Boron-deficiency-responsive micrornas and their targets in Citrus sinensis leaves

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1 Lu et l. BMC Plnt Biology (2015) 15:271 DOI /s y RESEARCH ARTICLE Open Access Boron-deficiency-responsive micrornas nd their trgets in Citrus sinensis leves Yi-Bin Lu 1,2, Yi-Ping Qi 3, Lin-Tong Yng 1,2, Peng Guo 1,2, Yn Li 1 nd Li-Song Chen 1,2,4,5* Astrct Bckground: MicroRNAs ply importnt roles in the dptive responses of plnts to nutrient deficiencies. Most reserch, however, hs focused on nitrogen (N), phosphorus (P), sulfur (S), copper (Cu) nd iron (Fe) deficiencies, limited dt re ville on the differentil expression of mirnas nd their trget genes in response to deficiencies of other nutrient elements. In this study, we identified the known nd novel mirnas s well s the oron (B)-deficiency-responsive mirnas from citrus leves in order to otin the potentil mirnas relted to the tolernce of citrus to B-deficiency. Methods: Seedlings of Xuegn [Citrus sinensis (L.) Oseck] were supplied every other dy with B-deficient (0 μm H 3 BO 3 ) or -sufficient (10 μm H 3 BO 3 ) nutrient solution for 15 weeks. Therefter, we sequenced two smll RNA lirries from B-deficient nd -sufficient (control) citrus leves, respectively, using Illumin sequencing. Results: Ninety one (83 known nd 8 novel) up- nd 81 (75 known nd 6 novel) down-regulted mirnas were isolted from B-deficient leves. The gret ltertion of mirna expression might contriute to the tolernce of citrus to B-deficiency. The dptive responses of mirnas to B-deficiency might relted to severl spects: () ttenution of plnt growth nd development y repressing uxin signling due to decresed TIR1 level nd ARF-medited gene expression y ltering the expression of mir393, mir160 nd mir3946; () mintining lef phenotype nd enhncing the stress tolernce y up-regulting NACs trgeted y mir159, mir782, mir3946 nd mir7539; (c) ctivtion of the stress responses nd ntioxidnt system through down-regulting the expression of mir164, mir6260, mir5929, mir6214, mir3946 nd mir3446; (d) decresing the expression of mjor fcilittor superfmily protein genes trgeted y mir5037, thus lowering B export from plnts. Also, B-deficiency-induced down-regultion of mir408 might ply role in plnt tolernce to B-deficiency y regulting Cu homeostsis nd enhncing superoxide dismutse ctivity. Conclusions: Our study revels some novel responses of citrus to B-deficiency, which increse our understnding of the dptive mechnisms of citrus to B-deficiency t the mirna (post-trnscriptionl) level. Keywords: Boron-deficiency, Citrus sinensis, Illumin sequencing, Leves, MicroRNA Bckground Boron (B), n essentil micronutrient for norml growth nd development of plnts, is involved in series of importnt physiologicl functions, including the structure of cell wlls, memrne integrity, cell division, phenol metolism, protein metolism nd nucleic cid metolism during growth nd development of higher plnts [1 5]. B-deficiency widespredly exists in mny * Correspondence: lisongchen2002@hotmil.com 1 College of Resource nd Environmentl Science, Fujin Agriculture nd Forestry University, Fuzhou , Chin 2 Institute of Horticulturl Plnt Physiology, Biochemistry nd Moleculr Biology, Fujin Agriculture nd Forestry University, Fuzhou , Chin Full list of uthor informtion is ville t the end of the rticle griculturl crops, including citrus. In Chin, B-deficiency is frequently oserved in citrus orchrds, nd often contriutes to the loss of productivity nd poor fruit qulity [3]. Li et l. reported tht up to 9.0 % nd 43.5 % of Gunximiyou pummelo (Citrus grndis) orchrds in Pinghe, Zhngzhou, Chin were deficient in lef B nd soil wter-solule B, respectively [6]. In plnts, pprox. 21-nucleotide-long micrornas (mirnas), one of the most undnt clsses of non-coding smllrnas(srnas),recrucilpost-trnscriptionlregultors of gene expression y repressing trnsltion or directly degrding mrnas in plnts [7]. Evidence shows tht mirnas ply key roles in plnt response to nutrient 2015 Lu et l. 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 ( cretivecommons.org/pulicdomin/zero/1.0/) pplies to the dt mde ville in this rticle, unless otherwise stted.

2 Lu et l. BMC Plnt Biology (2015) 15:271 Pge 2 of 15 deficiencies [8 13]. Identifiction of nutrient-deficiencyresponsive-mirnas nd their trget genes hs ecome one of the hottest topics in plnt nutrition. Plnts hve developed diverse strtegies to mintin phosphorus (P) homeostsis, including mirna regultions [11, 12]. MiR399, which is specificlly induced y P- deficiency in Aridopsis nd rice, cn regulte P homeostsis y negtively regulting its trget gene UBC24 [13, 14]. Like mir399, mir827 is lso highly nd specificlly induced y P-deficiency nd is involved in the regultion of plnt P homeostsis y downregulting its trget gene nitrogen limittion dpttion (NLA) inaridopsis [13]. In ddition, mny other P- deficiency-responsive mirnas (i.e., mir1510, mir156, mir159, mir166, mir169, mir2109, mir395, mir397, mir398, mir408, mir447 nd mir482) hve een isolted from vrious plnt species [15 21]. MiR397, mir398, mir408, nd mir857, which re induced y copper (Cu)-deficiency, hve een shown to ply role in the regultion of Cu homeostsis y downregulting genes encoding nonessentil Cu proteins such s Cu/Zn superoxide dismutse (SOD), lccses nd plntcynin, hence sving Cu for other essentil Cu proteins such s plstocynin, which is essentil for photosynthesis [10, 22, 23]. In Aridopsis, lef mir395 ws induced y sulfur (S)- deficiency. MiR395 trgets ATP sulfurylses (APS) nd sulfte trnsporter 2;1 (SULTR2;1), oth of which re involved in the S metolism. Their trnscripts re gretly down-regulted in mir395-over-expressing trnsgenic Aridopsis ccompnied y incresed ccumultion of S in the shoot ut not in the root. They concluded tht mir395 ply role in the regultion of plnt S ccumultion nd lloction y trgeting APS nd SULTR2;1 [24]. MiRNAs hve een shown to ply role in the dpttion of plnts to Fe-deficiency. Eight Fe-deficiencyresponsive conserved mirnas from five fmilies hd een identified in Aridopsis roots nd shoots nd their expression profiles differed etween the two orgns [25]. Vldés- López et l. isolted ten up- nd four down-regulted mirnas, five up- nd six down-regulted mirnas, nd seven up- nd four down-regulted mirnas from the leves, roots nd nodules of Fe-deficient common en [17]. Wters et l. otined eight differentilly expressed mirnas from seven conserved fmilies in the rosettes of Fe-deficient Aridopsis. Interestingly, Fe-deficiency led to incresed ccumultion of Cu in rosettes nd decresed expression levels of mir397, mir398 nd mir398/c, which regulte the mrna levels of genes encoding Cucontining proteins, implying links etween Fedeficiency with Cu homeostsis [26]. Mny N-deficiency-responsive mirnas hve een identified from Aridopsis, soyen, mize nd common en. These mirnas elong to t lest 27 conserved fmilies [10, 17, 27, 28]. In Aridopsis, the expression of mir169 ws inhiited y N-deficiency, while the expression levels of its trget genes [i.e., NFYA2 (Nucler Fctor Y, suunit A2), NFYA3, NFYA5 nd NFYA8] were incresed [10, 13, 27, 29]. Trnsgenic Aridopsis plnts over-expressing mir169 hd less ccumultion of N nd NFYA fmily memers, nd were more sensitive to N stress thn the wild type, demonstrting role for mir169 in the dpttion of plnts to N-deficiency [29]. It is worth noting tht some N-deficiency-responsive mirnas (e.g., mir169, mir172, mir394, mir395, mir397, mir398, mir399, mir827, mir408 nd mir857) re lso responsive to other nutrient stresses (i.e., B, P, Fe, S nd Cu deficiencies) in plnts [8, 10], indicting the involvement of mirnamedited crosstlk mong N, B, P, Fe, S nd Cu under N- deficiency. An incresing numer of nutrient-deficiency-responsive mirnas hve een identified with different techniques [8 14]. Most reserch, however, hs focused on N, P, S, Cu nd Fe deficiencies, limited dt re ville on the differentil expression of mirnas nd their trget genes in response to deficiencies of other nutrient elements. Recently, we investigted mirna expression profiles in response to B-deficiency in Citrus sinensis roots y Illumin sequencing nd identified 134 (112 known nd 22 novel) B-deficiency-responsive mirnas, suggesting the possile roles of mirnas in the tolernce of citrus plnts to B-deficiency [8]. Previous studies showed tht the responses of mirnas to nutrient deficiencies differed etween plnt roots nd shoots (leves) [12, 17, 25]. In ddition, there were gret differences in B-deficiencyinduced chnges in mjor metolites, ctivities of key enzymes involved in orgnic cid nd mino cid metolism, gs exchnge nd gene expression profiles etween roots nd leves of C. sinensis [4, 30]. Therefore, B- deficiency-induced chnges in mirna expression profiles should e different etween citrus roots nd leves. In this study, we sequenced two smll RNA lirries from B-deficient nd -sufficient (control) citrus leves, respectively, using Illumin sequencing, then identified the known nd novel mirnas s well s the B-deficiencyresponsive mirnas. Also, we predicted the trget genes of these known nd novel B-deficiency-responsive mirnas nd discussed their possile roles in the response to B-deficiency in citrus. The ojective of this study is to identify the potentil mirnas relted to the tolernce of citrus to B-deficiency. Results B nd Cu concentrtions in leves B concentrtion in 10 μm B-treted leves ws in the sufficient rnge of 30 to 100 μg g 1 DW, while the vlue in 0 μm B-treted leves ws much less thn 30 μg g 1 DW (Fig. 1) [31]. Visile B-deficient symptoms were

3 Lu et l. BMC Plnt Biology (2015) 15:271 Pge 3 of 15 Lef B content ( g g -1 DW) Lef Cu content ( g g -1 DW) Control B-deficiency Tretments Fig. 1 Effects of B-deficiency on B nd Cu concentrtion in leves. Brs represent men ± SE (n = 3). Different letters ove the rs indicte significnt difference t P <0.05 oserved only in 0 μm B-treted leves (dt not shown). Therefore, seedlings treted with 0 μm B re considered s B-deficient, nd those treted with 10 μm B re considered s B-sufficient. B-deficiency decresed lef concentrtion of Cu (Fig. 1). Sequencing nd nlysis of two smll RNA lirries from B-sufficient nd -deficient citrus leves As shown in Tle 1, 17,996,827 nd 18,223,948 rw reds were generted from the lirries of B-sufficient nd -deficient leves, respectively. After removl of the contminnt reds like dptors nd low qulity tgs, 17,597,008 nd 17,829,966 cler reds were otined from the lirries of B-sufficient nd -deficient leves, comprising 3,673,054 nd 4,654,829 unique cler reds, respectively. Among these reds, 11,726,078 clen reds (1,961,407 unique reds) from B-sufficient leves nd 11,372,875 clen reds (2,484,833 unique reds) from B-deficient leves were mpped to C. sinensis genome (JGIversion 1.1, pz/portl.html#!info?lis=org_csinensis) using SOAP [32]. Exon, intron, mirna, rrna, repet regions, snrna, snorna nd trna reds were nnotted, respectively. After removl of these nnotted reds, the remined unique reds tht were used to predict novel mirnas for B-sufficient nd -deficient leves were 3,237,407 nd 4,179,224 reds, respectively. Most of the cler sequences were within the rnge of nt, which ccounted for 89 % of the totl cler reds. Reds with the length of 24 nt were t the most undnt, followed y the reds with the length of 21, 22, 23 nd 20 nt (Additionl file 1). Overll, the size distriution of srnas grees with the results otined on roots of Tle 1 Sttisticl nlysis of srna sequencing dt from B-sufficient nd -deficient leves of Citrus sinensis B-sufficiency B-deficiency Unique srnas Totl srnas Unique srnas Totl srnas Rw reds 17,996,827 18,223,948 Cler reds 3,673,054 (100 %) 17,597,008 (100 %) 4,654,829 (100 %) 17,829,966 (100 %) Mpped to genomic 1,961,407 (53.40 %) 11,726,078 (66.64 %) 2,484,833 (53.38 %) 11,372,875 (63.79 %) Exon ntisense 28,626 (0.78 %) 134,009 (0.76 %) 42,754 (0.92 %) 157,929 (0.89 %) Exon sense 77,868 (2.12 %) 281,505 (1.60 %) 81,887 (1.76 %) 287,483 (1.61 %) Intron ntisense 36,541 (0.99 %) 244,148 (1.39 %) 46,940 (1.01 %) 248,094 (1.39 %) Intron sense 56,020 (1.53 %) 526,848 (2.99 %) 67,594 (1.45 %) 457,839 (2.57 %) mirna 44,496 (1.21 %) 3,858,007 (21.92 %) 46,800 (1.01 %) 2,639,999 (14.81 %) rrna 164,311 (4.47 %) 3,052,914 (17.35 %) 158,009 (3.39 %) 2,851,216 (15.99 %) repet 821 (0.02 %) 2009 (0.01 %) 1014 (0.02 %) 2718 (0.02 %) snrna 2420 (0.07 %) 8040 (0.05 %) 3547 (0.08 %) 10,269 (0.06 %) snorna 1167 (0.03 %) 3628 (0.02 %) 1270 (0.03 %) 4748 (0.03 %) trna 23,377 (0.64 %) 810,902 (4.61 %) 25,790 (0.55 %) 722,780 (4.05 %) Unnnotted srnas 3,237,407 (88.14 %) 8,674,998 (49.30 %) 4,179,224 (89.78 %) 10,446,891 (58.59 %)

4 Lu et l. BMC Plnt Biology (2015) 15:271 Pge 4 of 15 Citrus sinensis [8], fruits of C. sinensis [33] nd Citrus trifolit, nd flowers of C. trifolite [34]. This indictes tht the dt of srna lirries otined y the Illumin sequencing re relile. Identifiction of known nd novel mirnas in citrus leves Here, totl of 734 known mirnas were isolted from the two lirries (Additionl file 2). The count of reds ws normlized to trnscript per million (TPM) in order to compre the undnce of mirnas in the two lirries. The most undnt mirna isolted from B-sufficient nd -deficient lirries ws mir157 (86, nd 48, TPM, respectively), followed y mir166 (36, nd TPM, respectively) nd mir167 (24, nd 16, , respectively). In this study, only these known mirnas with normlized red-count more thn ten TPM in B-sufficient nd/or -deficient lef lirries were used for further nlysis in order to void flse results cused y the use of low expressed mirnas [8, 35]. After removl of these low expressed mirnas, the remined 321 known mirnas were used for further nlysis (Additionl file 3). After removl of these nnotted reds (i.e., exon, intron, mirna, rrna, repet regions, snrna, snorna nd trna), the remined 3,237,407 nd 4,179,224 reds from B-sufficient nd -deficient lirries, respectively were used to predict novel mirnas using the Mirep ( Bsed on the criteri for nnottion of plnt mirnas [7, 36], totl of 71 novel mirnas were isolted from the two lirries (Additionl file 4). Like the known mirnas, novel mirnas with normlized red-count less thn ten TPM were not included in the expression nlysis [7, 35]. After excluding these low expressed novel mirnas, the remined 28 mir- NAs were used for further nlysis (Additionl file 5). Identifiction of B-deficiency-responsive mirnas in citrus leves We identified 91 (83 known nd 8 novel) up- nd 81 (75 known nd 6 novel) down-regulted mirnas from B-deficient leves. The most pronounced up- nd downregulted known (novel) mirnas were mir5266 with fold-chnge of (novel_mir_95 with fold-chnge of 17.61) nd mir401 with fold-chnge of (novel_mir_236 with fold-chnge of 18.48), respectively (Additionl files 3 nd 5). Vlidtion of high-throughput sequencing results y qrt- PCR We nlyzed the expression of 27 known mirnas using stem-loop qrt-pcr in order to vlidte the mirna expression ptterns reveled y Illumin sequencing. The expression levels of ll these mirnas except for mir6214, mir5262 nd mir7841 were comprle in mgnitude to the expression ptterns otined y Illumion sequencing (Fig. 2). Oviously, the high-throughput sequencing llowed us to identify the differentilly expressed mirnas under B- deficiency. Identifiction of trgets for differentilly expressed mirnas nd GO nlysis In this study, we predicted 489 nd 17 trget genes from the 70 known nd 6 novel differentilly expressed mir- NAs, respectively (Additionl files 6 nd 7). GO ctegories were ssigned to ll these trget genes sed on the cellulr component, moleculr function nd iologicl process. These trget genes for the known nd novel mirnas were relted to 12 nd 3 components, respectively sed on the cellulr component. The most three GO terms for known mirnas were memrne, chloroplst nd plstid, while more thn 42 % of the trget genes for novel mirnas elonged to memrne (Fig. 3). Bsed on the moleculr function, the trget genes for the known nd novel mir- NAs genes were grouped into 11 nd 9 ctegories, respectively, the highest percentge of three ctegories were nucleic cid inding, metl ion inding nd trnscription fctor ctivity (Fig. 3). In the iologicl process, the trget genes were minly focused on response to stress nd developmentl process for known mirnas, nd nucleic cid metolic process, developmentl process, response to stress nd regultion of trnscription for novel mir- NAs, respectively (Fig. 3c). qrt-pcr vlidtion of trget genes To verify the expression of the trget genes nd how the mirnas regulte their trget genes, 77 genes trgeted y 14 down- nd 13 up-regulted mirnas were ssyed y qrt-pcr (Tle 2). Among the 77 genes, the expression chnges of 58 trget genes showed negtive correltion with their corresponding mirnas, implying tht mirnas might ply role in regulting gene expression under B-deficiency y cleving mrnas. However, the expression chnges of the remined 19 trget genes hd positive correltion with their corresponding mirnas, which might e the results of the interction of different trget genes. Discussion Evidence shows tht mirnas re involved in the dptive regultion of higher plnts to nutrient deficiencies [8, 13, 17, 19, 24, 27, 37]. Here, we isolted 91 (83 known nd 8 novel) up- nd 81 (75 known nd 6 novel) downregulted mirnas from B-deficient leves (Additionl files 3 nd 5), indicting tht B-deficiency gretly ffected the expression profiles of mirnas in leves. The differentilly expressed mirnas isolted from leves were more thn from roots [i.e., 52 (40 known nd 12 novel) up- nd 82 (72

5 Lu et l. BMC Plnt Biology (2015) 15:271 Pge 5 of 15 Reltive expression Reltive expression mir158 mir1446 B-deficiency Control mir159 mir164 mir2643 mir2648 mir3446 mir1535 mir160 mir2099 mir2928 mir393 known nd 10 novel) down-regulted mirnas] [8]. The mjority of the differentilly expressed mirnas were isolted only from B-deficient roots or leves, only 22 mirnas were isolted from the oth. Moreover, mong the 22 mir- NAs, 11 mirnas in roots nd leves displyed different responses to B-deficiency (Tle 3). In conclusion, mny differences existed in B-deficiency-induced chnges in mirna expression profiles etween roots nd leves. We found tht mir159 ws down-regulted in B- deficient leves (Tle 2), s previously otined on slt stressed sugrcne leves [38]. Ptde nd Suprsnn showed tht the up-regultion of MYB t 1 h of sltstressed sugrcne leves ws ccompnied y the down-regultion of mir159 [38]. However, the expression of mir159 ws up-regulted in P-deficient soyen (Glycine mx) roots nd leves [39]. MiR159 plys importnt roles in mintining lef phenotype y negtively regulting MYB trnscription fctors [40]. Di et l. reported tht the expression of OsMYB3R-2 ws induced y vrious iotic stresses, nd tht over-expression of OsMYB3R-2 enhnced tolernce to freezing, drought, nd slt stress in trnsgenic Aridopsis [41]. B-1deficiency ffects wter uptke into the root, trnsport through the shoot, nd loss of wter from the leves [42]. Thus, B- deficiency-induced down-regultion of mir159 might mir3946 mir408 mir5259 mir6260 mir3953 mir477 mir5037 mir5227 mir5266 mir7539 mir782 mir6214 mir7841 mir6025 mir5262 increse the expression of MYBs (Tle 2), thus improving the tolernce of plnts to B-deficiency. qrt-pcr showed tht ll the four MYBs trget genes (i.e., MYB domin protein 33, MYB domin protein 97, MYB-like HTH trnscriptionl regultor fmily protein nd MYB domin protein 65) were induced y B-deficiency except for the lst one. Similrly, the expression levels of MYB trnscription fctor (MYBML2) trgeted y mir782, MYB-like HTH trnscriptionl regultor fmily protein nd MYB domin protein 65 trgeted y mir3946, nd MYB-like HTH trnscriptionl regultor fmily protein nd MYB trnscription fctor (MYBML2) trgeted y mir7539 incresed in response to B-deficiency except for MYB domin protein 65 (Tle 2). B-deficiency-induced up-regultion of MYBs in citrus leves grees with the previous report tht the expression of MYB85, MYB63 nd MYB42 were upregulted t the slight corking veins nd the seriously corky split veins cused y B-deficiency in Newhll nvel ornge (Citrus sinensis) leves [43]. TIR1/AFB2 (TRANSPORT INHIBITOR RESPONSE1/ AUXIN SIGNALING F-BOX PROTEIN2) Auxin Receptor (TAAR) fmily F-ox proteins re involved in uxin perception nd signling. The expression of TAAR is regulted y mir393 [44]. MiR393 plys key role in mintining proper homeostsis of uxin signling [45]. Fig. 2 Reltive undnces of selected known mirnas in B-deficient nd control leves reveled y qrt-pcr. Brs represent men ± SD (n = 3). Significnt differences were tested etween control nd B-deficient leves for the sme mirna. Different letters ove the rs indicte significnt difference t P < All the vlues were expressed reltive to the control leves

6 Lu et l. BMC Plnt Biology (2015) 15:271 Pge 6 of 15 Percentge (%) Percentge (%) Nucleic cid inding c Percentge (%) Trnscription fctor ctivity Metl ion inding ATPse ctivity Trnsporter ctivity Trnsferse ctivity Known mirnas Novel mirnas Memrne Nucleus Chloroplst Plstid Golgi pprtus Complex Thylkoid Vcuole Cytoskeleton Extrcellulr region Endoplsmic reticulum Others 19.5 Protein inding Kinse ctivity Peptidse ctivity Other inding 13.8 Other ctivity Response to stress Regultion of trnscription Trnsport Developmentl process Cellulr process Nucleic cid metolic process Protein metolic process Signling Crohydrte ctolic process Oxidtion reduction Phosphorus metolic process Orgnic cid ctolic process Other metolic process Fig. 3 GO of the predicted trget genes for 70 (6) differentilly expressed known (novel) mirnas. Ctegoriztion of mirnas trget genes ws performed ccording to cellulr component (), moleculr function () nd iologicl process (c)

7 Lu et l. BMC Plnt Biology (2015) 15:271 Pge 7 of 15 Tle 2 qrt-pcr reltive expression of experimentlly determined or predicted trget genes of selected mirnas mirna Fold chnge of mirna Accession Homology Trget genes Reltive chnge of trget genes mir ** ornge1.1g022993m AT1G SPFH/Bnd 7/PHB domin-contining ** memrne-ssocited protein fmily AT2G03210 Fucosyltrnsferse ** ornge1.1g001709m AT3G07400 Lipse clss 3 fmily protein * mir ** ornge1.1g039708m AT5G MYB domin protein * ornge1.1g044979m AT4G Sporocyteless (SPL) ** ornge1.1g046419m AT4G MYB domin protein ** ornge1.1g011938m AT3G MYB domin protein ** ornge1.1g038795m AT3G MYB-like HTH trnscriptionl regultor ** fmily protein mir ** ornge1.1g004896m AT2G ARF ** ornge1.1g005075m AT4G ARF ** ornge1.1g008078m AT1G ARF ** mir ** ornge1.1g030909m AT1G NAC domin contining protein ** ornge1.1g047710m AT5G NAC domin trnscriptionl regultor superfmily ** protein ornge1.1g017827m AT5G NAC domin contining protein ** ornge1.1g017636m AT3G Protein of unknown function, DUF ** mir ** ornge1.1g022993m AT1G SPFH/Bnd 7/PHB domin-contining ** memrne-ssocited protein fmily AT2G03210 Fucosyltrnsferse ** ornge1.1g001709m AT3G07400 Lipse clss 3 fmily protein * mir ** ornge1.1g010049m AT3G B-S glucosidse ** ornge1.1g007916m At3g62980 TIR ** At4g03190 AFB ** ornge1.1g008325m At3g26810 AFB ** At1g12820 AFB ** mir ** ornge1.1g013075m At2g30210 Lccse ** ornge1.1g041358m At5g05390 Lccse ** At5g07130 Lccse * ornge1.1g048131m At2g02850 Plntcynin ** mir ** ornge1.1g018483m AT3G UDP-Glycosyltrnsferse superfmily protein ** mir ** HQ MYB trnscription fctor (MYBML2) ** ornge1.1g039969m NM_ Protein disulfide isomerse (PDIL5-1) ** mir ** ornge1.1g037028m AT1G GRAS fmily trnscription fctor fmily protein ** mir ** ornge1.1g001616m AT3G ATPse E1-E2 type fmily protein/hlocid ** dehlogense-like hydrolse fmily protein ornge1.1g015157m AT3G Ction efflux fmily protein ** mir ** ornge1.1g017694m AT3G Het shock trnscription fctor A6B ** mir ** ornge1.1g018307m AT1G Nucleotide-sugr trnsporter fmily protein ornge1.1g020050m AT5G Peroxidse superfmily protein ** mir ** ornge1.1g003798m AT5G Ction/H + exchnger ** mir ** ornge1.1g007099m AT4G WRKY fmily trnscription fctor ** ornge1.1g014735m AT4G WRKY DNA-inding protein ** ornge1.1g016623m AT1G WRKY fmily trnscription fctor **

8 Lu et l. BMC Plnt Biology (2015) 15:271 Pge 8 of 15 Tle 2 qrt-pcr reltive expression of experimentlly determined or predicted trget genes of selected mirnas (Continued) mir ** ornge1.1g004633m AT5G Mitogen-ctivted protein kinse kinse kinse ** ornge1.1g004928m AT2G Hydroxyproline-rich glycoprotein fmily protein ** ornge1.1g036074m AT4G Potssium trnsport 2/ ** mir ** ornge1.1g029573m AT5G Homeoox-leucine zipper protein 4 (HB-4)/HD-ZIP protein * ornge1.1g041705m AT4G Peroxidse superfmily protein ** ornge1.1g031837m AT1G Copper/zinc superoxide dismutse ** ornge1.1g016997m AT1G Endosoml trgeting BRO1-like domin-contining protein ** ornge1.1g014089m AT1G Endosoml trgeting BRO1-like domin-contining ** protein ornge1.1g027084m AT3G PDI-like * ornge1.1g017665m AT3G NAC domin contining protein ** ornge1.1g010076m AT3G Phosphte trnsporter 1; ** ornge1.1g034408m AT1G MATE efflux fmily protein ** ornge1.1g027612m AT1G Vesicle-ssocited memrne protein ** ornge1.1g027026m AT4G Het shock protein ** ornge1.1g020124m AT2G MYB-like HTH trnscriptionl regultor fmily protein ** ornge1.1g011938m AT3G MYB domin protein ** ornge1.1g005651m AT1G Bsic helix-loop-helix (HLH) DNA-inding fmily ** protein ornge1.1g012387m AT4G Bsic helix-loop-helix (HLH) DNA-inding superfmily ** protein ornge1.1g004509m AT2G Trnsketolse * ornge1.1g033760m AT2G SAUR-like uxin-responsive protein fmily ** mir ** ornge1.1g016435m AT5G NAC domin contining protein ** ornge1.1g017142m AT5G NAC domin contining protein * mir ** ornge1.1g013411m AT2G Mjor fcilittor superfmily protein ** ornge1.1g016066m AT2G Mjor fcilittor superfmily protein ** mir ** ornge1.1g031467m AT2G DnJ/Hsp40 cysteine-rich domin superfmily protein ** ornge1.1g018585m AT1G Zinc trnsporter 10 precursor ** mir ** ornge1.1g005832m AT1G Crotenoid isomerse ** ornge1.1g003885m AT5G Chloride chnnel C 0.844** mir ** ornge1.1g040022m AT4G Ammonium trnsporter 1; * mir ** ornge1.1g005910m AT5G Chperone DnJ-domin superfmily protein ** mir ** ornge1.1g005832m AT1G Crotenoid isomerse ornge1.1g023118m AT2G Shikimte kinse ** mir ** ornge1.1g037661m AT5G Chperone DnJ-domin superfmily protein ** mir ** ornge1.1g010903m AT5G WRKY DNA-inding protein ** ornge1.1g003752m AT5G Chperone DnJ-domin superfmily protein ** ornge1.1g041599m AT1G Hydroxyproline-rich glycoprotein fmily protein ** ornge1.1g029026m AT1G Mjor fcilittor superfmily protein 1.777** mir ** ornge1.1g002698m AT2G Phosphoenolpyruvte croxylse ** ornge1.1g020124m AT2G MYB-like HTH trnscriptionl regultor fmily protein ** mir ** ornge1.1g041450m AT3G H + -ATPse ** Both fold chnge of mirnas nd reltive chnge of trget genes re the rtio of B-deficient to sufficient leves. The vlue is n verge of t lest three iologicl replictes with three technicl replictes; Trget genes tht hd the expected chnges in mrna levels were mrked in old. * nd ** indicte significnt difference t P <0.05nd P < 0.01, respectively

9 Lu et l. BMC Plnt Biology (2015) 15:271 Pge 9 of 15 Tle 3 List of differentilly expressed mirnas present in oth roots nd leves MiRNA Fold chnge Roots Leves 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 Lu et l. [8]; ** indictes significnt difference t P < 0.01 Si-Ammour et l. showed tht mir393 down-regulted ll four TAAR genes y guiding the clevge of their mrnas, leding to the chnges in uxin perception nd some uxin-relted lef development [44]. Stress-induced increse in mir393 level my decrese the level of TIR1, positive regultor of growth nd development, therey resulting in ttenution in growth nd development during stress conditions [14]. Auxin response fctors (ARFs) ply role in relying uxin signling t the trnscriptionl level y inducing minly three groups of genes [i.e., Aux/IAA (Auxin/indole-3-cetic cid), GH3 nd smll uxin-up RNA (SAUR)] [46, 47]. MiR160 is predicted to trget ARF10, ARF16 nd ARF17. MiR160-directed regultion of Aridopsis ARF17 is necessry for the norml growth nd development of mny orgns, proper GH3-like gene expression nd perhps uxin distriution, while the ARF10 nd ARF16 knockout mutnts do not disply ovious developmentl nomlies [48]. Wekened plnt growth nd reduced metolic rte re common survivl strtegies employed to divert energy nd other resources to del with stress conditions. It hs een suggested tht the stressinduced up-regultion of mir393 nd mir160 might led to the ttenution of plnt growth nd development under stress y repressing uxin signling due to decresed TIR1 level nd y suppressing the ARF-medited gene expression, respectively, thus promoting plnt stress tolernce [47]. Therefore, B-deficiency-induced up-regultion of lef mir393 nd mir160 might e n dptive response of plnts to B-deficiency, ecuse the expression of the three genes trgeted y mir160 nd TIR1, AFB1, AFB2 nd AFB3 trgeted y mir393 ws downregulted y B-deficiency except for AFB3 (Tle 2). Similrly, the expression of SAUR-like uxin-responsive protein fmily trgeted y mir3946 ws down-regulted in B-deficient leves despite decresed expression of mir3946 (Tle 2). By contrst, root mir3946 ws up-regulted y B-deficiency [8]. Lef mir164 ws down-regulted y B-deficiency (Tle 2), s previously oserved on trnsient low nitrtestressed mize leves [28]. Wter stress led to decresed expression of mir164 in cssv (Mnihot esculent) leves, while its trget gene MesNAC (No Apicl Meristem) ws strongly induced [49]. As expected, the expression of NAC domin trnscriptionl regultor superfmily protein nd NAC domin contining protein 100 ws induced in B-deficient leves, while the expression of NAC domin contining protein 1 ws depressed (Tle 2). Over-expression of SNAC1 nd OsNAC6 conferred drought nd slt tolernce in rice [50, 51]. SINAC4-RNAi tomto plnts ecme less tolernt to slt nd drought stress [52]. Therefore, the down-regultion of mir164 in B-deficient leves might e involved in the B-deficiency tolernce of plnts y improving the expression of NAC. However, Xu et l. found tht mir164 ws up-regulted in mize leves under chronic N limittion, nd suggested tht mir164 might function in remoilizing the N from old to new leves to cope with the N-limiting condition vi ccelerting senescence due to decresed expression of NAC [28]. Lef mir408 ws down-regulted y B-deficiency (Tle 2), s previously reported on N-deficient seedlings of Aridopsis [27]. MiR408 trgets genes encoding Cu contining proteins such s Cu/Zn SODs (CSDs), plntcynin nd severl lccses [23]. Adel-Ghny nd Pilon oserved tht mir408 ws induced under Cu strvtion to down-regulte trget gene expression nd to sve Cu for the most essentil functionl protein, concluding tht might ply role in the regultion of Cu homeostsis [22]. Although B-deficiency decresed lef concentrtion of Cu, its level ws not lower thn the sufficiency rnge of Cu in citrus leves [53]. Thus, B-deficiency-induced decrese in mir408 might e dvntgeous to plnt survivl under B-deficiency y regulting Cu homeostsis nd improving ntioxidnt (SOD) ctivity, ecuse the expression of its four trget genes ws induced y B-deficiency

10 Lu et l. BMC Plnt Biology (2015) 15:271 Pge 10 of 15 except for lccse 12 (Tle 2). Indeed, SOD ctivity ws higher in B-deficient C. sinensis leves thn in B-sufficient ones [54]. Also, SOD expression ws up-regulted in B- deficient Medicgo trunctul root nodules [55]. Lef mir477 ws up-regulted y B-deficiency (Tle 2), s previously reported on slt-stressed Populus cthyn plntlets [56]. NAC nd GRAS trnscription fctors re trget genes of mir477. NAC is involved in developmentl process nd stress responses [56], while GRAS proteins ply role in signl trnsduction nd the mintennce nd development of meristems [57]. Also, GRAS is the trget gene of mir1446 (Tle 2), mir170 nd mir171 [58], nd NAC is the trget gene of mir164, mir3953 nd mir3946 (Tle 2). This indictes the complex regultion in plnt development nd stress response. WRKY proteins ply importnt roles in plnt responses to ()iotic stresses, llowing plnts to dpt to unfvorle environmentl conditions including B-deficiency [59, 60]. Our results showed tht lef trnscript of mir6260 decresed in response to B-deficiency ccompnied y incresed expression of its trget gene: WRKY DNA-inding protein 72 (Tle 2), which grees with the previous reports tht WRKY3 DNA inding protein expression ws induced in B-deficient M. trunctul root nodules [55] nd tht WRKY6 ws up-regulted in B-deficient Aridopsis roots [60]. Over-expression of vrious WRKY conferred tolernce to different iotic stresses in different plnt species, possile through the regultion of the rective oxygen species system [61, 62]. Trnsgenic Nicotin enthmin plnts over-expressing GhWRKY39 hd enhnced tolernce to slt nd oxidtive stress nd incresed expression of genes encoding ntioxidnt enzymes such s SOD, scorte peroxidse (APX), ctlse (CAT) nd glutthione-s-trnsferse (GST) [62]. Thus, lef expression levels of ntioxidnt enzyme genes might e incresed in response to B-deficiency. This grees with our report tht B-deficient citrus leves hd higher ctivities of SOD, APX, MDAR nd GR [54]. Het shock proteins (HSPs)/chperones function in protecting plnts ginst vrious stresses. As expected, the expression of mir6260 ws down-regulted in B-deficient leves ccompnied y incresed expression of its one trget gene: chperone DnJ-domin superfmily protein (Tle 2). Similrly, lef expression levels of mir5929 nd mir6214 were decresed y B-deficiency ccompnied y incresed expression levels of their corresponding trget genes: DnJ-domin superfmily protein (AT5G nd AT5G ; Tle 2). However, the expression of het shock trnscription fctor A6B trgeted y mir2099 were inhiited in B-deficient leves despite down-regulted expression of mir2099 (Tle 2). Hydroxyproline-rich glycoproteins (HRGPs) re the most undnt cell wll structurl proteins in dicotyledonous plnts [63]. Hll nd Cnnon demonstrted tht the cell wll HRGP RSH ws required for norml emryo development in Aridopsis [64]. Bonill et l. oserved tht B- deficiency-induced errnt cell wlls of en root nodules lcked covlently ound HRGPs [65]. Here, the expression of HRGP fmily protein (AT2G ), trget gene of mir3446, ws up-regulted in B-deficient leves (Tle 2), thus enhncing plnt tolernce to B-deficiency. However, mir3446 ws down-regulted in B-deficient leves, ut its trget gene (HRGP fmily protein; AT1G ) ws lso depressed (Tle 2). B-deficiency lowered lef expression level of mir158 (Tle 2), s previously otined on N-deficient Aridopsis seedlings [27] nd B-deficient citrus roots [8]. The downregultion of mir158 mens tht its trget genes: SPFH/ Bnd 7/PHB domin-contining memrne-ssocited protein fmily, fucosyltrnsferse 2 nd lipse clss 3 fmily protein might e up-regulted in B-deficient leves. However, qrt-pcr showed tht the expression of the former two trget genes ws induced y B-deficiency, while the lst one ws down-regulted (Tle 2). Lu et l. reported tht fucosyltrnsferse 2 nd lipse clss 3 fmily protein were down-regulted in B-deficient citrus roots ccompnied y decresed expression of mir158 [8]. The mjor fcilittor superfmily (MFS) is the lrgest group of trnsport crriers, which re often coupled to the movement of nother ion [66]. Ky et l. reported tht ATR1, which encodes multidrug resistnce trnsport protein of the MFS, ws responsile for most of the tolernce of high B in Scchromyces cerevisie, concluding tht ATR1 ws B exporter [67]. In this study, lef mir5037 ws induced y B-deficiency ccompnied y decresed expression of its trget gene: MFS protein (Tle 2), thus decresing B export from plnts nd improving plnt tolernce to B-deficiency. We found tht lef mir5266 ws induced y B- deficiency ccompnied y incresed expression of its trget gene: mmonium trnsporter 1;1 (Tle 2), which disgrees with our report tht the undnce of mir5266 ws lower in B-deficient citrus roots thn in controls, while the expression level of mmonium trnsporter 1;1 ws higher in the former [8]. We oserved tht mir3946 ws inhiited in B-deficient leves (Tle 2), which disgrees with the previous report tht mir3946 ws induced in B-deficient C. sinensis roots [8]. All the 17 trget genes trgeted y mir3946 were induced y B-deficiency except for homeoox-leucine zipper protein 4 (HB-4)/HD-ZIP protein, endosoml trgeting BRO1-like domin-contining protein (AT1G ), MYB domin protein 65 nd SAUR-like uxin-responsive protein fmily (Tle 2). Previous studies showed tht B-deficiency incresed the expression levels of some trnsport-relted genes nd the undnces of some trnsport-relted proteins in citrus roots [5, 8], thus improving the tolernce of plnts to B-deficiency. BOR1, n efflux-type B trnsporter for xylem loding,

11 Lu et l. BMC Plnt Biology (2015) 15:271 Pge 11 of 15 ply key role in the tolernce of plnts to low B. Aridopsis or1-1 mutnt ws more sensitive to B- deficiency thn the wild type [68]. Oryz stiv BOR1 hs een demonstrted to e required for B cquisition y roots nd trnsloction of B into shoots [69]. Thus, B-deficiency-induced up-regultion of lef endosoml trgeting BRO1-like domin-contining protein (AT1G ), phosphte trnsporter 1;7, MATE efflux fmily protein, vesicle-ssocited memrne protein 726 (trgeted y mir3946), potssium trnsport 2/3 (trgeted y mir3446), mmonium trnsporter 1;1 (trgeted y mir5266), Zn trnsporter 10 precursor (trgeted y mir5227) nd ction/h + exchnger 25 (trgeted y mir 2648) involved in cell trnsport (Tle 2) might contriute to the tolernce of citrus to B-deficiency. HD-ZIP trnscription fctors re found only in plnts. The expression of Hh-4, memer of Helinthus nnuus (sunflower) sufmily I, strongly incresed in wter-stressed sunflower [70]. Susequent study showed trnsgenic Aridopsis plnts over-expressing Hh-4 were more tolernt to drought y delying the onset of senescence [71]. Hung et l. demonstrted tht PtrHLH, sic helix-loop-helix trnscription fctor of Poncirus trifolit might ply crucil role in cold tolernce vi positively regulting peroxidse (POD)-medited ROS scvenging [72]. Trnsketolse is key enzyme of the pentose phosphte pthwy (PPP) in plnt cells. Our finding tht trnsketolse ws up-regulted in B-deficient leves grees with the report tht trnsketolse ctivity in mize modertely incresed in response to slt or oxidtive stress [73]. In citrus, PPP hs een suggested to ply role in the tolernce of plnts to B-deficiency y providing reducing power (NADPH) nd enhncing the ntioxidnt cpcity [4]. Protein disulfide isomerses (PDIs), which ct s moleculr chperones, ply role in the formtion of proper disulfide onds during protein folding [74]. Overexpression of protein disulfide isomerse-like protein (PDIL) gene conferred Hg tolernce in trnsgenic plnts, which hd higher ntioxidnt cpcity nd lower levels of superoxide nion rdicls, H 2 O 2 nd mlondildehyde (MDA) [75]. As shown in Tle 2, the expression level of PDIL5-3 trgeted y mir3946 ws incresed in B-deficient leves. To conclude, down-regultion of mir3946 in B- deficient leves might e n dptive response of plnts to B-deficiency. Crotenoid (Cr) isomerse (CRTISO), which ctlyzes the isomeriztion of poly-cis-crotenoids to ll trns-crotenoids in higher plnts, is regultory step for Cr iosynthesis. Aridopsis mutnts of crtiso hd incresed ccumultion of poly-cis-crotenoids nd reduced lutein concentrtion [76, 77]. Here, the expression of mir6025 ws incresed nd its one trget gene: CRTISO ws decresed in B-deficient leves (Tle 2), thus impiring Cr iosynthesis. This grees with our report tht B- deficient citrus leves hd lower Cr concentrtion [54]. Plnt phenolic secondry metolites nd their precursors re synthesized vi the pthwy of shikimte iosynthesis [78]. Shikimte kinse, key enzyme for the iosynthesis of polyphenols, ctlyzes the fifth rection of the shikimte pthwy. As shown in Tle 2, the expression level of shikimte kinse 1 ws down-regulted in B-deficient leves nd the expression of mir6025, which trgets the gene, ws up-regulted. This disgrees with our report tht B-deficient citrus leves displyed incresed ccumultion of phenolics [4]. Mitogen-ctivted protein kinse (MAPK) cscdes ply importnt roles in plnt response to vrious stresses. Ech MAPK cscde consists of MAPKs, MAPK kinses (MAPKKs), nd MAPKK kinses (MAPKKKs). In plnts, MAPKKKs hve een shown to e involved in vrious stresses. Ning et l. showed tht trnsgenic rice plnts over-expressing DSM1 ( puttive MAPKKK gene in rice) displyed higher tolernce to dehydrtion t the seedling stge y regulting ROS scvenging [79]. In this study, lef trnscript of mir3446 ws decresed y B-deficiency nd its trget gene (MAPKKK5) ws up-regulted under B- deficiency. This grees with the report tht MAPKKK genes were induced y drought, het, slt, cold, IAA nd jsmonic cid (JA) in Aridopsis [80]. Our finding tht lef expression level of mir7539 decresed in response to B-deficiency, nd its trget gene (phosphoenolpyruvte croxylse, PEPC) ws induced y B-deficiency (Tle 2). This grees with our report tht B-deficient citrus leves hd incresed ctivity of PEPC nd drk respirtion [4]. Conclusion We identified 734 known nd 71 novel mirnas from B-sufficient nd -deficient citrus leves using Illumin sequencing, nd otined 91 (83 known nd 8 novel) up- nd 81 (75 known nd 6 novel) down-regulted mirnas from B-deficient citrus leves. Oviously, the expression of mirnas ws gretly ltered in B-deficient leves, which might ply role in the tolernce of plnts to B-deficiency. In this study, we proposed model for the responses of lef mirnas to B-deficiency y integrting the present results with the dt ville in the previous litertures (Fig. 4). The dptive responses of lef mirnas to B-deficiency might e ssocited with severl spects: () ttenution of plnt growth nd development y down-regulting TIR1, ARF nd AFB due to up-regulted mir393 nd mir160, nd y lowering the expression of SAUR-like uxin-responsive protein fmily trgeted y mir3946, thus enhncing plnt stress tolernce; () improving the expression of NACs due to decresed expression mir159, mir782, mir3946 nd mir7539, hence mintining lef phenotype nd enhncing the

12 Lu et l. BMC Plnt Biology (2015) 15:271 Pge 12 of 15 Fig. 4 A potentil model for the roles of mirnas in the tolernce of citrus plnts to B-deficiency. VAMP 726: vesicle-ssocited memrne protein 726; CHE: ction/h + exchnger 25 stress tolernce; (c) ctivtion of the stress responses nd ntioxidnt system due to decresed expression of mir164, mir6260, mir5929, mir6214, mir3946 nd mir3446; (d) decresed expression of MFS resulting from incresed expression of mir5037, thus lowering B export from plnts. In ddition, B-deficiency-induced down-regultion of mir408 might e involved in the tolernce of plnts to B-deficiency y regulting Cu homeostsis nd enhncing SOD ctivity. In conclusion,ourstudyrevelssomedptivemechnismsof citrus to B-deficiency. Methods Plnt culture nd B tretments Both plnt culture nd B tretments were performed ccording to Yng et l. [5] nd Lu et l. [8]. Briefly, 15-week-old seedlings of Xuegn [Citrus sinensis (L.) Oseck] grown in 6 L pots (two seedlings per pot) contining fine river snd were supplied every other dy until dripping with B-deficient (0 μm H 3 BO 3 )or -sufficient (10 μm H 3 BO 3 ) nutrient solution for 15 weeks. There were 10 replictions per B tretment with 2 pots in completely rndomized design. At the end of the experiment, fully-expnded leves from different replictes nd tretments were collected t noon under full sun nd frozen immeditely in liquid N 2. Lef smples were stored t 80 C until extrction. It s worth mentioning tht C. sinensis is polyemryonic seed development, n pomictic process in which mny emryos re initited directly from the mternl nucellr cells surrounding the emryo sc contining developing zygotic emryo [81]. Isoltion of lef srnas, lirry construction nd Illumin sequencing Aout 0.1 g mixed frozen B-sufficient nd -deficient leves from five replictions were used to extrct RNA. Totl RNA ws extrcted from frozen leves using TRIzol regent (Invitrogen, Crlsd, CA) following mnufcturer s instructions. Two srna lirries were constructed ccording to Lu et l. [8]. High throughput sequencing ws performed on Solex sequencer (Illumin) t the Beijing Genomics Institute (BGI), Shenzhen, Chin. srna nnottion nd mirna identifiction Both srna nnottion nd mirna identifiction were performed ccording to Lu et l. [8]. Briefly, softwre developed y the BGI ws used to del with the rw dt from the Solex sequencing. Clen reds were then used to nlyze length distriution nd common/specific sequences. Therefter, the cler reds were mpped to C. sinensis genome (JGIversion 1.1, doe.gov/pz/portl.html#!info?lis=org_csinensis) using SOAP, only perfectly mpped sequences were retined nd nlyzed further. rrnas, trnas, snrnas nd snor- NAs 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 tht were not nnotted were used to predict novel mirnas using prediction softwre Mirep (

13 Lu et l. BMC Plnt Biology (2015) 15:271 Pge 13 of 15 sourceforge.net/projects/mirep/), which ws developed y the BGI, y exploring the secondry structure, the Dicer clevge site nd the minimum free energy of the unnnotted smll RNA tgs which could e mpped to genome. In ddition, we used MTide: n integrted tool for the identifiction of mirna-trget interction in plnts ( [82] nd DNAMAN 8 ( to predict novel mir NA. Only these mirna cndidtes tht were simultneously predicted y the three softwres were considered to e rel novel mirnas. Differentil expression nlysis of mirnas Both the fold chnge etween B-deficiency nd -sufficiency nd the P-vlue were clculted from the normlized expression of TPM [83]. A 1.5 log2-fold cut-off ws set to determine up- nd down-regulted mirnas in ddition to P-vlue of less thn 0.01 [8]. Trget prediction of mirnas This ws performed y RNAhyrid sed on rules suggested y Allen et l. [84] nd Schw et l. [85]. Functions of the potentil trgets of the differentilly expressed mirnas All trgets of the differentilly expressed mirnas were mpped to GO terms in the dtse ( nd clculted gene numers for ech term. The GO results were expressed s three ctegories: cellulr component, moleculr function, iologicl process [8]. Vlidtion of mirna expression y stem-loop qrt-pcr The detection of mirna expression ws performed using stem-loop qrt-pcr method, stem-loop primers for reverse trnscription nd primers for qrt-pcr were listed in Additionl file 8. Totl RNA ws reversetrnscried using Tqmn MicroRNA Reverse Trnscription Kit (USA), nd SYBR Premix Ex Tq II (Tkr, Jpn) kit ws used for qrt-pcr. MiRNA specil (forwrd) primers were designed ccording to the mirna sequence ut excluded the lst six nucleotides t 3 end of the mirna. A 5 extension of severl nucleotides, which ws chosen rndomly nd reltively GC-rich, ws dded to ech forwrd primer to increse the melting temperture [86]. All the primers were ssigned to Primer Softwre Version 5.0 (PREMIER Biosoft Interntionl, USA) to ssess their qulity. For qrt-pcr, 20 μl rection solution contined 10 μl redy-to-use SYBR Premix Ex TqTM II (Tkr, Jpn), 0.8 μl 10 μm mirna forwrd primer, 0.8 μl 10 μm Uni-miR qpcr primer, 2 μl cdna templte nd 6.4 μl dh 2 O. The cycling conditions were 60 s t 95 C, followed y 40 cycles of 95 C for 10 s, 60 C for 30 s. qrt-pcr ws performed on the ABI 7500 Rel Time System. Smples for qrt-pcr were run in t lest three iologicl replictes with two technicl replictes. Reltive mirna expression ws clculted using ddct lgorithm. For the normliztion of mirna expression, ctin (AEK ) ws used s n internl stndrd nd the leves from control plnts were used s reference smple, which ws set to 1. qrt-pcr nlysis of mirna trget gene expression Totl RNA ws extrcted from frozen B-sufficient nd -deficient leves using TRIzol regent (Invitrogen, Crlsd, CA) following mnufcturer s instructions. The sequences of the F nd R primers used were given in Additionl file 9. qrt-pcr nlysis of mirna trget gene expression ws performed using ABI 7500 Rel Time System ccording to Lu et l. [8]. Experimentl design nd sttisticl nlysis There were 20 pot seedlings per tretment in completely rndomized design. Experiments were performed with 3 replictes. Differences mong tretments were seprted y the lest significnt difference (LSD) test t P < 0.05 level. Avilility of dt nd mterils The dt set supporting the results of this rticle re ville in the Gene Expression Omnius repository under ccession no GSE72108 ( nih.gov/geo/query/cc.cgi?cc=gse72108). The mture mirna nd precursor sequences will e sumitted to mirbse registry nd ssigned finl nmes fter finl cceptnce of the mnuscript. Additionl files Additionl file 1: Length distriution of smll RNAs from control nd B-deficient leves of Citrus sinensis seedlings. (DOC 81 k) Additionl file 2: List of known mirnas in Citrus sinensis leves. (DOC 1525 k) Additionl file 3: List of known mirnas in Citrus sinensis leves fter removing these mirnas with normlized red-count less thn 10 TPM in the two mirna lirries constructed from control nd B-deficient leves. (DOC 452 k) Additionl file 4: List of novel mirnas in Citrus sinensis leves. (DOC 158 k) Additionl file 5: List of novel mirnas in Citrus sinensis leves fter removing these mirnas with normlized red-count less thn 10 TPM in two mirna lirries constructed from control nd B- deficient leves. (DOC 69 k) Additionl file 6: List of trget genes for prts of known mirnas in Citrus sinensis leves. (DOC 198 k) Additionl file 7: List of trget genes for prts of novel mirnas in Citrus sinensis leves. (DOC 33 k) Additionl file 8: List of stem loop qrt-pcr primers. (DOC 61 k) Additionl file 9: Specific primer pirs used for qrt-pcr expression nlysis of selected mirna trget genes. (DOC 160 k)