Gene 555 (2015) 186–193

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miR408 overexpression causes increased drought tolerance in chickpea

Mortaza Hajyzadeh a, Mine Turktas a, Khalid Mahmood Khawar b, Turgay Unver a,⁎a Cankiri Karatekin University, Faculty of Science, Department of Biology, Cankiri, Turkeyb Ankara University, Faculty of Agriculture, Department of Field Crops, Ankara, Turkey

Abbreviations:Cu, copper;miRNA,microRNA;NCBI, NInformation;Pre, precursor; qRT-PCR, quantitative reversetion factor; U, Unite.⁎ Corresponding author at: Faculty of Science, Departm

University, 18100 Cankiri, Turkey.E-mail address: turgayunver@gmail.com (T. Unver).

http://dx.doi.org/10.1016/j.gene.2014.11.0020378-1119/© 2014 Elsevier B.V. All rights reserved.

a b s t r a c t

a r t i c l e i n f o

Article history:Received 11 August 2014Received in revised form 28 October 2014Accepted 2 November 2014Available online 4 November 2014

Keywords:ChickpeaDrought stressmiR408DevelopmentCopper accumulationPlantacyanin

Drought stress limits yield severely in most of the crops. Plants utilize complex gene regulation mechanisms to tol-erate water deficiency as well as other abiotic stresses. MicroRNAs (miRNAs) are a class of small non-coding RNAsthat are progressively recognized as important regulators of gene expression acting at post-transcriptional level.miR408, conserved in terrestrial plants, targets copper related genes. Although, expression level of miR408 is influ-enced by various environmental factors including drought stress, the biological action of miR408 is still unclear. Toexamine the miR408 function upon drought stress in chickpea, transgenic lines overexpressing the miR408 weregenerated. Induced tolerance was observed in the plants with enhancedmiR408 expression upon 17-daywater de-ficiency. Expression levels of miR408 target gene together with seven drought responsive genes were measuredusing qRT-PCR. Here, the involvement of miR408 in drought stress response has been reported. The overexpressionleading plantacyanin transcript repression caused regulation of DREB and other drought responsive genes.

© 2014 Elsevier B.V. All rights reserved.

1. Introduction

Chickpea (Cicer arietinum L.) is an important legume crop grown inarid and semi-arid zones. Due to its long taproot, chickpea can with-stand water deficiency. However, 7% to 51% yield reduction due todrought has been reported in chickpea cultivars (Khodadadi, 2013).Drought stress restricts plant growth and development severely, whiletolerant plants are able to survive by several mechanisms such as con-suming small amount of water keeping their stomata closed at a highrate under drought conditions (Nunes et al., 2008), several genes, regu-latory RNAs and signaling molecules are involved to overcome droughtstress. The latest studies have shown that small RNAs act as importantplayers for stress response (Kurihara and Watanabe, 2004; Chen andRajewsky, 2007; Voinnet, 2009; Zhang and Li, 2013).

miRNAs are non-coding small (20–24 nucleotide long) RNAs, andshow high conservation among higher plants. They are involved inpost-transcriptional gene regulation via silencing mechanism. A givenmiRNA targets multiple genes or more common several miRNAs act to-gether in regulation of one target gene (Chen and Rajewsky, 2007;Eldem et al., 2013). miR408 is one of the highly conserved miRNAfamilies among 28,645 miRNAs deposited in miRBase v.21 (released in2014, June) (Kozomara and Griffiths-Jones, 2011). 114 homologous

ational Center for BiotechnologytranscriptionPCR; TF, transcrip-

ent of Biology, Cankiri Karatekin

sequences have been identified in more than 20 plants. It has been doc-umented that the expression of miR408 is regulated by environmentalstresses (Zhang and Li, 2013). Induced miR408 expression in responseto dehydration (Kantar et al., 2010; Trindade et al., 2010), mechanicalstress (Lu et al., 2005), and reactive oxygen species (Li et al., 2011) hasalso been reported. It was reported that expression level of miR408was up-regulated in Medicago truncatula (Trindade et al., 2010) and inHordeum vulgare (Kantar et al., 2010)while reducedmiR408 expressionwas observed in Prunus persica (Eldem et al., 2012) and in Oryza sativa(Zhou et al., 2010) upon drought stress.

Besides, altered miR408 expression was also measured in responseto phosphate (Melnikova et al., 2014) Ca2+ (Mutum et al., 2013), andseveral metal stresses (Valdés‐López et al., 2010; Lima et al., 2011;Gielen et al., 2012; Zhou et al., 2012). Plantacyanin, responsible for cop-per (Cu) binding, is directly regulated by target ofmiR408. Expression ofthe miR408 in response to Cu deficiency plays an important role in ad-justment of copper level (Abdel-Ghany and Pilon, 2008).

Here, the miR408 overexpressor lines were generated and expres-sion of several genes including miR408 target and seven droughtresponsive genes was measured by qRT-PCR. The regulatory role ofmiR408 on drought response was indicated. This study aimed to under-stand the function of miR408 in drought stress in chickpea.

2. Material and methods

2.1. Plant material and drought stress treatment

Seeds of chickpea (Cicer arietinum L.) cv. Gokce were provided byCentral Field Crops Research Institute, Ankara, Turkey. The stress

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treatment was performed for 17 days in a greenhouse while controlplants were watered regularly. The experimental data about morpho-logical characteristics of the treated plants were analyzed using IBM-SPSS v.20 with one-way ANOVA. The significance of the mean valueswas compared using Student's t-test.

2.2. Plasmid construction

DNA fragment of pre-miR408 was amplified from genomic DNA ofArabidopsis thaliana Col-5. The gene fragment was amplified by PCRusing primer including AttB sites on 5′ and 3′ end sites in the forwardprimer (Athpre-miR408 F: 5′ ggggACAAGTTTGTACAAAAAAGCAGGCTAAGGTTAGATTGGTA TTGCAA 3′) and reverse primer (Athpre-miR408R: 5′ ggggACCACTTTGTACAAGAAAGCTGGGT cgt tag cag tag caa taa aat3′). The PCR reaction was carried out in a total volume of 25 μL contain-ing 2 mMMgCl2, 0.5 mM of each dNTP, 0.5 μM of each primer (forwardand reverse), 100 ng genomic DNA and 1 U Taq polymerase under thefollowing conditions: Initial denaturation at 94 °C for 6 min; 35 cyclesof denaturation at 94 °C for 45 s, annealing at 52 °C for 45 s, and exten-sion at 72 °C for 45 s and final extension at 72 °C for 10min. The ampli-fied fragment was inserted into the pEarleyGate 103 plasmid byGateway cloning system according to the manufacturer's instructions.The insert was confirmed by sequencing. Kanamycin resistance ofpEarleyGate 103 was used as a marker for colony selection. In additionto pre-miR408, the right and left borders of T-DNA contained bar geneprotein that encodes phosphinothricin acetyltransferase (PAT) enzymewas used for plant selection. The gene was expressed under the CaMV35S promoter. Transformation was performed using Agrobacteriumtumefaciens strain LBA 4404 by electroporation. Empty pEarleyGate103 plasmid was used as vector control.

2.3. Chickpea transformation

Chickpea seeds were surface sterilized with 30% commercial bleachcontaining 5% NaOCl for 25min followed by 3 × 3min rinsing with dis-tilled sterilized water for 5 min. Thereafter, the seeds were kept in dou-ble distilled sterilized water overnight (12 h) to soften and imbibe theseeds for easy recovery of embryos. Mature embryo explants were ex-cised from seeds, damaged with surgical blade and transferred to inoc-ulation buffer for 30 min containing 100 mM of acetosyringone and10 mM MES. The bacterial OD at 600 nm was adjusted to 0.5. Theinoculated matured embryos were incubated in co-cultivation mediumadditionally containing the same concentrations of acetosyringoneand MES for 48 h. The embryos were transferred to regeneration medi-um containing 1/2 × MS salts, 500 mg/mL bacteriostatic augmentin(GlaxoSmithKline, Istanbul Turkey) and 3 mg/mL phosphinothricin(Sigma Aldrich) at 25 °C under 16 h photoperiod. The growing shootsalso regenerated roots on 1/2 × MS medium after three weeks. Sincethe developing root system was weak, it was strengthened by adding6 mg/L GA3 + 0.1 mg/L IBA in 3 mg/mL phosphinothricin containingMS medium. After 7 weeks, plants were transferred to soil for acclima-tization using peat moss. Finally putative transgenic chickpea plantswere shifted to a greenhouse, and the samples taken were subjectedto further molecular analyses.

2.4. Molecular analyses of transgenic plants

The To transgenic plants were detected by PCR followed by selectionwith DNA from non-transformed plants, no DNA (water) in place oftemplate DNA as negative control, and pEarleyGate 103 containingAth pre-miR408 as positive controlwas used to ascertain correct results.Genomic DNA was isolated using a Qiagen DNeasy Plant Mini Kit(Valencia, CA). Integrity of the isolated DNA was checked on 1% (w/v)agarose gel and the concentration was determined using a NanoDrop2000c spectrophotometer (Thermo Fisher Scientific, Lenexa, KS, USA).PCR amplification was achieved with vector primers of bar gene, 35S

promoter, and also a gene specific primer of Ath pre-miR408. PCRamplification was performed with 25 μL of final volume containing2.5 mM MgCl2, 0.5 mM of each dNTP, 0.4 μM of each primer (forwardand reverse), 1.4 × Taq DNA polymerase buffer (+KCl, −MgCl2),100ng genomic DNA and 1U Taq polymerase (Fermentas). The reactionconditions were as follows: Initial denaturation at 95 °C for 6 minfollowed by 30 cycles of denaturation at 94 °C for 35 s, annealing at50–60 °C for 35 s, extension at 72 °C for 50 s and final elongation at72 °C for 10 min.

2.5. RNA isolation

Total RNA from leaves of untreated non-transgenic sample, untreat-ed empty vector control sample, drought treated empty vector controlsample, and drought treated miR408 overexpressing sample was ex-tracted using the TRIzol reagent (Ambion, Austin, TX, USA) accordingto the manufacturer's instructions. The quality of RNA was checked on2% agarose gel, and the quantity of the RNA was measured using aNanoDrop 2000c spectrophotometer (Thermo Fisher Scientific, Lenexa,KS, USA).

2.6. Stem-loop reverse-transcription

Stem-loop RT primers for miR408 were designed according toVarkonyi-Gasic et al. (2007) (Table S1). The miRNA stem-loop reversetranscription was achieved using 1000 ng of total RNA samples ofnon-drought and drought treated vector control sample, also droughttreated transgenic miR408 leave samples. First-strand cDNA was syn-thesized using the miRNA First-Strand cDNA Kit (Invitrogen). The reac-tionwas carried outwith 10mMdNTPmix, stem-loop RTprimer (1 μM)by adding nuclease free water to 13.65 μL final volume, followed bymixing the components, incubation for 5 min at 65 °C, and then putonto ice for 2 min. Thereafter, 4 μL 5 × first single strand buffer, 2 μL1 M DTT (0.1 M), 0.1 μL RNAseOUT (40 units/mL), and 0.25 mL Super-Script III (200 units/mL) were added to each tube. The RT reactionswere achieved as 30 min at 16 °C followed by 60 cycles of 30 °C for30 s, 42 °C for 30 s and 50 °C for 1 s. The control tubes included all com-ponents without RT primer (no RT) and RNA template (no RNA).

2.7. Quantitative RT-PCR analysis for miRNA measurement

To verify the expression of miR408, qRT-PCR was performed. Thereactions were conducted using a SYBR Green I Master Kit (Roche,Germany) on a qRT-PCR LightCycler 480 Instrument II (Roche,Germany). qRT-PCR analysis was carried out in optical plate and in atotal volume of 20 μL including 2× Master mix, 100 pmol of each for-ward and universal reverse primers, and 2 μL RT stem-loop cDNA. For-ward primers were specifically designed for miR408, and 5′-GTGCAGGGTCCGAGGT-3′ was used as the universal reverse primer (Varkonyi-Gasic et al., 2007) (Table S1). The qRT-PCR conditions were setup asfollows: Initial denaturation at 95 °C for 5 min, followed by 41 cyclesat 95 °C for 10 s, 55 °C for 20 s, and 72 °C for 10 s. The melting curveswere adjusted as 95 °C for 5 s and 50 °C for 120 s. All reactions were re-peated three times for each sample (Unver and Budak, 2009). The rela-tive fold change for each collation was calculated by 2−Ct (Unver et al.,2009; Varkonyi-Gasic et al., 2007).

2.8. Quantitative RT-PCR analyses for mRNA measurement

Transcription levels of bar, plantacyanin and Dreb1A, Dreb2A, Rd17,Rd22, Rd29a, Rd29b, bhlh23, and ERF17 genes weremeasured by quanti-tative RT-PCR. Due to the absence of sequence information in chickpea,the primers were designed using conserved regions of orthologous se-quences in different organisms. Multiple alignments of the sequencesobtained from database were performed to find conserved nucleotidesof each selected gene by using ClustalW2 software. Sequences of the

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primerswere listed in Supplementary Table 1. The reverse transcriptionreaction was achieved by First-Strand cDNA Synthesis Kit (Fermentas,Thermo Fisher Scientific) according to the manufacturer′s instruction.PCR reactions were generated on the LightCycler 480 Instrument II(Roche, Germany). Optical plates were used for qRT-PCR, and the reac-tions were carried out in a final concentration of 20 μL including100 pmol gene specific primers, 2 μL of cDNA, and SYBR Green I MasterMix (Roche, Germany). 18S rRNA (GenBank ID: AF147501)was used asinternal control for each reaction. The reaction conditionswere setup asfollow: Preheating at 95 °C for 5 min; followed by 50 cycles of 95 °C for10 s; 53–55 °C (depending on the primer's annealing temperature) for20 s, and 72 °C for 10 s. The melting curves were generated at the endof the PCR cycles by heating the PCR products from 70 to 95 °C. Eachsample was measured in triplicate. Mean-signal intensity across thereplicates was taken to calculate the expression levels. The normalizedexpression levels were calculated by 2−ΔCt formula.

3. Results

3.1. Construct confirmation and chickpea transformation

After numerous trials, pre-miR408 could not be amplified in thechickpea, while the reactions were succeeded in A. thaliana. The resultsuggested that the sequence of chickpea pre-miR408wasdifferent com-pared to that of A. thaliana. Therefore, pre-miR408 A. thaliana was usedto prepare construct for overexpression. pEarleyGate 103 which con-tains 276 bp fragment of pre-miR408 gene construct was verified byPCR and sequencing of the amplicon. The results were confirmed withfull homology to A. thaliana pre-miR408 sequence.

The putative transgenic plants survived in soil and grew well andwere further subjected to molecular analysis for gene integration andexpression. A total of 160 To putative pre-miR408 containing transgenicplants were recovered after 4–5 weeks of culture, and gene specificprimers of pre-miR408 and 35S CaMV and bar gene confirmed the pres-ence of pre-miRNA408 in two primary transformants with transforma-tion efficiency of 0.012%. Additionally, 80 putative vector controltransformants containing bar gene were recovered. Three plants wereconfirmed for bar gene and also 35S CaMV promoter primers.

3.2. Phenotype surveillances of the samples

The confirmed transgenic plants were exposed to drought stress for17 days, alongwith vector control plants. After drought application, thegrowth of transgenic lines was different compared to vector controlplants with remarkable phenotypic changes. After the 8th day ofdrought stress treatment, the vector control plants began to show chlo-rosis, wilting and drying; whereas no such stress symptoms were de-tected on transgenic plants. After 17 days of treatment, vector controlplants were severely wilted, whereas, transgenic plants showed highstress tolerance (Fig. 1).

Additionally, the height of the transgenic miR408 plants showed asignificant decrease independent to drought treatment. On the otherhand, after stress treatment, increasing number of leaveswere observedonmiR408 overexpressed plant compared to both vector control plantsand pre-treated overexpressed miR408 plant (Figs. 2a, b). No morpho-logical change was recorded on the leaf area between transgenic andvector control plants.

3.3. Quantitative gene expression levels

OnemiR408 overexpressed plant and one vector control plant wereselected to examine the expression levels of miR408 and its targetplantacyanin, and bar gene using quantitative RT-PCR. The qRT-PCR re-sults showed that the expression of bar gene was detected in bothmiR408 overexpressing and vector control plant, whereas expressionsof those genes were not detected in non-transgenic plants (Fig. 3a).

Our result showed that the expression level of miR408 was inducedby drought stress in drought treated vector control plants comparing tonon-treated vector control plant. Also the expression level of miR408 inoverexpression plant was induced upon drought treatment comparingto both treated and non-treated vector control plant (Fig. 3b). Underdrought stress, expression level of miR408 targeted plantacyaningene was decreased in overexpressed miR408 plant compared to vectorcontrol plant under drought stress (Fig. 3c).

The function of miR408 in response to drought stress is not clear. Tounderstand a part of complex response to drought, the expression ofimportant drought tolerance genes was analyzed in overexpressedmiR408 plants and vector control plant under drought stress. Interest-ingly, BHLH23 transcription factor encoding low copper levels conditionwas observed down-regulated in miR408 overexpressed plant com-pared to vector control plant upon stress treatment (Fig. 3d). Similarly,another drought stress responsive gene APETALA2/Ethylene ResponseFactors (ERF/AP2) showed a reduced expression profile in miR408overexpressed chickpea plant compared to vector control plant underdrought stress (Fig. 4g).

Previous studies showed that Dehydration-Responsive ElementBinding Protein 2A (DREB2A) and 1A (DREB1A) genes were induced bycertain abiotic stresses such as low and high temperatures, drought,and salinity (Sakuma et al., 2002, 2006a; Vadez et al., 2007; Qin et al.,2008b; Maruyama et al., 2009). In our analyses, the expression levelsof DREB1A and DREB2A transcription factors were significantly in-creased after drought treatment (Figs. 4a and b). Since DREB1A andDREB2A transcription factors act on Rd17, Rd29a, and Rd29b genes,therefore, expression profiles of those genes were analyzed underdrought conditions. The results showed that Rd17 and Rd29a were in-creased in miR408 overexpressed transgenic plant compared to emptyvector control plant upon drought (Figs. 4c, d). On the other hand,Rd29b controlled by DREB2A transcription factor showed reduced ex-pression upon drought treatment (Fig. 4e), while another member ofRd gene family Rd22 (also important in drought tolerance) was foundto be up-regulated in transgenic miR408 plant compared to vector con-trol plant under drought stress (Fig. 4f).

4. Discussion

Chickpea (Cicer arietinum L.) is counted among the most essentialdevoured grain legumes with high economic value and approximatelymore than 95% production comes from arid in semi-arid rainfed regionsof the world. Comparing different factors reducing yield, unfavorabletemperature and cold are the most widely recognized abiotic stressesthat influence chickpea production. Chickpea cultivars show differinglevels of drought tolerance (Khodadadi, 2013). Due to dubious precipi-tation at a discriminating phase in the areas where chickpea is mostoften grown, field screening of chickpea germplasms against waterstress is blur (Yaqoob et al., 2013). It was reported that drought stressconstituted up to 51% yield loss in chickpea (Croser et al., 2003; Singhet al., 1994). To adapt to drought stress, plants developed very intricateand overall sharpened reactions that incorporate reprogramming geneexpression, straighteningmetabolism andwater relations ensuring pro-tection of cell structures from the harms caused by stresses (Zhu, 2002).The articulation of a large set of genes is generally influenced due todrought stress showing that stress responses are involved in stretch ofreactions including a complex regulatory network (Seki et al., 2002). In-side this system, gene representation is transiently and spatially com-posed at transcriptional, post-transcriptional, and translational levels.Several microRNAs that intercede post-transcriptional regulation uponwater deficiency have been studied in other crop species. They act ascritical post-transcriptional regulators of drought stress responsivegenes and control their expression. So far, a number of drought respon-sive miRNAs have been identified in diverse plant species by utilizinghigh-throughput small RNA deep sequencing. Studying detailed molec-ular functions, high-throughput techniques have revealed that miRNAs

Fig. 1. Transgenic miR408 to compare with vector control plants. A: before drought treated. B: 8 days after drought treated. C: 17 days after drought treated.

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compose an important part of these regulatory networks (Shalgi et al.,2007; Hobert, 2008; Yu et al., 2008; Zhang and Li, 2013). To solve thefunction ofmiR408, several in silico tools in land plantswere performed.The analyses revealed thatmiR408 potentially targets drought stress re-sponsive genes (Eldem et al., 2012; Kantar et al., 2010; Trindade et al.,2010; Zhou et al., 2010). On the other hand, there ismeager information

Fig. 2.Quantitative comparison of A: number of leaves and B: plant height before and after 17 dstandard error ratio were calculated by P b 0.01 versus control, by Student's t-test.

on functional verification of those results. Functional studies of miR408were mainly conducted to show its relation with copper related genes.Additionally, there are few studies available on relation betweenmiR408 and drought stress; however they have been studied only inA. thaliana (Gou et al., 2011; Li et al., 2008; Sunkar et al., 2006). So far,studies about drought stress have not been reported in economically

ays drought vector control and overexpression ofmiR408 plants. Error bars that presented

Fig. 3. qRT-PCR analysis of miR408. a: detected level of bar gene expression in both miR408 transgenic and vector control types compared to non-transgenic plant. b: detected level ofmiR408 expression in transgenic miR408 to comparewith drought treated and non-treated vector control plants. c: expression level of miR408 target gene plantacyanin inmiR408 trans-genic and vector control plants. d: Overexpression ofmiR408 reduced the expression level of BHLH23 transcription factor in transgenicmiR408 to comparewith vector control plant. Errorbars present SD genes D: s.

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important crops. Expression level of miR408 was up-regulated underdrought stress in cereals such as H. vulgare and M. truncatula (Kantaret al., 2010; Trindade et al., 2010) and also Arabidopsis (Liu et al.,2008). Additionally, during water stress, expression levels of miR408were reduced in drought susceptible indica rice varieties (Pusa Basmati1 and IR64), while they remained significantly elevated in the resistantgenotype (Mutum et al., 2013). Similarly, the results of this studyshowed increased expression level of miR408 during the droughtwhen compared to non-treated plant (Fig. 3b). Furthermore, uponstress treatment, the miR408 overexpressed plants showed normalgrowth, while the other samples struggled with severe symptoms ofstress (Fig. 1). All these results clearly showed that miR408 is involvedin drought stress, and increased level of miR408 is important to droughttolerance.

Fig. 4. qRT-PCR analysis of drought related genes a and b: expression level of DREB1A and DREBc, d, e and f: detected level of expression RD17, RD29A, RD29B, and RD22 in transgenic miR408

Previous studies have been reported that miR408 regulates thegenes encoding copper proteins and this suggested a coordination incopper homeostasis against response towater stresswith targeting cop-per related transcripts (Abdel-Ghany and Pilon, 2008; Trindade et al.,2010). Under drought stress, plants survive by consuming low amountof water keeping their stomata closed at a high rate (Nunes et al.,2008). This strategy reduces dehydration due to transpiration andhelps plants to survive under drought stress (Nunes et al., 2008). Duringdrought stress, water is not transported in xylems and transpiration isinhibited. In this way, micro- and macro-elements including copperions are not transported to leaves and organs which results in reducedphotosynthesis. However, this situation does not cause copper deficien-cy. miRNAs regulate the level of copper in response to stress withtargeting some of copper related transcripts (Abdel-Ghany and Pilon,

2A in transgenic miR408 plant to compare with vector control plant under drought stress.to compare with vector control plants under drought treated.

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2008; Trindade et al., 2010). miR408 has been reported to down-regulate the transcripts which are related to copper protein such asplantacyanin in Arabidopsis grown under copper starvation, as a mecha-nism to save this mineral for plastocyanin (Abdel-Ghany and Pilon,2008; Yamasaki et al., 2009; Trindade et al., 2010). Induced miR408level correlates with reduced target genes in drought stress(Abdel-Ghany and Pilon, 2008; Trindade et al., 2010). The results ofthis study showed that the Plantacyanin transcripts like the other pro-teins included copper which is a direct target of miR408 (Abdel-Ghany and Pilon, 2008) and conserved in many crops including chick-pea (C. arietinum) (Sunkar and Zhu, 2004) that were down-regulatedwith the increased miR408 in miR408 overexpressed plant compare tovector control plant.

It is known that DREB1A and DREB2A transcription factors areinvolved in drought tolerance. Improved dehydration tolerance inDREB1A and DREB2A overexpressed transgenic plants has been report-ed previously (Maruyama et al., 2009; Sakuma et al., 2006b). This studyreports expression level of DREB1A in transgenic plant overexpressingmiR408 that increased under drought stress compared to vector controlplant. Similarly, other studies showed that overexpression of DREB1A(a, b) under 35S CaMV promoter induced resistance to drought withinhibited growth in Arabidopsis plants indicating (Kasuga et al., 2004).DREB2A, an element that binds to protein2a during dehydrationresponse, was up-regulated under water scarcity in this analysis.DREB2A transcription factor controls induced gene expression in waterdeficiency and needs post-translational changes to regulate drought re-sponsive genes (Qin et al., 2008a). Overexpression ofDREB2A (DREB2A-CA) provided strong drought tolerance to Arabidopsis (Sakuma et al.,2006a). Confirming the previous reports, this study confirmed the reg-ulatory roles of DREB1A and DREB2A in drought tolerance. Sakumaet al. (2006a) also showed induction of RD17 and RD29A genes targetedby DREB1A and DREB2A in transgenic miR408 Arabidopsis plant underdrought stress. Kasuga et al. (1999) also found the similar results. Theresults of this study noted that RD17 and RD29A expression levelswere up-regulated in miR408 overexpressed plant compared to vectorcontrol plant. A relation betweenmiR408 and copper level has been re-ported by Zhang and Li (2013). They found that the miR408 expressionlevel was induced under copper deficiency to accumulate more copper.Besides, the Cu level regulated the drought responsive genes (DREB)(Zhang and Li, 2013). Additionally, Ban et al. (2011) confirmed thatDREB level was up-regulated upon excess Cu. The results of this studyconfirm that the miR408 overexpression results in repression ofplantacyanin caused copper level accumulation. Consequently, the ex-pression level of DREB was relatively induced in miR408 overexpressedplant compared to vector control upon drought stress. On the otherhand, expression level of RD29B which is a target gene of DREB2A wasfound to be down-regulated in miR408 overexpressed plant comparedto vector control. In previous studies, although an enhanced expressionof this genewas found in response to dehydration, it was not significantasmuch as the other RD genes (Kasuga et al., 1999; Sakuma et al., 2006a,2006b). All these results confirmed that Dreb1A and Dreb2A transcrip-tion factors and their target genes RD17 and RD29 provided plant toler-ance to survive in drought stress.

RD22 is a drought stress inducible gene that is popularly used as awater deficit marker in many plants including soybean and Arabidopsis(Pellegrineschi et al., 2004; Stolf-Moreira et al., 2010; Neves-Borgeset al., 2012). The level of RD22 mRNA was very low in fully hydratedplants andwith increasing drought stress the expression ratiowas also el-evated (Yamaguchi-Shinozaki and Shinozaki, 2006). Similarly, our resultsindicated that RD22 gene was induced due to drought stress in miR408overexpressed plant compared to vector control plant (Fig. 4f). ThemiR408 overexpressed transgenic plants also were depicted as increasedRD22 expression; which is a drought responding gene and induced bywater stresses (Yamaguchi-Shinozaki and Shinozaki, 1993).

Manymembers of large Basic Helix-Loop-Helix (BHLH) protein fam-ily are involved in abiotic stress responses. They are the most highly

conserved residues in plants (Carretero-Paulet et al., 2010) and involvedin dehydration (Lorenzo et al., 2004). A member of this family bhlh23transcription factor has been known to play a role in copper deficiencyin different tissues of plants. In copper deficiency conditions, expressionof bhlh23 was increased in both root and shoots (Bernal et al., 2012). Itmay be speculated that when miR408 targets copper related transcripts,copper ions are not used and accumulated in plants. In the second andthe more possible scenario, it was proposed that miR408 directly targetsbhlh23 gene. In any case, it is expected to observe a lower expression ofbhlh23 in miR408 overexpressed plant due to excess copper ions orsilencing of bhlh23 by miR408. The results in our analysis were obtainedto satisfy this scenario. Expression level of bhlh23 gene was significantlyreduced in transgenic miR408 plant compared to vector control plant.However, the hypotheses need further confirmations.

Members of AP2/ERF superfamily are highly conserved and knownin many plant species, like Arabidopsis (Sakuma et al., 2002; Nakanoet al., 2006), soybean (Zhang et al., 2008), rice (Nakano et al., 2006;Rashid et al., 2012), tomato (Sharma et al., 2010), maize (Zhuanget al., 2010), cucumber (Hu and Liu, 2011), and poplar (Zhuang et al.,2008). TheAP2/ERF transcription factors are candidates to improvediffer-ent stresses, including drought stress response anddevelopment in plants(Sakuma et al., 2002; Wessler, 2005; Riechmann and Meyerowitz, 1998;Shigyo et al., 2006; Licausi et al., 2011). Due to its function in drought tol-erance, it was expected that expression of AP2/ERF would be increasedupon stress treatment. On the contrary, this study noted that the ex-pression level of AP2/ERF was significantly decreased in miR408overexpressed plant compared to vector control plant under droughtstress. There are no sufficient data available suggesting a correlation be-tween this transcription factor and miR408. Recently, 29 members ofAP2/ERF in rubber tree were predicted to be a target of miR408 (Duanet al., 2013). Together with this result, it may be proposed that AP2/ERFgene would be a potential target of miR408.

Unexpectedly, the currentwork showed a strong effect ofmiR408onvegetative growth in chickpea. During over production by constitutive35S promoter, excessive miR408 resulted in strong growth inhibition.ThemiR408-overexpressedplants exhibited a shorter plant height com-pared to vector control plants (Fig. 2a). It was supposed that occurrenceof dwarf plants was irrespective to drought treatment, since droughttreated plants were even longer than the untreated plants. The resultsuggested that in addition to plantacyanin, miR408 also targets othergenes responsible for growth and development. So far, no study hasbeen reported that shows the role of miR408 on growth. To our knowl-edge, this is the first study showing inhibition effect of miR408 on veg-etative growth.

Additionally, occurrence of dwarf phenotype suggests that miR408has functions other than stress tolerance. In addition to plantacyanin,the known target of miR408, other seven drought responsive genesare under the indirect regulation of miR408. In our study, the miR408overexpression results in repression of plantacyanin causing copperlevel accumulation. Consequently, the expression level of DREB wasrelatively induced in miR408 overexpressed plant compared to vectorcontrol upon drought stress.

Further analysis of the miR408 mediated gene circuits will providedesired insight into well coordinated and convenient control of thegene percussion instruments that uphold plant growth and develop-ment in changing environments.

Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.gene.2014.11.002.

Authors' contribution statement

MH: Performed the experiments.MT: Analyzed the data and prepared the MS.KMK: Designed the experiments and analyzed the data.TU: Designed experiments and prepared the MS.

192 M. Hajyzadeh et al. / Gene 555 (2015) 186–193

Conflict of interest

The authors declare that they have no conflict of interest.

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