Mol Biol Rep DOI 10.1007/s11033-014-3333-0
Differential regulation of microRNAs in response to osmotic, salt and cold stresses in wheat Om Prakash Gupta • Nand Lal Meena Indu Sharma • Pradeep Sharma
•
Received: 21 January 2014 / Accepted: 14 March 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract MicroRNAs (miRNAs) are tiny non-coding regulatory molecules that modulate plant’s gene expression either by cleaving or repressing their mRNA targets. To unravel the plant actions in response to various environmental factors, identification of stress related miRNAs is essential. For understanding the regulatory behaviour of various abiotic stresses and miRNAs in wheat genotype C-306, we examined expression profile of selected conserved miRNAs viz. miR159, miR164, miR168, miR172, miR393, miR397, miR529 and miR1029 tangled in adapting osmotic, salt and cold stresses. The investigation revealed that two miRNAs (miR168, miR397) were downregulated and miR172 was up-regulated under all the stress conditions. However, miR164 and miR1029 were up-regulated under cold and osmotic stresses in contrast to salt stress. miR529 responded to cold alone and does not change under osmotic and salt stress. miR393 showed upregulation under osmotic and salt, and down-regulation
Om Prakash Gupta and Nand Lal Meena have contributed equally to this work.
Electronic supplementary material The online version of this article (doi:10.1007/s11033-014-3333-0) contains supplementary material, which is available to authorized users. O. P. Gupta N. L. Meena Quality and Basic Sciences Division, Directorate of Wheat Research, Karnal 132001, Haryana, India N. L. Meena Basic Sciences Division, Indian Institute of Pulses Research, Kanpur 208024, India N. L. Meena I. Sharma P. Sharma (&) Biotechnology Laboratory, Directorate of Wheat Research, Karnal 132001, India e-mail: [email protected]
under cold stress indicating auxin based differential cold response. Variation in expression level of studied miRNAs in presence of target genes delivers a likely elucidation of miRNAs based abiotic stress regulation. In addition, we reported new stress induced miRNAs Ta-miR855 using computational approach. Results revealed first documentation that miR855 is regulated by salinity stress in wheat. These findings indicate that diverse miRNAs were responsive to osmotic, salt and cold stress and could function in wheat response to abiotic stresses. Keywords
miRNAs Wheat Drought Cold Salt
Introduction In order to adopt under adverse climatic conditions, plants have developed complex mechanisms at various stages that increase tolerance level. Being a sessile organism, plants accomplish stressful ecological conditions to best fit under abiotic stress for their molecular response that might ultimately lead to their adjustment to the stresses. MicroRNAs (miRNAs) have emerged as important players in posttranscriptional gene regulation [1]. Tolerance to abiotic stresses involves a complex relationship of numerous molecular components. After discovery of miRNAs as a potent gene regulator, it has led to understand the expression behaviour of genes at post-transcriptional level [2–9]. Plant encodes 20–24 nucleotides tiny small RNA. Based on mode of small RNA biogenesis in plants, it has been classified into four categories viz. miRNAs, ra-siRNAs, nat-siRNAs and ta-siRNAs. However, another class of small RNAs, called long-siRNAs (30–40 ntds) has been added up recently. Amongst all these, miRNAs has been extensively studied in plants [7, 10, 11].
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Mol Biol Rep Table 1 Predicted target function of differentially expressed diverse miRNAs and their functional annotation [40]
miRNAs
miR159
Identified direction of expression Cold
Drought
Salt
;
:
;
Target genes
Function
MYB transcription factor (TF), 1-aminocyclopropane-1carboxylate synthase, melanocyte-specific gene related gene, oligopeptide transport gene, saur family protein
Signaling pathway and development Propanoate metabolism Regulation of gene expression Oligopeptide transporter Auxin response
miR164
:
:
;
NAC domain TF, phytosulfokines
Signaling pathway, root development, response to oxidative stress
miR168
;
;
;
Argonaute
Signal transduction, development, stress response
miR172
:
:
:
Apetala2-like TF, ARF, helix-loophelix DNA-binding domain containing protein
Signaling pathway, development, stress response
Signaling pathway
Signaling pathway, flower development BR regulation of plant development
miR393
;
:
:
bHLH TF, transport inhibitor response 1/auxin F-box
BR regulation of plant development Defense response, response to phosphate starvation, signaling pathway, flower/root development
miR397
;, downregulated; :, upregulated; –, no significant difference
;
;
;
Cold stress Copper homeostasis, response to water deprivation
miR529
:
–
;
Apetala2-like TF, squamosapromoter binding protein-like
Signaling pathway
miR1029
:
:
;
Apetala2-like TF, DREB TF
Signaling pathway
Literature suggests that miRNAs are playing key role in regulating adaptation mechanism at cellular level under abiotic stress such as cold, drought, salinity [11–13], UV-B radiation [14], heavy metals [15, 16], sulphate/phosphate starvation [17, 18], nitrogen starvation response [19] and copper deficiency [6]. Currently, the miRBase database has documented 24,521 miRNAs precursor having 30,424 mature miRNAs from 206 plant species (Release 20, http:// www.mirbase.org). Deep sequencing has helped to a greater extent to identify novel miRNAs in diverse plants [20, 21]. Comparative analysis of miRNAs in various plants has shown the differential behaviour of the same miRNA exposed to numerous abiotic stresses in different plants. This kind of regulatory behaviour of miRNAs shows their tight regulation. In the recent past, several miRNAs has been studied for their crucial role under abiotic and biotic stresses in various model plants [22]. However, reports in wheat miRNAs are
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Ice1 (inducer of CBF expression 1) TF, laccase
Plant development Abiotic stress
poorly documented [23–25]. In current study, we exploited the regulatory mechanism to characterize miRNAs that could be involved in mitigating abiotic stress tolerance in wheat. We analysed eight conserved miRNAs (Table 1) in C-306 wheat genotype with a view to capture miRNAs that might be controlled differentially under various abiotic stress conditions. The aim of this study was to validate the expression of eight conserved miRNAs (viz. miR159, miR164, miR168, miR172, miR393, miR397, miR529 and miR1029) in genotype C-306 exposed to osmotic, cold and salt stress to address their role in mitigating these stresses. In addition, miR529 has been reported in other plants but not in wheat while miR1029 has been reported only in Physcomitrella patens. We selected these two miRNAs to validate in wheat as they are involved in regulating abiotic stresses. Other miRNAs were selected owing to their role in various abiotic stress responses across the species. In addition, we have validated the expression of
Mol Biol Rep
computationally identified salt stress responsive TamiR855 in wheat [24].
Materials and methods Plant materials and treatment We selected drought tolerant wheat genotypes C306 (RGN/ CSK3//2*C59/3/C217/N14//C281) which released in the year 1965. It has multiple features like good chapatti quality, tolerance to abiotic stresses and have Lr34 resistance gene. The seeds of Triticum aestivum L. cv. C306 were initially treated with 1 % sodium hypochlorite for 10 min, rinsed in distilled water for three times, and germinated on moist filter papers. Seedlings were grown into hydroponic condition in full strength Hoagland’s liquid medium for 2 weeks. Two weeks old seedlings with similar heights were transferred to full strength Hoagland’s liquid medium containing 150 mM NaCl, polyethylene glycol 6000 (20 % v/v) for salt and osmotic stress treatment, respectively. For cold treatment, wheat seedlings were exposed to 4 °C. For expression analysis of miR855, samples were collected at 1, 12 and 24 h after salt stress at 150 mM NaCl. Control plants, corresponding to each treatment were raised in full strength Hoagland’s liquid solution at 25 °C. After 24 h of stress treatment whole seedling were harvested and transferred into liquid nitrogen. Three biological replications were performed independently for each treatment.
Fig. 1 Validation of abiotic stress-responsive miRNAs in C-306 wheat genotype. Agarose gel showing qRT-PCR products. Negative control 1 represents no-srcDNA and negative control 2 represents noRT primer. M-100 bp marker
Quantitative real-time PCR After srcDNA synthesis, each miRNAs were confirmed with PCR using conditions of 94 °C for 8 min, then 32 cycles of 94 °C for 15 s, 55 °C for 30 s and 72 °C for 30 s. PCR products were resolved using 3 % agarose gel (low EEO, Himedia) electrophoresis with a size of *100 bp (Fig. 1). qPCR was carried out using ABI PRISM 7500 Real-Time PCR System (ABI, USA). Total volume of the PCR reaction was 20 ll consisting of SYBR Green master mix (Roche), RTQ-UNIr primer (10 lM), forward primer (10 lM) and 10 ng/ll of srcDNA using above mentioned PCR protocol. Expression data generated was used for quantifying relative expression level of all the miRNAs using comparative 2-DDCt method.
Results and discussion Small RNA isolation and small RNA cDNA (srcDNA) library generation Small RNA was isolated from 100 mg of whole seedlings using mirVanaTM isolation Kit (Ambion) following manufacture’s instruction. Small RNA concentration was checked using Nano-Drop (NanoDrop technologies). For analysing expression patterns of selected miRNAs, srcDNA library was generated as described earlier [23]. Briefly, isolated small RNA was subjected to polyadenylation at 37 °C for 45 min. After tailing, samples were purified using mirVana Probe and Marker Kit (Ambion). Furthermore, expression of miRNAs by qPCR was carried out using miRNA specific forward primer and universal reverse primer (Supplementary Table 1). RTQ primer was used for cDNA synthesis and U6 snRNA was used as internal control for data normalization. All the primers were validated using thermal dissociation (Tm) curve of PCR amplicons which showed single peak.
Small-RNA discovery has opened a new avenue to detect and understand complex regulatory systems in plants in particular, those involved in abiotic stress. Plants suffer from a array of abiotic stresses; however, cold, drought, heat and salt stress are more frequently encountered. Recent studies in various plant species suggest that miRNAs play essential role in these stress tolerance. In the present study, we carried out expression analysis of eight conserved miRNAs viz. miR159, miR164, miR168, miR172, miR393, miR 397, miR529 and miR1029 in C-306 genotype exposed to osmotic, cold and salt stress to address their role in mitigating these stresses. Based upon expression pattern, above mentioned miRNAs were categorized into two groups: first group consists of miR159, miR164, miR393, miR529 and miR1029 showing differential expression pattern and second group comprised of miR168, miR172 and miR397 showing common expression pattern among all the stress conditions.
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Mol Biol Rep
Expression analysis of the tested miRNAs exposed to osmotic stress
involved in signalling pathway and abiotic stress responses. Our results showed up-regulation of miR1029 in wheat which might be involved in mediating osmotic stress tolerance.
Osmotic treatment of C-306 genotype has resulted in differential expression of five miRNAs (miR159, miR164, miR393, miR529 and miR1029). The accumulation of four miRNAs (miR159, miR164, miR393 and miR1029) was significantly higher while osmotic stress had no major effect on the expression of miR529 compared to control plants. Among over accumulated miRNAs, the expression of miR393 was maximum (*12.8-fold) followed by miR1029 (*9 fold), miR159 (*0.5-fold) and miR164 (*0.3-fold) (Fig. 2). Previously, efforts were made to validate the expression of miRNAs in numerous crop species under drought stress [12, 13, 26–31] however; work on wheat miRNA is very limited. Until date, there is only one report on miRNA expression behaviour in Triticum dicoccoides exposed to drought shock [32]. Up regulation of miR159 in C-306 wheat cultivar is in agreement with other studies like French bean [33], cotton [34], maize [35] suggesting its proactive involvement in regulating signal transduction pathways under osmotic stress. Over accumulation of miR164 in the seedlings of C-306 wheat cultivar in present study as well as in Brachypodium [36] and sugarcane [37] demonstrate role of NAC domain transcription factor in perturbation of signalling, root development and oxidative stress responses. The expression of miR393 is commonly up-regulated during drought stress in Oryza, Medicago, Pinguicula, Arabidopsis [38]. Our result of miR393 expression is in tune with these findings. miR393 modulates auxin mediated response by targeting TIR1 which promotes degradation of Aux/IAA repressor by ubiquitin ligase [39]. In addition, the role of miR1029 is reported to target DREB and AP2 transcription factor [40] which
Abiotic stress particularly salinity in wheat is a major problem which limits wheat yield in salt affected areas. Many researchers have documented the role of miRNAs in controlling developmental stages in plants under salinity stress [41–45]. However, the role of miRNAs in wheat under salinity stress is completely unexplored as there is only one report present till now [41] which urges strongly to look into. Interestingly, we could find only one miRNA i.e. miR393 to be up-regulated amongst miR159, miR164, miR393, miR529 and miR1029 in C-306 under 150 mM NaCl stress while miR159, miR164 and miR1029 were down-regulated. miR529 showed no significant alteration under salt stress (Fig. 3). Our result on accumulation of miR393 is in quite agreement with the previous findings in Arabidopsis [12, 27] suggesting similar regulatory response in Arabidopsis and wheat under salinity stress. Most of the down-regulated miRNAs are reported to be involved in root development, signal transduction, and abiotic stress response [40] signifying that they might be involved in regulating salt stress response in C-306 cultivar by allowing accumulation of their corresponding targets. Downregulation of miR159 in C-306 wheat cultivar under 150 mM NaCl is differing with the result of Lu et al. [41] where they reported up-regulation under 200 mM NaCl treatment which might be due difference in salt concentration.
Fig. 2 Relative miRNAs expression in genotype wheat C-306 exposed to osmotic stress. Bars represent mean and standard deviation of values obtained from three biological replicates. The relative expression level of all miRNA was calculated using the comparative 2-DDCt method. The data was normalized to U6 snRNA expression level as an internal control
Fig. 3 The expression of selected miRNAs in wheat genotype C-306 exposed to salinity. Bars represent mean and standard deviation of values obtained from three biological replicates. The relative expression level of all miRNA was calculated using the comparative 2-DDCt method. The data was normalized to U6 snRNA expression level as an internal control
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Expression behaviour of the tested miRNAs under salt stress
Mol Biol Rep
Fig. 4 The expression of miR855 in wheat genotype C-306 exposed to 150 mM NaCl stress. Bars represent mean and standard deviation of values obtained from three biological replicates. Samples were collected after 1, 12 and 24 h duration of salt treatment. The relative expression level of all miRNA was calculated using the comparative 2-DDCt method. The data was normalized to U6 snRNA expression level as an internal control
In our previous work, we have shown five new miRNAs (Ta-miR819 k, Ta-miR855, Ta-miR3708, Ta-miR5156 and Ta-miR5653) from wheat EST database [24]. Till date miR855 is only identified in Arabidopsis thaliana in Chromosome I to Chromosome V. The miR855 is mapped in wheat EST (BG313724) which is located in DSW03C5_Contig2094.1 with nine members having 6AS, 6BS, and 6DS chromosomal locations. Effect of salt stress on C-306 led to down-regulation of miR855 (approximately -4.5-fold) at 1 h followed by subsequent over accumulation at 12 h (*20-fold) and 24 h (*41.3-fold) of stress exposure (Fig. 4). miR855 was shown to be expressed in wheat sheath under salt stress condition and targets MYB TF which regulates leaf development. MYB transcription factor is also targeted by miR159, miR828 and miR858 which primarily regulate signal transduction and development of the plants under various stress conditions [40]. Down-regulation of miR855 in C-306 at 1 h might be linked with activation of MYB based signalling to cope up the salt stress whereas up-regulation at later stages (12 and 24 h) of salt exposure could be due to suppression of MYB based tolerance mechanism in C-306 wheat genotype. Up-regulation of miR159 in wheat under salinity stress [41] suggesting a coordinating link between miR159 based regulation of MYB TF. Although, miR855 showed differential response under salt stress in wheat, further work on functional validation along with the target is required. Expression array of the tested miRNAs under cold stress Although, C-306 wheat genotype not reported to be cold tolerant, we carried out expression analysis of same set of
Fig. 5 Relative miRNAs expression in genotype wheat C-306 exposed to cold. Bars represent mean and standard deviation of values obtained from three biological replicates. The relative expression level of all miRNA was calculated using the comparative 2-DDCt method. The data was normalized to U6 snRNA expression level as an internal control
miRNAs to observe any similarity with the salt and drought stressed seedlings (Fig. 5). Interestingly, the expression pattern of miR159 (down-regulated) from cold stressed (4 °C for 24 h) seedlings is similar to the salinity stressed seedlings while miR164 and miR1029 was similar with drought stressed seedlings indicating their common mode of regulation of these two stress response in wheat. In addition, miR393 which was up-regulated under drought and salt, showed down-regulation under cold stress indicating auxin based differential cold response. Osmotic, salt and cold stress induces miRNA mediated common regulatory pathway in wheat In order to understand common regulatory pathways operated in wheat under osmotic, salt and cold stresses, we were interested to capture common miRNAs showing similar response under all the stress condition in wheat genotype C-306. Surprisingly, we could encountered two miRNAs namely miR168 and miR397 showing downregulation while miR172 showed up-regulation under all the stress conditions (Fig. 6). miR168 has been reported to be up regulated under drought [27] and salt stress [46] in Arabidopsis while down-regulated in rice under cold stress [47]. In plants, AGO1, target of miR168, is the main effector protein for regulating the activity of miRNAs. However, it will be interesting to know whether any such changes of miR168 regulation lead to change in miRNA functions [48]. Similarly, the expression of miR397 under cold stress and dehydration response was up-regulated in Arabidopsis [12] which is in contrast with our result in wheat which could be due to variation in the experimental procedure and plant species. miR397 targets an upstream transcription factor ICE1, which regulates the expression of
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Mol Biol Rep Research, New Delhi. We acknowledge the support of Amit Kumar and Bharti. This paper is DWR contribution No. 15.
References
Fig. 6 Expression analyses of miRNA 168, miR172 and miR198 in wheat genotype C-306 exposed to osmotic, salinity and cold. Bars represent mean and standard deviation of values obtained from three biological replicates. The relative expression level of all miRNA was calculated using the comparative 2-DDCt method. The data was normalized to U6 snRNA expression level as an internal control
CBF genes during cold stress response in plants [40]. Down-regulation of miR397 under cold stress in C-306 cultivar would increase the accumulation of ice1, which might positively be regulating cold stress response in wheat. Expression of miR172 in C-306 is in agreement with the previous report in Arabidopsis [12, 47], Brachypodium [49], Prunus [50] under cold stress and in cotton under drought and salt stress [34]. These results are in contrast with that of rice where miR172 was down-regulated under drought stress [21]. Differential regulation of these three miRNAs indicates the evolutionary divergence of the crop species along with experimental procedure and treatment conditions. Here, we report the common expression pattern of these three miRNAs in C-306 wheat cultivar under osmotic, salt and cold stress condition suggesting their common mode of action however, this needs further validation. In conclusion, an understanding of post-transcriptional gene regulation by miRNAs under adverse climatic conditions is critical for understanding and improving stress tolerance in crop plants. Several studies have demonstrated that many plant miRNAs are highly conserved across the species. Therefore, it is tempting to speculate that miRNAs biological functionality might be similar. We validated the expression of eight stress-induced conserved miRNAs viz. miR159, miR164, miR168, miR172, miR393, miR 397, miR529 and miR1029, in wheat genotype C-306 exposed to osmotic, salt and cold treatments to address their role in mitigating cold, salt and drought stresses. In addition, miR855 that was identified previously was also shown to be differentially regulated under salt stress. Acknowledgments Authors acknowledge Grant-in-Aids under the project DWR/RP/10-5.3 of the Indian Council of Agricultural
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1. Kawaguchi R, Girke T, Bray EA, Bailey-Serres J (2004) Differential mRNA translation contributes to gene regulation under non-stress and dehydration stress conditions in Arabidopsis thaliana. Plant J 38:823–839 2. Bartel DP (2004) MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116:281–297 3. Borsani O, Zhu J, Verslues PE, Sunkar R, Zhu JK (2005) Endogenous siRNAs derived from a pair of natural cis-antisense transcripts regulates salt tolerance in Arabidopsis. Cell 123:1279–1291 4. Navarro L, Dunoyer P, Jay F, Arnold B, Dharmasiri N, Estelle M, Voinnet O, Jones JD (2006) A plant miRNA contributes to antibacterial resistance by repressing auxin signalling. Science 312:436–439 5. Brodersen P, Sakvarelidze-Achard L, Bruun-Rasmussen M, Dunoyer P, Yamamoto YY, Sieburth L, Voinnet O (2008) Widespread translational inhibition by plant miRNAs and siRNAs. Science 320:1185–1190 6. Abdel-Ghany SE, Pilon M (2008) MicroRNA-mediated systemic down-regulation of copper protein expression in response to low copper availability in Arabidopsis. J Biol Chem 28:15932–15945 7. Voinnet O (2009) Origin, biogenesis, and activity of plant microRNAs. Cell 36:669–687 8. Wu G, Park MY, Conway SR, Wang JW, Weigel D, Poethig RS (2009) The sequential action of miR156 and miR172 regulates developmental timing in Arabidopsis. Cell 138:750–759 9. Vazquez F, Legrand S, Windels D (2010) The biosynthetic pathways and biological scopes of plant small RNAs. Trends Plant Sci 15:337–345 10. Surekha-Katiyar A, Shang G, Adam VS, Hailing J (2007) A novel class of bacteria-induced small RNAs in Arabidopsis. Genes Dev 21:3123–3134 11. Jones-Rhoades MW, Bartel DP, Bartel B (2006) MicroRNAs and their regulatory roles in plants. Annu Rev Plant Biol 57:19–53 12. Sunkar R, Zhu JK (2004) Novel and stress-regulated microRNAs and other small RNAs from Arabidopsis. Plant Cell 16:2001–2019 13. Zhao B, Liang R, Ge L, Li W, Xiao H, Lin H, Ruan K, Jin Y (2007) Identification of drought-induced microRNAs in rice. Biochem Biophys Res Commun 354:585–590 14. Zhou X, Wang G, Zhang W (2007) UV-B responsive microRNA genes in Arabidopsis thaliana. Mol Syst Biol 3:103–112 15. Huang SQ, Peng J, Qiu CX, Yang ZM (2009) Heavy metalregulated new microRNAs from rice. J Inorg Biochem 3:282–287 16. Gupta OP, Sharma P, Gupta RK, Sharma I (2014) MicroRNA mediated regulation of metal toxicity in plants: present status and future perspectives. Plant Mol Biol 84:1–18 17. Jones-Rhoades MW, Bartel DP (2004) Computational identification of plant microRNAs and their targets, including a stressinduced miRNA. Mol Cell 14:787–799 18. Pant BD, Musialak-Lange M, Nuc P, May P, Buhtz A, Kehr J, Walther D, Scheible WR (2009) Identification of nutrientresponsive Arabidopsis and rapeseed microRNAs by comprehensive real-time polymerase chain reaction profiling and small RNA sequencing. Plant Physiol 150:1541–1555 19. Xu Z, Zhong S, Li X, Li W, Rothstein SJ, Zhang S, Bi Y, Xie C (2011) Genome-wide identification of microRNAs in response to low nitrate availability in maize leaves and roots. PLoS ONE 6:e28009
Mol Biol Rep 20. Yang X, Li L (2011) miRDeep-P: a computational tool for analyzing the microRNA transcriptome in plants. Bioinformatics 27:2614–2615 21. Zhou L, Liu Y, Liu Z, Kong D, Duan M, Luo L (2010) Genomewide identification and analysis of drought-responsive microRNAs in Oryza sativa. J Exp Bot 61:4157–4168 22. Gupta OP, Sharma P, Gupta RK, Sharma I (2014) Current status on role of miRNAs during plant–fungus interaction. Physiol Mol Plant Pathol 85:1–7 23. Gupta OP, Permar V, Koundal V, Singh UD, Praveen S (2012) MicroRNA regulated defense responses in Triticum aestivum L. during Puccinia graminis f. sp. tritici infection. Mol Biol Rep 39:817–822 24. Pandey B, Gupta OP, Pandey DM, Sharma I, Sharma P (2013) Identification of new stress-induced microRNA and their targets in wheat using computational approach. Plant Sig Behav 8:e23932 25. Xin MM, Wang Y, Yao YY, Xie CJ, Peng HR, Ni ZF, Sun QX (2010) Diverse set of microRNAs are responsive to powdery mildew infection and heat stress in wheat (Triticum aestivum L.). BMC Plant Biol 10:123–134 26. Reyes JL, Chua NH (2007) ABA induction of miR159 controls transcript levels of two MYB factors during Arabidopsis seed germination. Plant J 49:592–606 27. Liu HH, Tian X, Li YJ, Wu CA, Zheng CC (2008) Microarraybased analysis of stress-regulated microRNAs in Arabidopsis thaliana. RNA 14:836–843 28. Li WX, Ono Y, Zhu J, He XJ, Wu JM, Iida K, Lu XY, Cui X, Hin H, Zhu JK (2008) The Arabidopsis NFYA5 transcription factor is regulated transcriptionally and post-transcriptionally to promote drought resistance. Plant Cell 20:2238–2251 29. Trindade I, Capitao C, Dalmay T, Fevereiro MP, Santos DM (2010) miR398 and miR408 are up-regulated in response to water deficit in Medicago truncatula. Planta 231:705–716 30. Wang T, Chen L, Zhao M, Tian Q, Zhang WH (2011) Identification of drought-responsive microRNAs in Medicago truncatula by genome-wide high throughput sequencing. BMC Genomics 12:367–378 31. Eldem V, Celikkol-Akcay U, Ozhuner E, Bakir Y, Uranbey S, Akcay U, Unver T (2012) Genome-wide identification of miRNAs responsive to drought in peach (Prunus persica) by highthroughput deep sequencing. PLoS ONE 7:e50298 32. Kantar M, Lucas SJ, Budak H (2011) miRNA expression patterns of Triticum dicoccoides in response to shock drought stress. Planta 233:471–484 33. Nageshbabu R, Jyothi MN, Sharadamma N, Sarika S, Rai DV, Devaraj VR (2013) Expression of miRNAs regulates growth and development of frenchbean (Phaseolus vulgaris) under salt and drought stress conditions. Int Res J Biol Sci 2:52–56 34. Wang M, Wang Q, Zhang B (2013) Response of miRNAs and their targets to salt and drought stresses in cotton (Gossypium hirsutum L.). Gene 30:26–32
35. Li JS, Fu F, Ming AN, Feng SZ, Hui YS, Li WC (2013) Differential expression of microRNAs in response to drought stress in maize. J Integr Agric 12:1414–1422 36. Bertolini E, Verelst W, Horner DS, Gianfranceschi L, Piccolo V, Inze D, Pe ME, Mica E (2013) Addressing the role of microRNAs in reprogramming leaf growth during drought stress in Brachypodium distachyon. Mol Plant 6:423–443 37. Ferreira TH, Gentile A, Vilela RD, Costa GGL, Dias L, Endres L, Menossi MM (2012) MicroRNAs associated with drought response in the bioenergy crop sugarcane (Saccharum spp.). PLoS ONE 7:e46703 38. Sunkar R (2010) MicroRNAs with macro-effects on plant stress responses. Semin Cell Dev Biol 21:805–811 39. Dharmasiri N, Dharmasiri S, Estelle M (2005) The F-box protein TIR1 is an auxin receptor. Nature 435:441–445 40. Sun G (2012) MicroRNAs and their diverse functions in plants. Plant Mol Biol 80:17–36 41. Lu W, Li J, Liu F, Gu J, Guo C, Xu L, Zhang H, Xiao K (2011) Expression pattern of wheat miRNAs under salinity stress and prediction of salt-inducible miRNAs targets. Front Agric China 5:413–422 42. Kong YM, Elling AA, Chen B, Deng XW (2010) Differential expression of microRNAs in maize inbred and hybrid lines during salt and drought stress. Am J Plant Sci 1:69–76 43. Li B, Duan H, Li J, Deng XW, Yin W, Xia X (2013) Global identification of miRNAs and targets in Populus euphratica under salt stress. Plant Mol Biol 81:525–539 44. Macovei A, Tuteja N (2012) MicroRNAs targeting DEAD-box helicases are involved in salinity stress response in rice (Oryza sativa L.). BMC Plant Biol 12:183–195 45. Min W, Qinglian W, Zhang B (2013) Response of miRNAs and their targets to salt and drought stresses in cotton (Gossypium hirsutum L.). Gene 530:26–32 46. Ding D, Zhang L, Wang H, Liu Z, Zhang Z, Zheng Y (2009) Differential expression of miRNAs in response to salt stress in maize roots. Ann Bot 103:29–38 47. Lv DK, Bai X, Li Y, Ding XD, Ge Y, Cai H, Ji W, Wu N, Zhu YM (2012) 0) Profiling of cold-stress-responsive miRNAs in rice by microarrays. Gene 459:39–47 48. Vaucheret H, Vazquez F, Crete P, Bartel DP (2004) The action of AGRONAUTE1 in the miRNA pathway and its regulation by the miRNA pathway are crucial for plant development. Genes Dev 18:1187–1197 49. Zhang J, Xu Y, Huan Q, Chong K (2009) Deep sequencing of Brachypodium small RNAs at the global genome level identifies microRNAs involved in cold stress response. BMC Genomics 10:449–465 50. Barakat A, Sriram A, Park J, Zhebentyayeva T, Main D, Abbott A (2012) Genome wide identification of chilling responsive microRNAs in Prunus persica. BMC Genomics 13:481–488
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Differential regulation of microRNAs in response to osmotic, salt and cold stresses in wheat Om Prakash Gupta • Nand Lal Meena Indu Sharma • Pradeep Sharma
•
Received: 21 January 2014 / Accepted: 14 March 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract MicroRNAs (miRNAs) are tiny non-coding regulatory molecules that modulate plant’s gene expression either by cleaving or repressing their mRNA targets. To unravel the plant actions in response to various environmental factors, identification of stress related miRNAs is essential. For understanding the regulatory behaviour of various abiotic stresses and miRNAs in wheat genotype C-306, we examined expression profile of selected conserved miRNAs viz. miR159, miR164, miR168, miR172, miR393, miR397, miR529 and miR1029 tangled in adapting osmotic, salt and cold stresses. The investigation revealed that two miRNAs (miR168, miR397) were downregulated and miR172 was up-regulated under all the stress conditions. However, miR164 and miR1029 were up-regulated under cold and osmotic stresses in contrast to salt stress. miR529 responded to cold alone and does not change under osmotic and salt stress. miR393 showed upregulation under osmotic and salt, and down-regulation
Om Prakash Gupta and Nand Lal Meena have contributed equally to this work.
Electronic supplementary material The online version of this article (doi:10.1007/s11033-014-3333-0) contains supplementary material, which is available to authorized users. O. P. Gupta N. L. Meena Quality and Basic Sciences Division, Directorate of Wheat Research, Karnal 132001, Haryana, India N. L. Meena Basic Sciences Division, Indian Institute of Pulses Research, Kanpur 208024, India N. L. Meena I. Sharma P. Sharma (&) Biotechnology Laboratory, Directorate of Wheat Research, Karnal 132001, India e-mail: [email protected]
under cold stress indicating auxin based differential cold response. Variation in expression level of studied miRNAs in presence of target genes delivers a likely elucidation of miRNAs based abiotic stress regulation. In addition, we reported new stress induced miRNAs Ta-miR855 using computational approach. Results revealed first documentation that miR855 is regulated by salinity stress in wheat. These findings indicate that diverse miRNAs were responsive to osmotic, salt and cold stress and could function in wheat response to abiotic stresses. Keywords
miRNAs Wheat Drought Cold Salt
Introduction In order to adopt under adverse climatic conditions, plants have developed complex mechanisms at various stages that increase tolerance level. Being a sessile organism, plants accomplish stressful ecological conditions to best fit under abiotic stress for their molecular response that might ultimately lead to their adjustment to the stresses. MicroRNAs (miRNAs) have emerged as important players in posttranscriptional gene regulation [1]. Tolerance to abiotic stresses involves a complex relationship of numerous molecular components. After discovery of miRNAs as a potent gene regulator, it has led to understand the expression behaviour of genes at post-transcriptional level [2–9]. Plant encodes 20–24 nucleotides tiny small RNA. Based on mode of small RNA biogenesis in plants, it has been classified into four categories viz. miRNAs, ra-siRNAs, nat-siRNAs and ta-siRNAs. However, another class of small RNAs, called long-siRNAs (30–40 ntds) has been added up recently. Amongst all these, miRNAs has been extensively studied in plants [7, 10, 11].
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Mol Biol Rep Table 1 Predicted target function of differentially expressed diverse miRNAs and their functional annotation [40]
miRNAs
miR159
Identified direction of expression Cold
Drought
Salt
;
:
;
Target genes
Function
MYB transcription factor (TF), 1-aminocyclopropane-1carboxylate synthase, melanocyte-specific gene related gene, oligopeptide transport gene, saur family protein
Signaling pathway and development Propanoate metabolism Regulation of gene expression Oligopeptide transporter Auxin response
miR164
:
:
;
NAC domain TF, phytosulfokines
Signaling pathway, root development, response to oxidative stress
miR168
;
;
;
Argonaute
Signal transduction, development, stress response
miR172
:
:
:
Apetala2-like TF, ARF, helix-loophelix DNA-binding domain containing protein
Signaling pathway, development, stress response
Signaling pathway
Signaling pathway, flower development BR regulation of plant development
miR393
;
:
:
bHLH TF, transport inhibitor response 1/auxin F-box
BR regulation of plant development Defense response, response to phosphate starvation, signaling pathway, flower/root development
miR397
;, downregulated; :, upregulated; –, no significant difference
;
;
;
Cold stress Copper homeostasis, response to water deprivation
miR529
:
–
;
Apetala2-like TF, squamosapromoter binding protein-like
Signaling pathway
miR1029
:
:
;
Apetala2-like TF, DREB TF
Signaling pathway
Literature suggests that miRNAs are playing key role in regulating adaptation mechanism at cellular level under abiotic stress such as cold, drought, salinity [11–13], UV-B radiation [14], heavy metals [15, 16], sulphate/phosphate starvation [17, 18], nitrogen starvation response [19] and copper deficiency [6]. Currently, the miRBase database has documented 24,521 miRNAs precursor having 30,424 mature miRNAs from 206 plant species (Release 20, http:// www.mirbase.org). Deep sequencing has helped to a greater extent to identify novel miRNAs in diverse plants [20, 21]. Comparative analysis of miRNAs in various plants has shown the differential behaviour of the same miRNA exposed to numerous abiotic stresses in different plants. This kind of regulatory behaviour of miRNAs shows their tight regulation. In the recent past, several miRNAs has been studied for their crucial role under abiotic and biotic stresses in various model plants [22]. However, reports in wheat miRNAs are
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Ice1 (inducer of CBF expression 1) TF, laccase
Plant development Abiotic stress
poorly documented [23–25]. In current study, we exploited the regulatory mechanism to characterize miRNAs that could be involved in mitigating abiotic stress tolerance in wheat. We analysed eight conserved miRNAs (Table 1) in C-306 wheat genotype with a view to capture miRNAs that might be controlled differentially under various abiotic stress conditions. The aim of this study was to validate the expression of eight conserved miRNAs (viz. miR159, miR164, miR168, miR172, miR393, miR397, miR529 and miR1029) in genotype C-306 exposed to osmotic, cold and salt stress to address their role in mitigating these stresses. In addition, miR529 has been reported in other plants but not in wheat while miR1029 has been reported only in Physcomitrella patens. We selected these two miRNAs to validate in wheat as they are involved in regulating abiotic stresses. Other miRNAs were selected owing to their role in various abiotic stress responses across the species. In addition, we have validated the expression of
Mol Biol Rep
computationally identified salt stress responsive TamiR855 in wheat [24].
Materials and methods Plant materials and treatment We selected drought tolerant wheat genotypes C306 (RGN/ CSK3//2*C59/3/C217/N14//C281) which released in the year 1965. It has multiple features like good chapatti quality, tolerance to abiotic stresses and have Lr34 resistance gene. The seeds of Triticum aestivum L. cv. C306 were initially treated with 1 % sodium hypochlorite for 10 min, rinsed in distilled water for three times, and germinated on moist filter papers. Seedlings were grown into hydroponic condition in full strength Hoagland’s liquid medium for 2 weeks. Two weeks old seedlings with similar heights were transferred to full strength Hoagland’s liquid medium containing 150 mM NaCl, polyethylene glycol 6000 (20 % v/v) for salt and osmotic stress treatment, respectively. For cold treatment, wheat seedlings were exposed to 4 °C. For expression analysis of miR855, samples were collected at 1, 12 and 24 h after salt stress at 150 mM NaCl. Control plants, corresponding to each treatment were raised in full strength Hoagland’s liquid solution at 25 °C. After 24 h of stress treatment whole seedling were harvested and transferred into liquid nitrogen. Three biological replications were performed independently for each treatment.
Fig. 1 Validation of abiotic stress-responsive miRNAs in C-306 wheat genotype. Agarose gel showing qRT-PCR products. Negative control 1 represents no-srcDNA and negative control 2 represents noRT primer. M-100 bp marker
Quantitative real-time PCR After srcDNA synthesis, each miRNAs were confirmed with PCR using conditions of 94 °C for 8 min, then 32 cycles of 94 °C for 15 s, 55 °C for 30 s and 72 °C for 30 s. PCR products were resolved using 3 % agarose gel (low EEO, Himedia) electrophoresis with a size of *100 bp (Fig. 1). qPCR was carried out using ABI PRISM 7500 Real-Time PCR System (ABI, USA). Total volume of the PCR reaction was 20 ll consisting of SYBR Green master mix (Roche), RTQ-UNIr primer (10 lM), forward primer (10 lM) and 10 ng/ll of srcDNA using above mentioned PCR protocol. Expression data generated was used for quantifying relative expression level of all the miRNAs using comparative 2-DDCt method.
Results and discussion Small RNA isolation and small RNA cDNA (srcDNA) library generation Small RNA was isolated from 100 mg of whole seedlings using mirVanaTM isolation Kit (Ambion) following manufacture’s instruction. Small RNA concentration was checked using Nano-Drop (NanoDrop technologies). For analysing expression patterns of selected miRNAs, srcDNA library was generated as described earlier [23]. Briefly, isolated small RNA was subjected to polyadenylation at 37 °C for 45 min. After tailing, samples were purified using mirVana Probe and Marker Kit (Ambion). Furthermore, expression of miRNAs by qPCR was carried out using miRNA specific forward primer and universal reverse primer (Supplementary Table 1). RTQ primer was used for cDNA synthesis and U6 snRNA was used as internal control for data normalization. All the primers were validated using thermal dissociation (Tm) curve of PCR amplicons which showed single peak.
Small-RNA discovery has opened a new avenue to detect and understand complex regulatory systems in plants in particular, those involved in abiotic stress. Plants suffer from a array of abiotic stresses; however, cold, drought, heat and salt stress are more frequently encountered. Recent studies in various plant species suggest that miRNAs play essential role in these stress tolerance. In the present study, we carried out expression analysis of eight conserved miRNAs viz. miR159, miR164, miR168, miR172, miR393, miR 397, miR529 and miR1029 in C-306 genotype exposed to osmotic, cold and salt stress to address their role in mitigating these stresses. Based upon expression pattern, above mentioned miRNAs were categorized into two groups: first group consists of miR159, miR164, miR393, miR529 and miR1029 showing differential expression pattern and second group comprised of miR168, miR172 and miR397 showing common expression pattern among all the stress conditions.
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Expression analysis of the tested miRNAs exposed to osmotic stress
involved in signalling pathway and abiotic stress responses. Our results showed up-regulation of miR1029 in wheat which might be involved in mediating osmotic stress tolerance.
Osmotic treatment of C-306 genotype has resulted in differential expression of five miRNAs (miR159, miR164, miR393, miR529 and miR1029). The accumulation of four miRNAs (miR159, miR164, miR393 and miR1029) was significantly higher while osmotic stress had no major effect on the expression of miR529 compared to control plants. Among over accumulated miRNAs, the expression of miR393 was maximum (*12.8-fold) followed by miR1029 (*9 fold), miR159 (*0.5-fold) and miR164 (*0.3-fold) (Fig. 2). Previously, efforts were made to validate the expression of miRNAs in numerous crop species under drought stress [12, 13, 26–31] however; work on wheat miRNA is very limited. Until date, there is only one report on miRNA expression behaviour in Triticum dicoccoides exposed to drought shock [32]. Up regulation of miR159 in C-306 wheat cultivar is in agreement with other studies like French bean [33], cotton [34], maize [35] suggesting its proactive involvement in regulating signal transduction pathways under osmotic stress. Over accumulation of miR164 in the seedlings of C-306 wheat cultivar in present study as well as in Brachypodium [36] and sugarcane [37] demonstrate role of NAC domain transcription factor in perturbation of signalling, root development and oxidative stress responses. The expression of miR393 is commonly up-regulated during drought stress in Oryza, Medicago, Pinguicula, Arabidopsis [38]. Our result of miR393 expression is in tune with these findings. miR393 modulates auxin mediated response by targeting TIR1 which promotes degradation of Aux/IAA repressor by ubiquitin ligase [39]. In addition, the role of miR1029 is reported to target DREB and AP2 transcription factor [40] which
Abiotic stress particularly salinity in wheat is a major problem which limits wheat yield in salt affected areas. Many researchers have documented the role of miRNAs in controlling developmental stages in plants under salinity stress [41–45]. However, the role of miRNAs in wheat under salinity stress is completely unexplored as there is only one report present till now [41] which urges strongly to look into. Interestingly, we could find only one miRNA i.e. miR393 to be up-regulated amongst miR159, miR164, miR393, miR529 and miR1029 in C-306 under 150 mM NaCl stress while miR159, miR164 and miR1029 were down-regulated. miR529 showed no significant alteration under salt stress (Fig. 3). Our result on accumulation of miR393 is in quite agreement with the previous findings in Arabidopsis [12, 27] suggesting similar regulatory response in Arabidopsis and wheat under salinity stress. Most of the down-regulated miRNAs are reported to be involved in root development, signal transduction, and abiotic stress response [40] signifying that they might be involved in regulating salt stress response in C-306 cultivar by allowing accumulation of their corresponding targets. Downregulation of miR159 in C-306 wheat cultivar under 150 mM NaCl is differing with the result of Lu et al. [41] where they reported up-regulation under 200 mM NaCl treatment which might be due difference in salt concentration.
Fig. 2 Relative miRNAs expression in genotype wheat C-306 exposed to osmotic stress. Bars represent mean and standard deviation of values obtained from three biological replicates. The relative expression level of all miRNA was calculated using the comparative 2-DDCt method. The data was normalized to U6 snRNA expression level as an internal control
Fig. 3 The expression of selected miRNAs in wheat genotype C-306 exposed to salinity. Bars represent mean and standard deviation of values obtained from three biological replicates. The relative expression level of all miRNA was calculated using the comparative 2-DDCt method. The data was normalized to U6 snRNA expression level as an internal control
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Expression behaviour of the tested miRNAs under salt stress
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Fig. 4 The expression of miR855 in wheat genotype C-306 exposed to 150 mM NaCl stress. Bars represent mean and standard deviation of values obtained from three biological replicates. Samples were collected after 1, 12 and 24 h duration of salt treatment. The relative expression level of all miRNA was calculated using the comparative 2-DDCt method. The data was normalized to U6 snRNA expression level as an internal control
In our previous work, we have shown five new miRNAs (Ta-miR819 k, Ta-miR855, Ta-miR3708, Ta-miR5156 and Ta-miR5653) from wheat EST database [24]. Till date miR855 is only identified in Arabidopsis thaliana in Chromosome I to Chromosome V. The miR855 is mapped in wheat EST (BG313724) which is located in DSW03C5_Contig2094.1 with nine members having 6AS, 6BS, and 6DS chromosomal locations. Effect of salt stress on C-306 led to down-regulation of miR855 (approximately -4.5-fold) at 1 h followed by subsequent over accumulation at 12 h (*20-fold) and 24 h (*41.3-fold) of stress exposure (Fig. 4). miR855 was shown to be expressed in wheat sheath under salt stress condition and targets MYB TF which regulates leaf development. MYB transcription factor is also targeted by miR159, miR828 and miR858 which primarily regulate signal transduction and development of the plants under various stress conditions [40]. Down-regulation of miR855 in C-306 at 1 h might be linked with activation of MYB based signalling to cope up the salt stress whereas up-regulation at later stages (12 and 24 h) of salt exposure could be due to suppression of MYB based tolerance mechanism in C-306 wheat genotype. Up-regulation of miR159 in wheat under salinity stress [41] suggesting a coordinating link between miR159 based regulation of MYB TF. Although, miR855 showed differential response under salt stress in wheat, further work on functional validation along with the target is required. Expression array of the tested miRNAs under cold stress Although, C-306 wheat genotype not reported to be cold tolerant, we carried out expression analysis of same set of
Fig. 5 Relative miRNAs expression in genotype wheat C-306 exposed to cold. Bars represent mean and standard deviation of values obtained from three biological replicates. The relative expression level of all miRNA was calculated using the comparative 2-DDCt method. The data was normalized to U6 snRNA expression level as an internal control
miRNAs to observe any similarity with the salt and drought stressed seedlings (Fig. 5). Interestingly, the expression pattern of miR159 (down-regulated) from cold stressed (4 °C for 24 h) seedlings is similar to the salinity stressed seedlings while miR164 and miR1029 was similar with drought stressed seedlings indicating their common mode of regulation of these two stress response in wheat. In addition, miR393 which was up-regulated under drought and salt, showed down-regulation under cold stress indicating auxin based differential cold response. Osmotic, salt and cold stress induces miRNA mediated common regulatory pathway in wheat In order to understand common regulatory pathways operated in wheat under osmotic, salt and cold stresses, we were interested to capture common miRNAs showing similar response under all the stress condition in wheat genotype C-306. Surprisingly, we could encountered two miRNAs namely miR168 and miR397 showing downregulation while miR172 showed up-regulation under all the stress conditions (Fig. 6). miR168 has been reported to be up regulated under drought [27] and salt stress [46] in Arabidopsis while down-regulated in rice under cold stress [47]. In plants, AGO1, target of miR168, is the main effector protein for regulating the activity of miRNAs. However, it will be interesting to know whether any such changes of miR168 regulation lead to change in miRNA functions [48]. Similarly, the expression of miR397 under cold stress and dehydration response was up-regulated in Arabidopsis [12] which is in contrast with our result in wheat which could be due to variation in the experimental procedure and plant species. miR397 targets an upstream transcription factor ICE1, which regulates the expression of
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Mol Biol Rep Research, New Delhi. We acknowledge the support of Amit Kumar and Bharti. This paper is DWR contribution No. 15.
References
Fig. 6 Expression analyses of miRNA 168, miR172 and miR198 in wheat genotype C-306 exposed to osmotic, salinity and cold. Bars represent mean and standard deviation of values obtained from three biological replicates. The relative expression level of all miRNA was calculated using the comparative 2-DDCt method. The data was normalized to U6 snRNA expression level as an internal control
CBF genes during cold stress response in plants [40]. Down-regulation of miR397 under cold stress in C-306 cultivar would increase the accumulation of ice1, which might positively be regulating cold stress response in wheat. Expression of miR172 in C-306 is in agreement with the previous report in Arabidopsis [12, 47], Brachypodium [49], Prunus [50] under cold stress and in cotton under drought and salt stress [34]. These results are in contrast with that of rice where miR172 was down-regulated under drought stress [21]. Differential regulation of these three miRNAs indicates the evolutionary divergence of the crop species along with experimental procedure and treatment conditions. Here, we report the common expression pattern of these three miRNAs in C-306 wheat cultivar under osmotic, salt and cold stress condition suggesting their common mode of action however, this needs further validation. In conclusion, an understanding of post-transcriptional gene regulation by miRNAs under adverse climatic conditions is critical for understanding and improving stress tolerance in crop plants. Several studies have demonstrated that many plant miRNAs are highly conserved across the species. Therefore, it is tempting to speculate that miRNAs biological functionality might be similar. We validated the expression of eight stress-induced conserved miRNAs viz. miR159, miR164, miR168, miR172, miR393, miR 397, miR529 and miR1029, in wheat genotype C-306 exposed to osmotic, salt and cold treatments to address their role in mitigating cold, salt and drought stresses. In addition, miR855 that was identified previously was also shown to be differentially regulated under salt stress. Acknowledgments Authors acknowledge Grant-in-Aids under the project DWR/RP/10-5.3 of the Indian Council of Agricultural
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1. Kawaguchi R, Girke T, Bray EA, Bailey-Serres J (2004) Differential mRNA translation contributes to gene regulation under non-stress and dehydration stress conditions in Arabidopsis thaliana. Plant J 38:823–839 2. Bartel DP (2004) MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116:281–297 3. Borsani O, Zhu J, Verslues PE, Sunkar R, Zhu JK (2005) Endogenous siRNAs derived from a pair of natural cis-antisense transcripts regulates salt tolerance in Arabidopsis. Cell 123:1279–1291 4. Navarro L, Dunoyer P, Jay F, Arnold B, Dharmasiri N, Estelle M, Voinnet O, Jones JD (2006) A plant miRNA contributes to antibacterial resistance by repressing auxin signalling. Science 312:436–439 5. Brodersen P, Sakvarelidze-Achard L, Bruun-Rasmussen M, Dunoyer P, Yamamoto YY, Sieburth L, Voinnet O (2008) Widespread translational inhibition by plant miRNAs and siRNAs. Science 320:1185–1190 6. Abdel-Ghany SE, Pilon M (2008) MicroRNA-mediated systemic down-regulation of copper protein expression in response to low copper availability in Arabidopsis. J Biol Chem 28:15932–15945 7. Voinnet O (2009) Origin, biogenesis, and activity of plant microRNAs. Cell 36:669–687 8. Wu G, Park MY, Conway SR, Wang JW, Weigel D, Poethig RS (2009) The sequential action of miR156 and miR172 regulates developmental timing in Arabidopsis. Cell 138:750–759 9. Vazquez F, Legrand S, Windels D (2010) The biosynthetic pathways and biological scopes of plant small RNAs. Trends Plant Sci 15:337–345 10. Surekha-Katiyar A, Shang G, Adam VS, Hailing J (2007) A novel class of bacteria-induced small RNAs in Arabidopsis. Genes Dev 21:3123–3134 11. Jones-Rhoades MW, Bartel DP, Bartel B (2006) MicroRNAs and their regulatory roles in plants. Annu Rev Plant Biol 57:19–53 12. Sunkar R, Zhu JK (2004) Novel and stress-regulated microRNAs and other small RNAs from Arabidopsis. Plant Cell 16:2001–2019 13. Zhao B, Liang R, Ge L, Li W, Xiao H, Lin H, Ruan K, Jin Y (2007) Identification of drought-induced microRNAs in rice. Biochem Biophys Res Commun 354:585–590 14. Zhou X, Wang G, Zhang W (2007) UV-B responsive microRNA genes in Arabidopsis thaliana. Mol Syst Biol 3:103–112 15. Huang SQ, Peng J, Qiu CX, Yang ZM (2009) Heavy metalregulated new microRNAs from rice. J Inorg Biochem 3:282–287 16. Gupta OP, Sharma P, Gupta RK, Sharma I (2014) MicroRNA mediated regulation of metal toxicity in plants: present status and future perspectives. Plant Mol Biol 84:1–18 17. Jones-Rhoades MW, Bartel DP (2004) Computational identification of plant microRNAs and their targets, including a stressinduced miRNA. Mol Cell 14:787–799 18. Pant BD, Musialak-Lange M, Nuc P, May P, Buhtz A, Kehr J, Walther D, Scheible WR (2009) Identification of nutrientresponsive Arabidopsis and rapeseed microRNAs by comprehensive real-time polymerase chain reaction profiling and small RNA sequencing. Plant Physiol 150:1541–1555 19. Xu Z, Zhong S, Li X, Li W, Rothstein SJ, Zhang S, Bi Y, Xie C (2011) Genome-wide identification of microRNAs in response to low nitrate availability in maize leaves and roots. PLoS ONE 6:e28009
Mol Biol Rep 20. Yang X, Li L (2011) miRDeep-P: a computational tool for analyzing the microRNA transcriptome in plants. Bioinformatics 27:2614–2615 21. Zhou L, Liu Y, Liu Z, Kong D, Duan M, Luo L (2010) Genomewide identification and analysis of drought-responsive microRNAs in Oryza sativa. J Exp Bot 61:4157–4168 22. Gupta OP, Sharma P, Gupta RK, Sharma I (2014) Current status on role of miRNAs during plant–fungus interaction. Physiol Mol Plant Pathol 85:1–7 23. Gupta OP, Permar V, Koundal V, Singh UD, Praveen S (2012) MicroRNA regulated defense responses in Triticum aestivum L. during Puccinia graminis f. sp. tritici infection. Mol Biol Rep 39:817–822 24. Pandey B, Gupta OP, Pandey DM, Sharma I, Sharma P (2013) Identification of new stress-induced microRNA and their targets in wheat using computational approach. Plant Sig Behav 8:e23932 25. Xin MM, Wang Y, Yao YY, Xie CJ, Peng HR, Ni ZF, Sun QX (2010) Diverse set of microRNAs are responsive to powdery mildew infection and heat stress in wheat (Triticum aestivum L.). BMC Plant Biol 10:123–134 26. Reyes JL, Chua NH (2007) ABA induction of miR159 controls transcript levels of two MYB factors during Arabidopsis seed germination. Plant J 49:592–606 27. Liu HH, Tian X, Li YJ, Wu CA, Zheng CC (2008) Microarraybased analysis of stress-regulated microRNAs in Arabidopsis thaliana. RNA 14:836–843 28. Li WX, Ono Y, Zhu J, He XJ, Wu JM, Iida K, Lu XY, Cui X, Hin H, Zhu JK (2008) The Arabidopsis NFYA5 transcription factor is regulated transcriptionally and post-transcriptionally to promote drought resistance. Plant Cell 20:2238–2251 29. Trindade I, Capitao C, Dalmay T, Fevereiro MP, Santos DM (2010) miR398 and miR408 are up-regulated in response to water deficit in Medicago truncatula. Planta 231:705–716 30. Wang T, Chen L, Zhao M, Tian Q, Zhang WH (2011) Identification of drought-responsive microRNAs in Medicago truncatula by genome-wide high throughput sequencing. BMC Genomics 12:367–378 31. Eldem V, Celikkol-Akcay U, Ozhuner E, Bakir Y, Uranbey S, Akcay U, Unver T (2012) Genome-wide identification of miRNAs responsive to drought in peach (Prunus persica) by highthroughput deep sequencing. PLoS ONE 7:e50298 32. Kantar M, Lucas SJ, Budak H (2011) miRNA expression patterns of Triticum dicoccoides in response to shock drought stress. Planta 233:471–484 33. Nageshbabu R, Jyothi MN, Sharadamma N, Sarika S, Rai DV, Devaraj VR (2013) Expression of miRNAs regulates growth and development of frenchbean (Phaseolus vulgaris) under salt and drought stress conditions. Int Res J Biol Sci 2:52–56 34. Wang M, Wang Q, Zhang B (2013) Response of miRNAs and their targets to salt and drought stresses in cotton (Gossypium hirsutum L.). Gene 30:26–32
35. Li JS, Fu F, Ming AN, Feng SZ, Hui YS, Li WC (2013) Differential expression of microRNAs in response to drought stress in maize. J Integr Agric 12:1414–1422 36. Bertolini E, Verelst W, Horner DS, Gianfranceschi L, Piccolo V, Inze D, Pe ME, Mica E (2013) Addressing the role of microRNAs in reprogramming leaf growth during drought stress in Brachypodium distachyon. Mol Plant 6:423–443 37. Ferreira TH, Gentile A, Vilela RD, Costa GGL, Dias L, Endres L, Menossi MM (2012) MicroRNAs associated with drought response in the bioenergy crop sugarcane (Saccharum spp.). PLoS ONE 7:e46703 38. Sunkar R (2010) MicroRNAs with macro-effects on plant stress responses. Semin Cell Dev Biol 21:805–811 39. Dharmasiri N, Dharmasiri S, Estelle M (2005) The F-box protein TIR1 is an auxin receptor. Nature 435:441–445 40. Sun G (2012) MicroRNAs and their diverse functions in plants. Plant Mol Biol 80:17–36 41. Lu W, Li J, Liu F, Gu J, Guo C, Xu L, Zhang H, Xiao K (2011) Expression pattern of wheat miRNAs under salinity stress and prediction of salt-inducible miRNAs targets. Front Agric China 5:413–422 42. Kong YM, Elling AA, Chen B, Deng XW (2010) Differential expression of microRNAs in maize inbred and hybrid lines during salt and drought stress. Am J Plant Sci 1:69–76 43. Li B, Duan H, Li J, Deng XW, Yin W, Xia X (2013) Global identification of miRNAs and targets in Populus euphratica under salt stress. Plant Mol Biol 81:525–539 44. Macovei A, Tuteja N (2012) MicroRNAs targeting DEAD-box helicases are involved in salinity stress response in rice (Oryza sativa L.). BMC Plant Biol 12:183–195 45. Min W, Qinglian W, Zhang B (2013) Response of miRNAs and their targets to salt and drought stresses in cotton (Gossypium hirsutum L.). Gene 530:26–32 46. Ding D, Zhang L, Wang H, Liu Z, Zhang Z, Zheng Y (2009) Differential expression of miRNAs in response to salt stress in maize roots. Ann Bot 103:29–38 47. Lv DK, Bai X, Li Y, Ding XD, Ge Y, Cai H, Ji W, Wu N, Zhu YM (2012) 0) Profiling of cold-stress-responsive miRNAs in rice by microarrays. Gene 459:39–47 48. Vaucheret H, Vazquez F, Crete P, Bartel DP (2004) The action of AGRONAUTE1 in the miRNA pathway and its regulation by the miRNA pathway are crucial for plant development. Genes Dev 18:1187–1197 49. Zhang J, Xu Y, Huan Q, Chong K (2009) Deep sequencing of Brachypodium small RNAs at the global genome level identifies microRNAs involved in cold stress response. BMC Genomics 10:449–465 50. Barakat A, Sriram A, Park J, Zhebentyayeva T, Main D, Abbott A (2012) Genome wide identification of chilling responsive microRNAs in Prunus persica. BMC Genomics 13:481–488
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