boot- strap 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. 4 A. Paul, S. Kumar / Plant Gene 1 (2015) 1–7

(95 bp) and a long 3′ untranslated region (380 bp) (Fig. 1a). The 1.0 open reading frame encoded for a protein of 173 amino acids with 0.8 a predicted molecular weight of 18.44 kDa and pI of 6.50. The * ProtParam analysis suggested that the protein is hydrophilic with * 0.6 GRAVY value of −0.334. The database search using the amino acid sequence as query showed homology to several characterized AN1/ 0.4 A20-ZFPs from plants including the Solanum lycopersicum Stress Associated Protein 5 (SlSAP5) (Solanke et al., 2009), Oryza sativa 0.2 Stress Associated Protein 8 (OsSAP8) (Kanneganti and Gupta, 2008), Solanum pennellii Stress Associated Protein 5 (SpSAP5) Relative gene expression fi 0.0 (Solanke et al., 2009)andSaccharum of cinarum Stress Associated TAB ML FB Fruit Stem Root 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 Fig. 2. Expression of CsZfp in apical bud and the associated two leaves (TAB, two and a region (Fig. 1b). An AN1-type zinc-finger region is present at the C- bud), mature leaf (ML; leaf at node position 5th with reference to the apical bud), flower terminus of the protein stretching between amino acids 113 and bud (FB), fruit, stem and root of a field grown tea bush. Transcriptional change values, as 151. It has a consensus sequence of Cx2-4Cx9-12Cx2Cx4Cx2Hx5HxC, calculated by relative expression software tool (REST), are shown in Supplementary Table 1a. Data represent the average of three biological replicates. Statistical significance where x represents any amino acid. The conserved cysteine and of differences was determined by one way ANOVA. Vertical bars ± SD; *P b 0.05, signifi- histidine residues might help to form a zinc finger (indicated in cant difference. 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 3.3. CsZfp is responsive to ABA and GA3 atGT(25±3°C)andLT(4±2°C), CsZFP with other plant A20/AN1-ZFPs revealed that CsZFP showed respectively close similarity to Malus domestica Stress-Associated Protein 8-like (MdSAP8), Prunus mume Stress-Associated Protein 8-like (PmSAP8) The expression kinetics of CsZfp was investigated in response to and Prunus persica Protein (PpZFP) (Fig. 1c). Although abscisic acid and gibberellic acid in PAG and WD tissues placed at LT MdSap8 and PmSap8 are not characterized yet, an expressed and GT. These cues were selected based upon their possible involve- sequence tag (EST) of PpZfp was reported to be enriched in dormant ment in the process of WD and gene modulation (Paul and Kumar, buds of P. persica (Leida et al., 2010). 2011). Expression studies revealed that CsZfp was significantly upregu- Comparison of full-length cDNA of CsZfp with a genomic frag- lated in response to ABA in PAG and WD tissues kept at GT; whereas at ment generated by PCR amplification and sequencing revealed LT, the expression was the same as the untreated controls (Fig. 3a,c). In- the coding region of the genomic clone of CsZfp to be continuous, terestingly, significant upregulation was also observed in response to without an intron (Supplementary Fig. 2). The indica rice, GA3 in both PAG and WD tissues kept at LT, though at GT the expression O. sativa Stress associated protein 1 (OsSap1), identified as the first was not affected (Fig. 3b,d). The expression was not modulated by LT plant protein having A20 and AN1 zinc-fingers present at the N- either in the tissue harvested during PAG or during WD (Fig. 3). and C-terminus, respectively, was also an intron-less gene Study of the interactive effects of temperature, osmotic stress, and (Mukhopadhyay et al., 2004). Southern blotting analysis of tea ge- ABA in the regulation of expression of a transgene (RD29A-Luc) nomic DNA digested with DraI, EcoRI, EcoRVandSpeIwithaprobe consisting of the firefly luciferase coding sequence (Luc)drivenbythe encompassing the full-length cDNA of CsZfp resulted in multiple stress-responsive RD29A promoter in A. thaliana seedlings indicated bands (Fig. 1d). This finding indicates the presence of the CsZfp that both positive and negative interactions existed among the studied gene family in the genome of tea. This was in agreement with the stress factors in regulating gene expression (Xiong et al., 1999). At result of Vij and Tyagi (2008), who reported the presence of 14, normal growth temperature (22 °C), osmotic stress and ABA acted 18, 10, 19, 10 and 18 A20/AN1-Zfp in Arabidopsis thaliana, synergistically to induce the transgene expression. LT inhibited the re- O. sativa, Physcomitrella patens, Populus trichocarpa, Vitis vinifera, sponse to osmotic stress or to combined treatment of osmotic stress and Sorghum bicolor, respectively. 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 3.2. CsZfp is ubiquitously expressed in various organs with highest stress. Previously, Paul and Kumar (2011) also reported shared gene expression in root networks in response to WD, temperature and plant hormones in tea.

In O. sativa,aGA3 inducible A20/AN1-Zfp (OsDog: O. sativa dwarf rice Expression of CsZfp was ubiquitous in all the tissues, with the with overexpression of gibberellin-induced gene)wassuggestedtofunc- highest expression in root followed by fruit, FB, ML, TAB and stem tion in regulating gibberellins (GA) homeostasis and in negative main- (Fig. 2) suggesting that CsZFP might play an essential role in root de- tenance of plant cell elongation (Liu et al., 2011). Transgenic O. sativa velopment. In rice, most of the A20/AN1-Zfp genes, Zfp175, Zfp176, overexpressing the OsDog showed dwarf phenotypes, deficient in cell Zfp177, Zfp184 and Zfp185, exhibited high constitutive expression elongation. Further, the overexpressing line showed decreased levels in roots, leaves, culms and spikes; whereas the expression of of GA1 concomitantly with reduced expression of GA3ox2 (encodes GA Zfp161 and Zfp181 was higher in roots and in leaves, respectively. 3-oxidase, which converts GA20 to GA1) and enhanced expression of Tissue specificity of A20/AN1-Zfp was also observed, as Zfp157 was GA2ox1 and GA2ox3 (GA2ox1 and GA2ox3 encode GA 2-oxidases, specifically expressed in leaves. However the expression of Zfp156, which transfers GA1 and its immediate precursors into inactive Zfp168 and Zfp229 was below the detection level in all the tissues forms). Several studies suggested an involvement of GA in different (Huang et al., 2008). The indica rice OsSap1 transcript was also kinds of abiotic stresses (Achard et al., 2008; Magome et al., 2008). LT detected at higher levels in root and pre-pollination stage panicle reduces GA content through enhanced expression of GA-inactivating (Mukhopadhyay et al., 2004). SoSap1,aS. officinarum A20/AN1-Zfp, GA 2-oxidase genes (Achard et al., 2008). Low levels of GA in turn in- exhibited higher expression in mature stalk as compared to imma- crease stability of DELLA proteins (Castillon et al., 2007). DELLA proteins ture stems (Li et al., 2011). SoSap1 was suggested to function in are a class of nuclear proteins that belong to the GRAS (GAI, RGA, and sugarcane maturation processes. SCARECROW) family of plant transcriptional regulators (Magome A. Paul, S. Kumar / Plant Gene 1 (2015) 1–7 5 et al., 2008). DELLA proteins play an important role in regulation of sen- 1.2 0 h 1 h 4 h 24 h 48 h * sitivity to GA, given that they were involved in negative GA signaling. 1.0 DELLA proteins also conferred tolerance to salt stress by reducing reac- tive oxygen species (ROS) accumulation through the upregulation of 0.8 genes encoding for ROS detoxification (Achard et al., 2008). Similarly in tea, regulating GA homeostasis at LT might be a desirable feature to * 0.6 mitigate LT mediated cell injury, where CsZfp might play key role. 0.4 * * * * 3.4. CsZfp is upregulated by oxidative and salinity stress, whereas it is 0.2 non-responsive to osmotic stress Relative gene expression 0.0 A20/AN1-Zfps were modulated by various abiotic stresses (Huang et al., 2008; Kang et al., 2011; Kanneganti and Gupta, 2008; 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 upregula- tion was observed at 1 and 24 h of sodium chloride and hydrogen peroxide treatments, respectively. Similar differential expression was observed with O. sativa Zfp177 Fig. 4. Expression of CsZfp in response to polyethylene glycol-8000 (10%), sodium chloride fi (100 mM) and hydrogen peroxide (0.25%) in the apical bud and the associated two leaves (Huang et al., 2008). OsZfp177 was signi cantly induced by cold and (TAB) harvested during the period of active growth (PAG) as measured by quantitative heat stresses, but downregulated by salt stress. For polyethylene real-time PCR. Each experiment was carried out over a period of 48 h and the analysis glycol-6000 stimulated drought stress, OsZfp177 was upregulated at was performed at different time points. Stem cuttings kept in deionized water served as 2 h followed by downregulation at 6 h of polyethylene glycol-6000 the control. Transcriptional change values are shown in Supplementary Table 1c. Data rep- resent the average of three biological replicates. Statistical significance of differences be- treatment. Further, overexpression of OsZfp177 in tobacco plants tween the control and treatment groups was determined by one way ANOVA. Vertical increased tolerance to both high and LT and hydrogen peroxide stress bars ± SD; *P b 0.05, significant difference. 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, 3.5. CsZFP exhibits upregulation during winter dormancy wounding, polyethylene glycol-6000, but not by LT or ultraviolet light (Martin et al., 2012). FaZnf was shown to influence transcription of Tea plant experiences abiotic stresses during winter months (Kumar oxidative stress pathway genes in F. arundinacea calli overexpressing et al., 2012; Paul et al., 2014; Vyas et al., 2007). The pattern of transcript FaZnf. accumulation in response to seasonal variation was examined in field-

a PAG + GT c WD + GT 1.0 1.0 0 h 1 h 4 h 24 h 48 h 0 h 1 h 4 h 24 h 48 h * 0.8 0.8

0.6 0.6

0.4 * 0.4 0.2 0.2 Relative gene expression 0.0 Relative gene expression 0.0 Control Abscisic acid Gibberellic acid Control Abscisic acid Gibberellic acid

b PAG + LT d WD + LT 1.0 1.0 0 h 1 h 4 h 24 h 48 h * 0 h 1 h 4 h 24 h 48 h * * 0.8 0.8

0.6 0.6

0.4 * * 0.4

0.2 0.2 Relative gene expression 0.0 Relative gene expression 0.0 Control Abscisic acid Gibberellic acid Control Abscisic acid Gibberellic acid

Fig. 3. Effect of abscisic acid (ABA, 100 μM) and gibberellic acid (GA3,100μM) 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 exper- iments 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 sig- nificance of differences between the control and treatment group was determined by one way ANOVA. Vertical bars ± SD; *P b 0.05, significant difference. 6 A. Paul, S. Kumar / Plant Gene 1 (2015) 1–7 grown tea bushes (Fig. 5). CsZfp expression was the lowest during PAG A20/AN1-ZFPs might have ubiquitination activity and might be in- while the highest expression was observed during WD. This coincided volved in downregulating the pathway associated with abiotic stress as- with the growth cessation of TAB. The expression declined during sociated injuries, such as cell death, by ubiquitinating the key proteins dormancy release (DR), when TAB resumed growth (Fig. 5). The phylo- and targeting them for degradation (Vij and Tyagi, 2008). AtSAP5, a genetic tree (Fig. 1c) suggested a close similarity of CsZFP to PpZFP. member of A. thaliana A20/AN1-ZFP was shown to have E3 li- Leida et al. (2010) reported upregulation of PpZfp in the dormant bud gase activity in vitro (Kang et al., 2011). The E3 ubiquitin ligase activity of P. persica. Further, expression of PpZfp showed an initial gradual was mediated primarily through the AN1 zinc-finger motif. Transgenic decrease by about a month in ‘Zincal 5’, a cultivar with a shorter period A. thaliana plants, overexpressing AtSAP5 exhibited increased tolerance of dormancy, as compared to ‘Springlady’, a cultivar with longer dor- to abiotic stresses such as salt stress, osmotic stress and water deficit, mancy period. Though CsZfp was upregulated during WD, the expres- suggesting that AtSAP5 acts as a positive regulator of stress responses sion of the gene was not modulated by LT and polyethylene glycol- mediated through ubiquitination in A. thaliana. 8000, but hydrogen peroxide treatment did modulate its expression CsZFP might act through ubiquitin related mechanisms, through (Fig. 4), suggesting that oxidative stress might regulate the expression interactions with protein kinases, or through interactions with other of this gene. In fact tea does experience oxidative stress during winters A20/AN1-ZFP, non-A20/AN1-ZFP proteins or transcription factors. It is and a positive association of WD with oxidative stress does exist in this quite possible that CsZFP might have many functions as an abiotic stress species (Vyas et al., 2007). factor as do many stress related proteins. For survival in adverse conditions, plants activate a stress response Supplementary data to this article can be found online at http://dx. which involves several signaling and regulatory proteins. Genes of the doi.org/10.1016/j.plgene.2014.12.003. A20/AN1-Zfp family have been shown to be involved in animal immune responses and plant stress responses (Heyninck and Beyaert, 2005; Acknowledgment Huang et al., 2008; Kang et al., 2011; Kanneganti and Gupta, 2008; Mukhopadhyay et al., 2004). It has been suggested that A20/AN1-ZFP The authors are thankful to the Director, CSIR-IHBT for providing might act as regulatory protein by DNA or protein binding through necessary facilities and Council of Scientific and Industrial Research A20 and/or AN1 zinc-fingers and regulates the activity of other key (CSIR) for providing financial assistance through network projects stress-related genes such as receptor-like cytoplasmic kinases (RLCKs; BSC-0107, and BSC-0109. AP is grateful to CSIR for awarding Junior RLCKs are variants of receptor-like kinases without transmembrane and Senior Research Fellowships. MS represents CSIR-IHBT publication domain) during stress and helps in imparting subsequent tolerance number 3758. (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. 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