MicroRNA-mediated responses to longterm magnesium-deficiency in Citrus sinensis roots revealed by Illumina sequencing

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1 Ling et l. BMC Genomics (2017) 18:657 DOI /s RESEARCH ARTICLE Open Access MicroRNA-medited responses to longterm mgnesium-deficiency in Citrus sinensis roots reveled y Illumin sequencing Wei-Wei Ling 1, Jing-Ho Hung 1,2, Chun-Ping Li 1, Lin-Tong Yng 1, Xin Ye 1, Dn Lin 1 nd Li-Song Chen 1,3,4* Astrct Bckground: Mgnesium (Mg)-deficiency occurs most frequently in strongly cidic, sndy soils. Citrus re grown minly on cidic nd strong cidic soils. Mg-deficiency cuses poor fruit qulity nd low fruit yield in some Citrus orchrds. For the first time, we investigted Mg-deficiency-responsive mirnas in Xuegn (Citrus sinensis) roots using Illumin sequencing in order to otin some mirnas presumly responsile for Citrus Mg-deficiency tolernce. Results: We otined 101 (69) mirnas with incresed (decresed) expression from Mg-strved roots. Our results suggested tht the dpttion of Citrus roots to Mg-deficiency ws relted to the severl spects: () inhiiting root respirtion nd relted gene expression vi inducing mir158 nd mir2919; () enhncing ntioxidnt system y downregulting relted mirnas (mir780, mir6190, mir1044, mir5261 nd mir1151) nd the dpttion to low-phosphorus (mir6190); (c) ctivting trnsport-relted genes y ltering the expression of mir6190, mir6485, mir1044, mir5029 nd mir3437; (d) elevting protein uiquitintion due to decresed expression levels of mir1044, mir5261, mir1151 nd mir5029; (e) mintining root growth y regulting mir5261, mir6485 nd mir158 expression; nd (f) triggering DNA repir (trnscription regultion) y regulting mir5176 nd mir6485 (mir6028, mir6190, mir6485, mir5621, mir160 nd mir7708) expression. Mg-deficiency-responsive mirnas involved in root signl trnsduction lso hd functions in Citrus Mg-deficiency tolernce. Conclusions: We otined severl novel Mg-deficiency-responsive mirnas (i.e., mir5261, mir158, mir6190, mir6485, mir1151 nd mir1044) possily contriuting to Mg-deficiency tolernce. These results reveled some novel clues on the mirna-medited dpttion to nutrient deficiencies in higher plnts. Keywords: Citrus sinensis, Illumin sequencing, Mgnesium-deficiency, mirna, Root Bckground Mgnesium (Mg)-deficiency, common prolem in mny griculturl crops, occurs most frequently in strongly cidic, sndy soils, where Mg is very prone to leching [1]. Citrus re grown minly on cidic nd strong cidic soils nd Mg-deficiency is responsile for * Correspondence: lisongchen2002@hotmil.com; lisongchen@ffu.edu.cn 1 Institute of Plnt Nutritionl Physiology nd Moleculr Biology, College of Resources nd Environment, Fujin Agriculture nd Forestry University, Fuzhou , Chin 3 Fujin Provincil Key Lortory of Soil Environmentl Helth nd Regultion, College of Resources nd Environment, Fujin Agriculture nd Forestry University, Fuzhou , Chin Full list of uthor informtion is ville t the end of the rticle the poor fruit qulity nd the reduction in fruit yield in some Citrus orchrds [2]. According to our investigtion in 2011, over 90% nd 77% of Citrus grndis orchrd soils from Pinghe county, Fujin province hd ph less thn 5.0 nd soil exchnge Mg content less thn the optimum rnge, respectively [3]. Wht s worse, crop Mgdeficiency, which is ecoming more nd more populr due to soil cidifiction nd improper frmer prctices such s intensive crop production systems nd highly fortified rottion, hs een considered to e n urgent griculturl prolem [3, 4]. Although Mg is one of the most importnt nutrients in higher plnts nd plys essentil roles in numerous cellulr processes such s The Author(s) Open Access This rticle is distriuted under the terms of the Cretive Commons Attriution 4.0 Interntionl License ( which permits unrestricted use, distriution, nd reproduction in ny medium, provided you give pproprite credit to the originl uthor(s) nd the source, provide link to the Cretive Commons license, nd indicte if chnges were mde. The Cretive Commons Pulic Domin Dediction wiver ( pplies to the dt mde ville in this rticle, unless otherwise stted.

2 Ling et l. BMC Genomics (2017) 18:657 Pge 2 of 16 chlorophyll iosynthesis, gs exchnges [2, 5 7], conformtionl stiliztion of proteins, nucleic cids, cell wlls nd memrnes [8], prtitioning nd utiliztion of photossimiltes [7, 9], ctivtion of enzymes [9, 10] nd rective oxygen species (ROS) genertion [9]. Despite the importnt roles of Mg in higher plnts, Mg hs een less pid ttention y gronomists nd otnists reltive to the other nutrients nd is considered to e the forgotten element [4, 11]. Therefore, it is very importnt to elucidte the moleculr mechnisms on Mg-deficiency impirments nd tolernce in higher plnts. To our knowledge, such dt re rre [10, 12, 13]. Evidence demonstrtes tht microrna (mirna)-medited posttrnscriptionl regultion of gene expression plys role in plnt dptive responses to deficiencies of phosphorus (P), potssium (K), nitrogen (N), sulfur (S), mngnese (Mn), oron (B), zinc (Zn) nd iron (Fe) [14 20]. Numerous differentilly expressed mirnas hve een isolted from P-strved Aridopsis, white lupin, Medicgo trunctul, common en, rice, rley, tomto nd soyen [21 25]. The roles of P-deficiencyinduced up-regultion of plnt mir399 nd mir827 in the mintennce of P homeostsis vi inhiiting their trgets uiquitin-conjugting enzyme E2 24 (UBC24) nd N limittion dpttion (NLA), respectively hve een well chrcterized [14, 23, 26, 27]. Nitrogen-deficiency-induced ltertions of mirna profiles hve een reported on severl higher plnts including mize, Aridopsis, soyen, common en [20, 24, 25, 28, 29]. Severl N-deficiency-responsive mirnas hve een chrcterized in some detils. For exmple, root modultion under N-deficiency ws coordinted y mir160, mir167 nd mir171 nd root growth ws promoted y down-regulting mir167 expression nd up-regulting mir160 nd mir171 expression [24, 28]. N-deficiency-induced down-regultion of mir169 hs een demonstrted to e n dptive strtegy of plnts to N-strvtion vi N-uptke nd remoiliztion [24, 30]. Despite the vitl roles of K in higher plnts, little is known out K-deficiency-responsive mirnas. In study, Yn et l. [31] exmined K-deficiency-induced ltertions in expression of mir444 nd its trgets (i.e., MADS-57, MADS-27, MADS-27 nd MADS-23) in rice roots, nd found tht mir444 ws slightly down-regulted nd MADS-23 ws gretly up-regulted. In ddition, mny differentilly expressed mirnas hve een identified in B-deprived C. sinensis roots nd leves [17, 18], Cu-strved [32] nd Fe-deficient [33] Aridopsis, S-deprived Brssic rpus [34], Mn-limited Phseolus vulgris [25] nd Zn-deficient Sorghum icolor [35]. Although the effects of nutrient deficiencies on mirna expression in higher plnts hve een explored y some workers, most of these studies hve een pid to herceous plnts. Little is known out Mgdeficiency-induced ltertions of mirna expression in woody plnts. Previously, we exmined Mg-deficiencyresponsive mirnas in C. sinensis leves reveled y Illumin sequencing nd identified 71 down- nd 75 up-regulted mirnas, implying the potentil roles of mirnas in Citrus Mg-deficiency tolernce [36]. On this sis, we used Illumin sequencing to sequence two smll RNA lirries from Mg-sufficient (control) nd -deficient C. sinensis roots in order to distinguish the differences in Mg-deficiency-induced ltertions of mirna profiles etween C. sinensis roots nd leves nd to otin some mirnas presumly responsile for Citrus Mg-deficiency tolernce. Results Root dry weight (DW) nd root nd lef Mg Root DW nd root nd lef Mg levels were lower in 0 mm Mg-treted seedlings thn in 1 mm Mg-treted ones, nd Mg level in leves from 0 mm Mg-treted seedlings ws much less thn the sufficient rnge (Fig. 1) [37]. Bsed on these dt nd our previous reports [6, 12], these seedlings sumitted to 0 nd 1 mm Mg were regrded s Mg-deficient nd -sufficient (control), respectively. Illumin sequencing nd mirna nnottion Using high-throughput sequencing, we got 20,726,716 (22,139,574) rw reds from srna lirry constructed from control (Mg-deficient) roots. After the dptors, low qulity tgs nd contminnts eing removed, the control nd Mg-deficient root srna lirries generted 20,325,777 (5,561,214) nd 21,783,568 (6,124,980) cler reds (unique reds), respectively (Tle 1). As shown in Fig. 2, the mjority of the cler reds fell within the rnge of nt. The most undnt cler reds were 24 nt length, followed y 21, 22, 23 nd 20 nt length. This grees with the previous dt otined on leves, roots [17, 18] nd fruits [38] of C. sinensis, nd fruits nd flowers of Citrus trifolit [39]. Therefore, these dt otined vi high-throughput sequencing of srna lirries re relile. Mg-deficiency incresed nd decresed the undnces of 24 nd 21 nt reds, respectively. Here, 13,624,836 clen reds (3,077,845 unique reds) from Mg-sufficient roots nd 14,510,776 cler reds (3,378,231 unique reds) from Mg-deficient roots were mpped to C. sinensis genome (JGIversion1.1, phytozome.jgi.doe.gov/pz/portl.html#!info?lis=org_c sinensis) using SOAP [40]. Therefter, we used the unnnotted 5,050,734 nd 5,550,820 unique reds from Mg-sufficient nd -deficient roots, respectively to predict novel mirnas (Tle 1).

3 Ling et l. BMC Genomics (2017) 18:657 Pge 3 of 16 A ) -1 Root DW (g plnt in Mg-deficient roots with TPM vlue ten in Mgdeficient nd/or -sufficient roots. Vlidtion of sequencing dt y stem-loop qrt-pcr The expression levels of 27 Mg-deficiency-responsive mirnas were ssyed y stem-loop qrt-pcr. Except for mir1222, the expression ptterns of ll mirnas otined y stem-loop qrt-pcr nd Illumion sequencing were similr (Fig. 3 nd Tle 2). Thus, the results produced y Illumion sequencing were relile. B R oot Mg ( g g - 1 DW ) C L ef Mg ( g g - 1 DW ) Control Mg-deficiency Fig. 1 Root DW (), root () nd lef (c) Mg concentrtions in response to Mg-deficiency. Brs represent men ± SD (n = 5 for root nd lef Mg nd 9 for root DW). Different letters ove the rs indicte significnt difference t P <0.05 Identifiction nd prediction of root mirnas As shown in Additionl file 1, we identified 733 known mirnas in C. sinensis roots. To vert flse results due to the use of low undnt mirnas, these known mirnas with trnscript per million (TPM) vlue <10 in oth Mg-sufficient nd -deficient roots were not utilized for further nlysis [17, 41]. The remined 300 mirnas with TPM vlue 10 in Mg-sufficient nd/or -deficient roots were utilized for Mg-deficiency-responsive mirna nlysis (Additionl file 2). As shown in Additionl file 3, we otined 71 up- nd 54 down-regulted known mirnas from Mgdeficient roots. As shown in Additionl files 4, 5, nd 6, we identified 181 novel mirnas in oth Mg-sufficient nd -deficient roots, nd 30 up- nd 15 down-regulted novel mirnas Prediction nd GO nlysis of trgets for Mg-deficiencyresponsive mirnas Here, we predicted 239 nd 130 trget genes from the 46 known nd 15 novel Mg-deficiency-responsive-miR- NAs, respectively (Additionl files 7 nd 8). As shown in Fig. 4, the trgets for known (novel) Mg-deficiencyresponsive mirnas were ssocited with 12 (nine) iologicl processes. The most undnt three GO terms were response to stress, trnsport nd protein process for known mirna trgets nd response to stress, regultion of trnscription nd trnsport for novel mirna trgets, respectively. On the sis of the moleculr function, the highest percentges of three groups for known nd novel mirna trgets were nucleic cid inding, other ctivity nd kinse ctivity, nd other ctivity, metl ion inding nd trnsporter ctivity, respectively (Fig. 4). As shown in Fig. 4c, the trgets for known (novel) Mg-deficiency-responsive mirnas were relted to 12 (eight) cellulr components. The most undnt component for known nd novel mir- NAs ws nucleus. Vlidtion of trget genes y qrt-pcr As shown in Tle 2, 105 trgets for 11 up- nd 16 down-regulted mirnas were vlidted y qrt-pcr. As expected, we found tht 65 (61.9%) trgets nd their corresponding mirnas displyed opposite trends in expression profiles in Mg-deprived roots, suggesting tht mirnas might ply role in gene regultion y cleving mrnas. However, 34 (32.4%) trgets displyed the sme expression profiles with their corresponding mirnas in Mg-deficient roots or were not significntly ffected y Mg-deficiency. The remining 6 (0.06%) trgets were not detected in roots. It is worth mentioning tht 4 trget genes (i.e., rnge1.1g005482m, ornge1.1g004896m, ornge1.1g005075m nd ornge1.1g008078m) elonging to uxin responsive fctor (ARF) fmily hve een vlidted y us in C. sinensis [42], suggesting tht the trget prediction ws ccurte. Discussion Little is known out the possile roles of mirnas in plnt Mg homeostsis [36, 43]. Here, we first

4 Ling et l. BMC Genomics (2017) 18:657 Pge 4 of 16 Tle 1 Summry of srna sequencing dt from Mg-sufficient nd -deficient Citrus sinensis roots Mg-sufficiency Mg-deficiency Unique srnas Totl srnas Unique srnas Totl srnas Rw reds 20,726,716 22,139,574 Cler reds 5,561,214(100%) 20,325,777(100%) 6,124,980(100%) 21,783,568(100%) Mpped to genomic 3,077,845(55.34%) 13,624,836(67.03%) 3,378,231(55.15%) 14,510,776(66.61%) Exon ntisense 47,462(0.85%) 176,617(0.87%) 50,014(0.82%) 177,516(0.81%) Exon sense 105,888(1.90%) 358,233(1.76%) 117,753(1.92%) 372,942(1.71%) Intron ntisense 65,331(1.17%) 281,377(1.38%) 71,109(1.16%) 294,537(1.35%) Intron sense 88,455(1.59%) 520,695(2.56%) 94,926(1.55%) 557,578(2.55%) mirna 54,043(0.97%) 3,125,403(15.37%) 53,522(0.87%) 3,364,650(15.45%) rrna 125,351(2.25%) 2,205,674(10.85%) 157,937(2.58%) 2,558,877(11.74%) repet 1384(0.02%) 3652(0.02%) 1587(0.03%) 3946(0.02%) snrna 2722(0.05%) 9423(0.05%) 3188(0.05%) 10,142(0.05%) snorna 1667(0.03%) 6123(0.03%) 1833(0.03%) 6354(0.03%) trna 18,177(0.33%) 767,772(3.78%) 22,291(0.36%) 660,793(3.03%) Unnnotted srnas 5,050,734(90.82%) 12,870,808(63.32%) 5,550,820(90.63%) 13,776,233(63.24%) investigted the Mg-deficiency-induced ltertions of mirna profiles in Citrus roots nd otined 101 upnd 69 down-regulted mirnas (Additionl files 3 nd 6), demonstrting tht mirnas might e involved in Mg-deficiency responses. We otined similr mount of mirnas (71 mirnas) with decresed expression, ut less mount of mirnas (75 mirnas) with incresed expression from Mg-deficient C. sinensis leves compred with Mg-deficient C. sinensis roots [36]. Moreover, most of these mirnas were isolted only from Mg-deprived roots or leves, only 30 Mg-deficiency-responsive mirnas were shred y the two. Among the 30 overlpping mir- NAs, only 15 mirnas displyed similr expression trends in Mg-deprived roots nd leves (Tle 3). Thus, gret differences existed in Mg-deficiencyinduced ltertions of mirna profiles etween roots nd leves. This grees with our report tht the (%) srnas Percentge of mm Mg 1 mm Mg Length of srnas (nt) Fig. 2 Smll RNA length distriution from Mg-deficient nd -sufficient C. sinensis roots physiologicl nd iochemicl responses to long-term Mg-deficiency gretly differed etween C. sinensis roots nd leves [7]. We oserved tht mir158 ws induced in Mgdeprived roots (Tle 2). Similr results hve een otined on Mg-deficient C. sinensis leves [36], P- deficient tomto roots nd leves [22], nd B-strved C. sinensis roots nd leves [17, 18]. As expected, its trget gene SPFH (stomtins, prohiitins, flotillins nd HflK/ C)/Bnd 7/PHB domin-contining memrnessocited protein fmily (AT5G62740) ws repressed in Mg-deprived roots. Wng et l. [44] found tht Aridopsis ph3 3 mutnts were less sensitive to slt-stressinduced inhiition of primry root growth. Thus, the down-regultion of AT5G62740 might contriute to Citrus Mg-deficiency tolernce vi lleviting Mgdeficiency-induced inhiition of root growth (Fig. 1). Gehl et l. [45] oserved tht the sl tissue respirtion rte in stomtin-like protein 1 (slp1) knockout Aridopsis roots ws reduced y 30% compred with wild-type. In ddition, mir2919 expression ws induced nd its trget: phosphoenolpyruvte croxylse 3 (PEPC3) ws inhiited in Mg-deprived C. sinensis roots (Tle 2). Therefore, root respirtion might e decresed in Mg-strved C. sinensis roots. This grees with our reports tht the undnces of pyruvte decroxylse (gi 255,579,310) nd phosphoglycerte kinse (gi 332,195,235) in glycolysis nd the ctivities of key enzymes in glycolysis nd tricroxylic cid (TCA) cycle were reduced in Mgdeprived C. sinensis roots ccompnied y decresed ccumultion of crohydrtes nd lower respirtion [7, 12]. Both root mir6278 nd its trgets: NB-ARC domincontining disese resistnce protein involved in disese resistnce nd DnJ/Hsp40 cysteine-rich domin

5 Ling et l. BMC Genomics (2017) 18:657 Pge 5 of 16 A Reltive expression B Reltive expression Mg-deficiency Control mir7821 mir418 mir6150 mir7121 mir158 mir6278 mir3437 mir780 mir414 mir394 mir5176 mir1847 mir2919 mir1222 mir6028 mir1151 mir5198 mir6219 mir6190 mir160 mir6446 mir6485 mir1044 mir7708 mir5261 mir5029 mir3438 Fig. 3 Reltive expression levels of selected Mg-deficiency-responsive known mirnas in Mg-deficient nd control roots reveled y qrt-pcr. Brs represent men ± SD (n = 3). For the sme mirna, different letters ove the rs indicte significnt difference t P < All the vlues were expressed reltive to the control roots superfmily protein (AT3G ) were induced y Mgdeficiency. In ddition, NB-ARC domin-contining disese resistnce protein (AT4G ) trgeted y mir780 ws induced in Mg-depprived roots (Tle 2). Similrly, the undnces of Grp94 (HSP; gi 23,477,636) nd disese resistnce protein (gi 227,438,123) ws incresed in Mg-strved C. sinensis roots [12]. Thus, disese resistnce might e elevted in these roots with incresed levels of C nd K [46], which contriute to plnt disese resistnce [47, 48]. MiR414 minly trgets trnscriptionl regultors including MYB, ZIP fmily trnscription fctors, WRKY nd screcrow nd might hve key roles in plnt growth nd development [49]. As expected, mir414 ws up-regulted nd its trget gene: poly(a) polymerse 1 ws inhiited in Mg-deprived roots (Tle 2). This ws lso supported y our reports tht the undnces of trnscription fctor homolog BTF3-like protein (gi 33,945,882), spliceosome RNA helicse BAT1 (gi 226,528,292) nd RNA polymerse β chin (gi 90,403,817) were lowered in Mg-deficient C. sinensis roots [12]. We found tht mir1847 ws inhiited in Mg-deprived roots (Tle 2). This grees with the results otined on B-deficient roots [17] nd disgrees with the dt otined on B-deficient leves [18]. As expected, its trget genes: riosoml protein S3 fmily proteins were upregulted in these roots. In ddition, VALRS trgeted y mir6485 were induced or ws little ffected in Mgstrved roots (Tle 2). Thus, protein iosynthesis might not e lowered in Mg-strved roots, s shown y unchnged concentrtion of totl solule proteins in Mg-deprived C. sinensis roots [7, 12]. Also, the reduced dilution due to the decrese in root DW (Fig. 1) might ccount for the unchnged protein level. MiR5176 ws induced in Mg-deprived roots (Tle 2), s found on B-strved C. sinensis roots [17]. DNA mismtch repir (MMR) system is required for the correction of DNA iosynthetic errors [50]. MUTL-homologue 1 (MLH1) prticiptes in DNA MMR, correcting DNA dmge nd insertion-deletion loops rising from DNA repliction [51]. MLH1 trgeted y mir5176 were induced rther thn inhiited in Mg-deprived roots (Tle 2). Thus, MMR system might e up-regulted in these roots, thus enhncing Citrus Mg-deficiency tolernce vi correcting DNA iosynthetic errors. Similrly, DNA repir nd meiosis protein (Mre11) trgeted y mir5261 ws induced in Mg-deprived roots (Tle 2). Plnt leucine-rich repet receptor-like kinse proteins ply crucil roles in iotic stresses [52]. MiR5198 nd its trget gene: leucine-rich receptor-like protein kinse (LR-RLK) fmily protein were repressed nd induced in Mg-strved roots, respectively (Tle 2). Similrly, leucine-rich repet receptor-like protein kinse (ACN ) ws up-regulted in Mg-strved C.

6 Ling et l. BMC Genomics (2017) 18:657 Pge 6 of 16 Tle 2 qrt-pcr nlysis of predicted trget genes for selected Mg-deficiency-responsive known mirnas in C. sinensis roots mirna Fold chnge of mirna Accession Homology Trget genes Potentil roles Reltive chnge of trget genes mir ** ornge1.1g022993m AT5G SPFH/Bnd 7/PHB domin-contining memrne-ssocited protein fmily mir ** ornge1.1g037429m AT4G NB-ARC domin-contining disese resistnce protein Stress response 0.73** Disese resistnce protein mir ** ornge1.1g002089m AT3G Phosphoenolpyruvte croxylse 3 Crohydrte metolism mir ** ornge1.1g040557m AT1G Leucine-rich repet trnsmemrne protein kinse mir ** ornge1.1g010745m AT1G Puttive dipose-regultory protein (Seipin) Trnsmemrne signl trnsduction Tricylglycerol ccumultion nd LD prolifertion mir ** ornge1.1g000114m AT1G U5 smll nucler rionucleoprotein mrna processing 1.48 helicse, puttive mir ** ornge1.1g004767m AT1G Poly(A) polymerse 1 mrna processing 0.76 ornge1.1g006232m AT1G Poly(A) polymerse 1 mrna processing 0.72** mir ** ornge1.1g003146m AT1G Senescence-ssocited E3 uiquitin ligse 1 Ul conjugtion pthwy mir ** ornge1.1g009434m AT5G Peroxin 14 Protein import into peroxisome mtrix, docking ornge1.1g009573m AT5G Peroxin 14 Protein import into peroxisome mtrix, docking ornge1.1g018459m AT3G ATPse, V0/A0 complex, suunit C/D mir ** ornge1.1g005896m AT3G NB-ARC domin-contining disese resistnce protein ornge1.1g030696m AT5G DnJ/Hsp40 cysteine-rich domin superfmily protein ATP hydrolysis coupled proton trnsport Disese resistnce protein ND 0.53** 0.65** ** 2.36* ** Stress response 1.56** mir ** ornge1.1g026316m AT5G Riosoml protein S3 fmily protein Trnsltion 2.76** ornge1.1g026835m AT5G Riosoml protein S3 fmily protein Trnsltion 1.84* ornge1.1g029201m AT2G Riosoml protein S3 fmily protein Trnsltion 1.91 mir ** ornge1.1g005923m AT2G LysM-contining receptor-like kinse 5 Trnsmemrne 0.67** signl trnsduction ornge1.1g034040m AT5G Uiquitin-conjugting enzyme 18 Protein uiquitintion ND ornge1.1g021729m AT4G Bsic helix-loop-helix (HLH) Trnscription fctor ND DNA-inding superfmily protein ornge1.1g026539m AT1G Enhncer of polycom-like Trnscription regultion ND trnscription fctor protein ornge1.1g045123m AT4G RNA polymerse II lrge suunit mrna synthesis ND ornge1.1g003175m AT4G Origin recognition complex 1 DNA synthesis 0.57** nd repliction ornge1.1g006076m AT3G Tetrtricopeptide repet 6.01** (TPR)-like superfmily protein ornge1.1g029970m AT3G LOB domin-contining protein ** ornge1.1g028357m AT2G AT-hook motif nucler-loclized Trnscription fctor 6.12** protein 9 (AHL9) mir ** ornge1.1g005789m AT4G MUTL-homologue 1 DNA mismtch repir 3.21** ornge1.1g008397m AT4G MUTL-homologue 1 DNA mismtch repir 2.87** ornge1.1g010846m AT4G MUTL-homologue 1 DNA mismtch repir 3.13** ornge1.1g012406m AT4G MUTL-homologue 1 DNA mismtch repir 5.39*

7 Ling et l. BMC Genomics (2017) 18:657 Pge 7 of 16 Tle 2 qrt-pcr nlysis of predicted trget genes for selected Mg-deficiency-responsive known mirnas in C. sinensis roots (Continued) mir ** ornge1.1g005267m AT1G Receptor like protein 12 Hormone-medited 0.66** signling pthwy ornge1.1g005542m AT1G Receptor like protein 12 Hormone-medited 0.77** signling pthwy ornge1.1g008628m AT1G Receptor like protein 12 Hormone-medited signling 1.76** pthwy ornge1.1g002167m AT5G Receptor like protein 53 Hormone-medited signling 5.01** pthwy ornge1.1g012980m AT5G O-cyltrnsferse (WSD1-like) fmily protein Lipid nd ftty-cid 0.86 metolism ornge1.1g013532m AT5G O-cyltrnsferse (WSD1-like) fmily protein Lipid nd ftty-cid 0.72** metolism ornge1.1g027358m AT5G Phosphtidic cid phosphtse (PAP2) Dephosphoryltion 0.53** fmily protein ornge1.1g027353m AT5G Phosphtidic cid phosphtse (PAP2) Dephosphoryltion 0.56** fmily protein mir ** ornge1.1g029300m AT5G Ortholog of humn splicing fctor SC35 SR protein 2.65** ornge1.1g017284m AT5G Purple cid phosphtse 26 Phosphte ion homeostsis 1.82** ornge1.1g002842m AT4G Sec23/Sec24 protein trnsport fmily protein mir ** ornge1.1g016909m AT5G Thimin diphosphte-inding fold (THDP-inding) superfmily protein ornge1.1g023827m AT5G Thimin diphosphte-inding fold (THDP-inding) superfmily protein Intrcellulr protein trnsport Lipid nd ftty-cid metolism Lipid nd ftty-cid metolism ornge1.1g001557m AT5G Sucrose phosphte synthse 1F C-compound nd 3.51** crohydrte metolism ornge1.1g002665m AT5G Sucrose phosphte synthse 1F C-compound nd 3.93** crohydrte metolism mir ** ornge1.1g001969m AT5G Trnscriptionl fctor B3 fmily protein / Trnscription fctor 1.41* uxin-responsive fctor AUX/IAA-relted ornge1.1g011274m AT3G Plnt protein of unknown function (DUF828) 1.68** with plnt pleckstrin homology-like region ornge1.1g031218m AT1G HSP20-like chperones superfmily protein Stress response 0.88 ornge1.1g009779m AT1G Ction exchnger 11 Trnsport 0.57** ornge1.1g029454m AT5G Ankyrin repet fmily protein 0.20** ornge1.1g013633m AT1G SnRNA ctivting complex fmily protein Auxin signling pthwy 2.27** ornge1.1g017698m AT1G SnRNA ctivting complex fmily protein Auxin signling pthwy 1.33** ornge1.1g042988m AT5G Nodulin MtN3 fmily protein Trnsport 0.59** ornge1.1g007868m AT1G My fmily trnscription fctor TRFL6 Trnscription fctor 4.39** ornge1.1g046667m AT2G Phosphte trnsporter 1;4 Phosphte trnsport 0.91 ornge1.1g001289m AT1G Vlyl-tRNA synthetse / vline-trna Protein iosynthesis 1.75** ligse (VALRS) ornge1.1g001303m AT1G Vlyl-tRNA synthetse / vline-trna Protein iosynthesis 1.99** ligse (VALRS) ornge1.1g001757m AT1G Vlyl-tRNA synthetse / vline-trna Protein iosynthesis 1.11 ligse (VALRS) ornge1.1g024117m AT2G Kinse intercting (KIP1-like) fmily protein 0.21** ornge1.1g036588m AT4G Leucine-rich repet trnsmemrne protein kinse Trnsmemrne signl trnsduction ornge1.1g003591m AT5G Nucler pore complex protein NUP88 mrna trnsport, protein trnsport 1.64** 3.23** 2.56** 4.54* 1.59*

8 Ling et l. BMC Genomics (2017) 18:657 Pge 8 of 16 Tle 2 qrt-pcr nlysis of predicted trget genes for selected Mg-deficiency-responsive known mirnas in C. sinensis roots (Continued) mir ** ornge1.1g001378m AT1G NF-X-like 1 Protein uiquitintion 2.00** ornge1.1g001376m AT1G NF-X-like 1 Protein uiquitintion 2.61* ornge1.1g047796m AT2G Peroxidse superfmily protein Stress response 1.97** ornge1.1g042193m AT5G ATPse, AAA-type, CDC48 protein Cell cycle, cell division, protein trnsport, trnsport 4.02* ornge1.1g019546m AT2G Integrse-type DNA-inding superfmily protein mir ** ornge1.1g002063m AT1G Leucine-rich receptor-like protein kinse fmily protein Ascisic cid signling pthwy Trnsmemrne signl trnsduction mir ** ornge1.1g012168m AT5G OBP3-responsive gene ** ornge1.1g026587m AT4G Protesome suunit et type-6 (PBA1) Protein uiquitintion 2.26** ornge1.1g029964m AT4G Protesome suunit et type-6 (PBA1) Protein uiquitintion 2.21** ornge1.1g030788m AT4G Protesome suunit et type-6 (PBA1) Protein uiquitintion 2.91** ornge1.1g014625m AT3G Cyclopropne-ftty-cyl-phospholipid synthse Lipid nd ftty-cid metolism ornge1.1g018123m AT3G Outer memrne OMP85 fmily protein Trnsmemrne trnsport 1.43* mir ** ornge1.1g018132m AT3G DHHC-type zinc finger fmily protein 1.94** ornge1.1g010695m AT3G RNA inding (RRM/RBD/RNP motifs) mrna processing 2.40** fmily protein ornge1.1g011967m AT3G RNA inding (RRM/RBD/RNP motifs) mrna processing 2.80** fmily protein ornge1.1g031636m AT1G Lojp-relted protein 2.41** ornge1.1g033883m AT1G Lojp-relted protein 2.03** ornge1.1g004959m AT5G Mitogen-ctivted protein kinse kinse Intrcellulr signlling 1.97* kinse 5 ornge1.1g043928m AT2G Polynucleotidyl trnsferse, rionuclese 3-5 exonuclese ctivity 3.32** H-like superfmily protein ornge1.1g037980m AT2G Polynucleotidyl trnsferse, rionuclese 3-5 exonuclese ctivity 2.17** H-like superfmily protein ornge1.1g004713m AT5G DNA repir nd meiosis protein (Mre11) DNA dmge, 4.47** DNA repir, meiosis ornge1.1g010785m AT3G Protein phosphtse 2A regultory Intrcellulr signlling 2.01* B suunit fmily protein ornge1.1g000012m AT1G Uiquitin-protein ligse 1 Protein uiquitintion 2.05** (E3 uiquitin-protein ligse UPL1) ornge1.1g000013m AT1G Uiquitin-protein ligse 1 Protein uiquitintion 2.82** (E3 uiquitin-protein ligse UPL1) ornge1.1g029528m AT5G RING/U-ox superfmily protein Protein uiquitintion 4.05* ornge1.1g029508m AT1G UDP-glucosyl trnsferse 85A2 Flvonoid iosynthetic process 1.99** mir ** ornge1.1g000163m AT1G RNA polymerse II trnscription meditors 2.29 mir ** ornge1.1g018149m AT5G F-ox fmily protein Protein uiquitintion 1.82* ornge1.1g018125m AT5G F-ox fmily protein Protein uiquitintion 2.69* ornge1.1g023739m AT2G Remorin fmily protein 4.00** ornge1.1g027436m AT2G Remorin fmily protein 2.11** ornge1.1g023033m AT2G oxoglutrte (2OG) nd Fe(II)-dependent oxygense superfmily protein Oxidoreductse 2.03* 0.60** 1.84** 2.44** ornge1.1g026453m AT1G Senescence-relted gene 1 Oxidoreductse 2.31** ornge1.1g020233m AT2G oxoglutrte (2OG) nd Fe(II)-dependent oxygense superfmily protein 3.73* ornge1.1g037473m AT5G KAR-UP oxidoreductse 1 Oxidoreductse 1.92

9 Ling et l. BMC Genomics (2017) 18:657 Pge 9 of 16 Tle 2 qrt-pcr nlysis of predicted trget genes for selected Mg-deficiency-responsive known mirnas in C. sinensis roots (Continued) mir ** ornge1.1g010903m AT5G WRKY DNA-inding protein 72 Trnscription fctor 1.65** mir ** ornge1.1g023136m AT1G Nodulin MtN21 /EmA-like 2.14** trnsporter fmily protein mir ** ornge1.1g044623m AT5G Phytoene synthse Crotenoid iosynthesis 2.15* ornge1.1g030826m AT2G Phospholipse A 2A Lipid degrdtion ND ornge1.1g004573m AT4G NB-ARC domin-contining Disese resistnce 1.58** disese resistnce protein mir ** ornge1.1g005482m AT4G Auxin response fctor 16 Auxin signling pthwy 4.54* ornge1.1g004896m AT2G Auxin response fctor 10 Auxin signling pthwy 22.94** ornge1.1g005075m AT4G Auxin response fctor 16 Auxin signling pthwy 4.01** ornge1.1g008078m AT1G Auxin response fctor 17 Auxin signling pthwy 2.90* The reltive chnges of trget genes re the rtio of Mg-deficient to -sufficient roots. The vlue for reltive chnge of trget gene ws men of three iologicl replictes with two technicl replictes; Trget genes tht hd the expected chnges in mrna levels were mrked in old; * nd ** indicte significnt difference t P <0.05ndP < 0.01, respectively. ND, not detected reticult roots [13]. Thus, mir5198 might e involved in Citrus Mg-deficiency responses. Root mir780 ws repressed y Mg-deficiency (Tle 2), s found on B-strved C. sinensis roots [17]. As expected, its trgets: NB-ARC domin-contining disese resistnce protein nd phytoene synthse (PSY) were up-regulted in Mg-strved roots (Tle 2). Vrious trnsgenic plnts over-expressing cteril or plnt gene encoding PSY, mjor rte-limiting crotenoid (Cr) enzyme, displyed incresed Cr level [53, 54]. Therefore, Cr iosynthesis might e enhnced in Mg-strved C. sinensis roots, thus incresing their ntioxidnt ility. We oserved tht mir7121 nd its trget gene: phosphtidic cid phosphtse (PAP2) fmily protein ws up- nd down-regulted in Mg-strved roots, respectively (Tle 2). Nkno et l. [55] demonstrted tht the inhiition of PAP2 expression or function conferred resistnce to Rlstoni solncerum vi rpidly triggering plnt defenses in Nicotin enthmin. Thus, the down-regultion of PAP2 fmily protein might contriute to plnt diseseresistnce. As shown in Tle 2, mir6190 ws down-regulted nd its trget genes [i.e., purple cid phosphtse 26 (PPAP26), Sec23/Sec24 protein trnsport fmily protein nd ortholog of humn splicing fctor SC35, lso known s serine/rginine-rich (SR) splicing fctor SC35] were up-regulted in Mg-deficient roots. SR proteins re required for regulting lterntive splicing. In higher plnts, gret ltertions in lterntive splicing due to vrious iotic stresses demonstrte the roles of SR proteins in the dpttion to environmentl stress [56]. Induction of cid phosphtses (APses) y P-strvtion is well-documented mechnism of plnt P-deficiency tolernce. Hurley et l. [57] demonstrted tht AtP- PAP26 ws the mjor contriutor to P-deficiencyinducile APse ctivity. In ddition, AtPPAP26 lso showed lkline peroxidse (POD) ctivity. Mg-deficiencyinduced up-regultion of root PPAP26 (Tle 2) grees with the report tht AtPPAP26 ws induced in P-deficient Aridopsis roots, shoots nd suspension cells [58] ecuse C. sinensis lef, stem nd root P levels were reduced y Mg-deficiency [46]. Cot protein complex II (COPII) vesicles ply n essentil role for the export of secretory crgo from the endoplsmic reticulum (ER) to the Golgi complex in ll eukryotes [59]. Mg-deficiency-induced upregultion of root gene encoding Sec23/Sec24 protein trnsport fmily protein (Tle 2), suset of the COPII components, grees with our report tht the undnce of Sec23/Sec24 protein trnsport fmily protein ws elevted in B-deficient roots [60]. In Aridopsis, mirna160 negtively regultes the repressor uxin response fctor (ARF) fmily: ARF17 [61], ARF16 [62] nd ARF10 [63]. The repression of these genes y mir160 is required for seed germintion nd the norml development of roots, stems nd leves. Li et l. [64] demonstrted tht soyen mir160 negtively regulted the progress of lef senescence vi repressing its trgets: ARF10, ARF16 nd ARF17. We found tht mir160 ws induced in Mg-deficient roots (Tle 2), s otined on P-strved Lupinus lus roots [65] nd N-deficient mize roots [66]. Therefore, the induction of root mir160 y Mg-deprivtion might e n dptive response. Unexpectedly, its trgets: ARF10, ARF16 nd ARF17 were up-regulted in Mg-deprived C. sinensis roots (Tle 2). Endogenous trget mimics (etms) cn impede the interction etween mirnas nd their uthentic trgets vi inding to mirnas [67, 68]. Lin et l. [69] demonstrted tht etms repressed mir160-medited clevge of ARF10, ARF16 nd ARF17 during longn somtic emryogenesis. No negtive correltions were oserved mong the levels of mir160 nd ARF10, ARF16 nd ARF17 trnscripts in longn vegettive nd genertive tissues. Thus, the correltions

10 Ling et l. BMC Genomics (2017) 18:657 Pge 10 of 16 A Known Novel 25.3 (%) Percentge B (%) Percentge C (%) Percentge Response to stress Trnsport Protein metolic process Regultion of trnscription Developmentl process Nucleic cid metolic process Signl trnsduction Oxidtion reduction Crohydrte metolic process Cellulr process Lipid metolic process Other metolic process Nucleic cid inding Kinse ctivity Metl ion inding Trnscription fctor ctivity trnsferse ctivity Protein inding Trnsporter ctivity Nuclese ctivity Ligse ctivity Oxidoreductse inding ATPse ctivity Other inding Other ctivity Nucleus Memrne Chloroplst Cytoplsm Complex Plsmodesm Cytosol Cell wll Vcuole Extrcellulr region Golgi pprtus Others Fig. 4 GO ctegories of the predicted trget genes for 46 (15) Mg-deficiency-responsive known (novel) mirnas in Citrus sinensis roots. MiRNAs trget genes were grouped sed on iologicl process (), moleculr function () nd cellulr component (c) etween mir160 nd its trgets in C. sinensis roots cn e explined in this wy. Root mir6485 ws repressed nd its severl trget genes were up-regulted y Mg-deprivtion (Tle 2). Li et l. [70] reported tht ARF7 (AT5G20730) is necessry for oth uxin signling nd ethylene responses in Aridopsis roots. Okushim et l. [71] oserved tht lterl root formtion ws dly dmged in Aridopsis rf7 rf19 doule knockout mutnt, concluding tht ARFs directly ctivted LATERAL ORGAN BOUNDAR- IES DOMAIN/ASYMMETRIC LEAVES2-LIKE (LBD/ ASL) genes, thus regulting lterl root formtion. Thus, the induction of root trnscriptionl fctor B3 fmily protein/uxin-responsive fctor AUX/IAA-relted y Mgdeficiency might ply prt in Mg-deficiency tolernce vi mintining lterl root formtion. Similrly, SnRNA ctivting complex fmily protein (SDR2), which is ssocited with uxin-ctivted signling pthwy, ws induced in Mg-deprived roots (Tle 2). Ohtni et l. [72] reported tht srd2 muttion repressed the expression of PIN-FORMED proteins, which might ccount for the filure to generte n uxin grdient, thus leding

11 Ling et l. BMC Genomics (2017) 18:657 Pge 11 of 16 Tle 3 List of Mg-deficiency-responsive known mirnas shred y oth C. sinensis roots nd leves MiRNA Fold chnge Leves Roots mir ** ** mir ** ** mir ** ** mir ** ** mir ** ** mir ** ** mir ** ** mir ** ** mir ** ** mir ** ** mir ** ** mir ** ** mir ** ** mir ** ** mir ** ** mir ** ** mir ** ** mir ** ** mir ** ** mir ** ** mir ** ** mir ** ** mir ** ** mir ** ** mir ** ** mir ** ** mir ** ** mir ** ** mir ** ** mir ** ** Dt from Additionl file 3 nd M et l. [36]; **indictes significnt difference t P < 0.01 to different normlities in root morphogenesis in Aridopsis mutnt. Nucler pore complex protein NUP88 is necessry for systemic cquired resistnce nd R protein-medited defense [73]. The induction of root NUP88 y Mg-deficiency (Tle 2) grees with the ove inference tht tht disese-resistnce ws improved in Mg-deficient roots. Root mir1044 ws repressed nd its trget genes [i.e., NF-X-like 1 (NFXL1), POD superfmily protein nd ATPse, AAA-type, CDC48 protein] were induced y Mg-deprivtion except for integrse-type DNA-inding superfmily protein (Tle 2). Lisso et l. [74] oserved tht AtNFXL1 ws induced in roots under slt nd osmotic stress, nd tht oth AtNFXL1-ntisense plnts nd tnfxl1 1 knock-out mutnts hd lower growth nd survivl rtes thn wild-type plnts when exposed to slt or osmotic stress. CDC48, memer of AAA-ATPse fmily proteins tht provides energy for plnt development vi regulting ATPse, is required for plnt cell division, expnsion nd differentition [75]. Wng et l. [76] suggested tht the induction of PpCDC48II y low temperture plyed key role in cold-induced freezing tolernce of Physcomitrell ptens cells. MiR5261 nd its trget genes were repressed nd induced in Mg-strved roots, respectively (Tle 2). The induction of root RNA inding (RRM/RBD/RNP motifs) fmily protein y Mg-deficiency grees with our report tht the undnce of RNA inding (RRM/RBD/RNP motifs) fmily protein ws elevted in B-deficient roots [60]. A typicl mitogen-ctivted protein kinse (MAPK) cscde is composed of three sequentilly ctivted protein kinses, nmely MAPK, MAPK kinse (MAPKK) nd MAPKK kinse (MAPKKK). Stress-tolernce of some plnts such s Aridopsis, tocco nd cerels hs een enhnced y geneticlly ltering the undnces nd/or the ctivities of some MAPK components [77, 78]. The induction of root protein phosphtse 2A (PP2A) regultory B suunit fmily protein y Mgdeficiency (Tle 2) grees with the report tht whet root PP2AB"-α ws up-regulted when exposed to vrious iotic stresses. Trnsgenic whet lines overexpressing TPP2AB"-α displyed etter lterl root development, especilly under NCl nd mnnitol stresses [79]. Uiquitintion-protesoml pthwy hs een shown to function in plnt senescence nd in stress response y fcilitting the degrdtion of ulk proteins for N recycling [80]. Trnsgenic tocco lines over-expressing mize gene encoding E3 uiquitin ligse (UPL) displyed incresed drought tolernce ccompnied y higher ctivities of superoxide dismutse (SOD) nd ctlse, more ccumultion of proline nd less ccumultion of mlondildehyde (MDA) nd ROS when exposed to drought stress [81]. Over-expression of TFBA1 encoding F-ox protein conferred drought nd oxidtive stress tolernce in tocco plnts vi up-regulting the ctivities of SOD, ctlse, scorte peroxidse (APX) nd POD, nd lowering the levels of ROS nd MDA [82, 83]. Thus, up-regultion of UPL1 nd RING/U-ox superfmily protein trgeted y mir5261, NFXL1 trgeted y mir1044, F-ox fmily protein trgeted y mir1151 nd protesome suunit et type-6 (PBA1) trgeted y mir5029 in Mg-strved roots (Tle 2) might confer stress-tolernce, thus contriuting to Mg-deficiency tolernce in Citrus plnts. Similrly, the expression levels of UPL5 (XP ) nd F-ox fmily protein (XP

12 Ling et l. BMC Genomics (2017) 18:657 Pge 12 of ) in C. reticult roots [13] nd the undnces of puttive protesome suunit lph type (gi 255,584,432) in C. sinensis roots [12] were elevted y Mg-deficiency. Conclusions For the first time, we used Illumin sequencing to identify 71 known nd 30 novel mirnas with incresed expressed, nd 54 known nd 15 novel mirnas with decresed expression in Mg-deficient C. sinensis roots, demonstrting tht mirnas might e involved in Citrus Mg-deficiency tolernce. Through integrting our findings with the previous dt, we put forwrd potentil scheme for the responses of mirnas to Mgdeficiency in Citrus roots (Fig. 5). Here, we otined severl novel Mg-deficiency-responsive mirnas (i.e., mir5261, mir158, mir6190, mir6485, mir1151 nd mir1044) possily responsile for Citrus Mg-deficiency tolernce. Our findings results not only incresed our knowledge on the functions of plnt mirnas under nutrient deficiencies, ut lso estlished foundtion to improve Mg-deficiency tolernce vi mnipulting the ctions of mirnas. Methods Citrus sinensis Seedling culture nd long-term Mgdeficient tretments Seedling culture nd long-term Mg-deficient tretments were crried out s descried previously [12]. In short, 15-week-old Xuegn [Citrus sinensis (L.) Oseck] seedlings, which were grown in 6 L pots (two seedlings per pot) filled with clen river snd in greenhouse under nturl photoperiod t Fujin Agriculture nd Forestry University, Fuzhou, were supplied every other dy until dripping with nutrient solution t Mg concentrtion of 0 mm (Mg-deficiency) or 1 mm (Mg-sufficiency, control) from MgSO 4. S t the nutrient solution ws kept t constnt level y dding equivlent moles of N 2 SO 4 in replce of MgSO 4. After 16 weeks, ~ 5-mm-long root pices from new white firous roots were hrvested nd immeditely frozen in liquid N 2,thenstoredt 80 C until extrction. The seedlings not eing smpled were used for the mesurements of root DW, lef nd root Mg. Root DW nd root nd lef Mg For ech tretment, roots from nine seedlings (one seedling per pot) were tken. Root DW ws mesured fter eing dried t 70 C to constnt weight (~ 48 h). Firous roots nd ~7-week-old leves (midris nd petioles removed) were hrvested nd then dried t 70 C to constnt weight. Dried roots nd leves were ground to pss 40 mesh sieve, finlly digested with 1 N HCl [84]. Mg concentrtion in the solution ws mesured y tomic sorption spectroscopy. Root srnas lirry construction, high-throughput sequencing, nnottion nd mirna identifiction Equl mounts of frozen root pices from five seedlings (one seedling per pot) were pooled s iologicl replicte. There ws one iologicl replicte for ech tretment. Approximtely 0.1 g mixed frozen Mg-deficient or control root pices were used to extrct totl RNA with TRIzol regent (Invitrogen, Crlsd, CA). Construction of srna lirries ws performed s descried y Lu et l. [17]. Illumin sequencing ws crried out with Solex sequencer t the Beijing Genomics Institute (BGI), Shenzhen, Chin. Both srna nnottion nd mirna identifiction were mde s descried previously [17, 18]. After rw dt eing nlyzed with softwre developed y BGI, clen Fig. 5 A potentil scheme for responses of C. sinensis roots mirnas to Mg-deficiency. LRRTPK: Leucine-rich repet trnsmemrne protein kinse; LYK5: LysM-contining receptor-like kinse 5; PEX14: Peroxin 14; RBFP: RNA inding (RRM/RBD/RNP motifs) fmily protein; RLP: Receptor like protein; RPS3: Riosoml protein S3 fmily protein; WRKY72: WRKY DNA-inding protein 72

13 Ling et l. BMC Genomics (2017) 18:657 Pge 13 of 16 reds were then utilized to ssy length distriution. Finlly, the cler reds were mpped to C. sinensis genome (JGIversion 1.1, portl.html#!info?lis=org_csinensis) using SOAP, only perfectly mpped sequences were retined nd nlyzed further. rrnas, trnas, snrnas nd snornas were removed from the srnas sequences through BLASTn serch using NCBI Genenk dtse ( nd Rfm (12.0) dtse ( rfm.html) (e = 0.01). The remining sequences were ligned with known plnt mirnas from mirbse 21 ( Only the perfectly mtched sequences were considered to e conserved mirnas. Reds not eing nnotted were used for the prediction of novel mirnas using Mirep ( projects/mirep/), softwre developed y BGI. Also, oth DNAMAN 8 ( nd MTide ( [85] were used for the prediction of novel mirnas. Only these mirna cndidtes eing simultneously predicted y the three softwres were regrded to e novel mirnas. Differentilly expressed mirnas nd trget prediction Both the fold chnge etween Mg-deficient nd -sufficient roots nd the P-vlue were clculted from the normlized expression of TPM [86]. A mirna ws regrded to e differentilly expressed when it hd oth P-vlue <0.01 nd log2-fold chnge >1.5 [17]. Trget prediction of mirnas ws crried out y RNAhyrid ccording to the rules proposed y Schw et l. [87] nd Allen et l. [88]. Ctegories of the potentil trgets predicted from Mgdeficiency-responsive mirnas All trget genes predicted from Mg-deficiency-responsive mirnas were mpped to GO terms in the dtse ( nd gene numers for ech term ws clculted. All these trgets were grouped into three ctegories: iologicl process, moleculr function, cellulr component [17]. Vlidtion of Mg-deficiency-responsive mirnas y stemloop qrt-pcr nd of trget genes y qrt-pcr Stem-loop qrt-pcr nlysis of mirnas ws crried out s descried previously [18]. Stem-loop primers for reverse trnscription nd primers for qrt-pcr were summrized in Additionl file 9. qrt-pcr nlysis of trget genes ws crried out with n ABI 7500 Rel Time System s descried y Lu et l. [17]. The sequences of the F nd R primers used were given in Additionl file 10. Equl mounts of frozen root pices from five seedlings (one seedling per pot) were pooled s iologicl replicte. For ech tretment, there were three iologicl replictes nd two technicl replictes. Reltive mirna expression ws clculted using ddct lgorithm. Actin (AEK ) ws used s n internl stndrd nd the roots from Mg-sufficient seedlings were used s reference smple, which ws set to 1. Experimentl design nd dt nlysis For ech tretment, there were 20 pot seedlings in completely rndomized design. Experiments were crried out with 3 replictes except for high-throughput sequencing (n = 1), root nd lef Mg (n =5),ndrootDW(n =9). Unpired t-test ws performed for the significnt test etween two mens (Mg-sufficiency nd -deficiency). Additionl files Additionl file 1: List of known mirnas in C. sinensis roots. (DOC 995 k) Additionl file 2: List of known mirnas in C. sinensis roots fter removing these mirnas with normlized red-count less thn 10 TPM in the two mirna lirries constructed from Mg-sufficient nd -deficient roots. (DOC 447 k) Additionl file 3: List of Mg-deficiency-responsive known mirnas in C. sinensis roots. (DOC 205 k) Additionl file 4: List of novel mirnas in C. sinensis roots. (DOC 288 k) Additionl file 5: List of novel mirnas in C. sinensis roots fter removing these mirnas with normlized red-count less thn 10 TPM in the two mirna lirries constructed from Mg-sufficient nd -deficient roots. (DOC 160 k) Additionl file 6: List of Mg-deficiency-responsive novel mirnas in C. sinensis roots. (DOC 105 k) Additionl file 7: List of trget genes for prts of known mirnas in C. sinensis roots. (DOC 262 k) Additionl file 8: List of trget genes for prts of novel mirnas in C. sinensis roots. (DOCX 25 k) Additionl file 9: Stem loop primer sequences for qrt-pcr nlysis of mirnas. (DOCX 17 k) Additionl file 10: Specific primer pirs used for qrt-pcr expression nlysis of selected mirna trget genes. (DOCX 31 k) Arevitions AHL9: AT-hook motif nucler-loclized protein 9; APse: Acid phosphtse; APX: Ascorte peroxidse; ARF: Auxin responsive fctor; Cr: Crotenoid; CDC48: A memer of AAA-ATPse fmily proteins; COPII: Cot protein complex II; ER: endoplsmic reticulum; etms: Endogenous trget mimics; LR-RLK: Leucine-rich receptor-like protein kinse; MAPK: Mitogen-ctivted protein kinse; MAPKK: MAPK kinse; MAPKKK: MAPKK kinse; MDA: Mlondildehyde; Mg: Mgnesium; MLH1: MUTL-homologue 1; MMR: DNA mismtch repir; NUP88: Nucler pore complex protein; NFXL1: NF-X-like 1; NLA: Nitrogen limittion dpttion; PAP: Phosphtidic cid phosphtse; PBA1: Protesome suunit et type-6; PEPC: Phosphoenolpyruvte croxylse; PHB: Prohiitin; PLA2A: Phospholipse A 2A; POD: Peroxidse; PP2A: Protein phosphtse 2A; PPAP: Purple cid phosphtse; PSY: Phytoene synthse; ROS: Rective oxygen species; SC35: Ortholog of humn splicing fctor; SDR2: SnRNA ctivting complex fmily protein; SLP1: Stomtin-like protein 1; SOD: Superoxide dismutse; SPFH: Stomtins, prohiitins, flotillins nd HflK/C; TPM: Trnscript per million; UCB24: Uiquitin-conjugting enzyme E2 24; UPL1: Uiquitin-protein ligse 1 (E3 uiquitin-protein ligse UPL1); VALRS: Vlyl-tRNA synthetse / vline-trna ligse.