Genome-wide analysis and molecular dissection of the SPL gene family in Salvia miltiorrhiza

Linsu Zhang,Bin Wu,Degang Zhao,Caili Li,Fenjuan Shao and Shanfa Lu＊

1Center for Research and Development of Fine Chemicals,Guizhou University,Guiyang 550025,China,2Institute of Medicinal Plant Development,the Chinese Academy of Medical Sciences and Peking Union Medical College,Beijing 100193,China,3Qiannan Medical College for Nationalities,Duyun 558003,China.＊Correspondence:sflu@implad.ac.cn

INTRODUCTION

Plant gene expression is regulated by various factors,such as environmental signals,transcription factors(TFs),microRNAs(miRNAs),etc.TFs are a large class of regulators controlling gene expression at the transcriptional level,and usually serve as an on-off switch in the developmental process of eukaryotic organisms(Sun and Oberley 1996).The typical structure of TFs includes the DNA-binding domain,transcriptional regulatory domain,oligomerization domain,and the nuclear localization signal(NLS)(Liu et al.1999).The DNA-binding domain is relatively conserved and can specifically bind to cis-elements.SPL(SQUAMOSA promoter binding protein-like)is a family of plant-specific transcription factors(Chen et al.2010).It was first identified in snapdragon(Klein et al.1996)and later found to be conserved in plants,such as Arabidopsis(Cardon et al.1999),rice(Xie et al.2006),and Populus trichocarpa(Lu et al.2011).SPL contains a conserved SBP(SQUAMOSA promoter binding protein)domain having two zinc-binding sites assembled as Cys-Cys-Cys-His and Cys-Cys-His-Cys,respectively(Yamasaki et al.2004),and a NLS partially overlapping with the second Zn-finger located at the C-terminal of the SBP domain.SPL is encoded by a large gene family in plants.For instance,there are 16 SPL genes in Arabidopsis thaliana(Cardon et al.1999),19 in rice(Xie et al.2006),and at least 17 in P.trichocarpa(Lu et al.2011).These genes play significant regulatory roles in various plant developmental processes,such as vegetative phase change(Cardon et al.1997),male fertility(Xing et al.2010),GA biosynthesis(Zhang et al.2007),plant architecture(Stone et al.2005),and in plant response to stress(Stone et al.2005).

MiRNAs are the other class of vital regulators of gene expression in plants,animals,and viruses(Lee et al.1993).They are small RNA molecules of about 21 nucleotides in length derived from long primary transcripts(pri-miRNAs).In plants,miRNAs regulate plant development and response to biotic and abiotic stresses through direct cleavage of transcripts,translational repression,or epigenetic modification(Jones-Rhoades et al.2006;Sunkar and Zhu 2007;Chen 2012).miR156/157 is one of the miRNA families highly conserved in plants(Axtell and Bowman 2008).It regulates plant development through direct cleavage of SPL transcripts.Among 16 Arabidopsis SPLs,10 are miR156/157 targets.It includes AtSPL2,AtSPL3,AtSPL4,AtSPL5,AtSPL6,AtSPL9,AtSPL10,AtSPL11,AtSPL13,and AtSPL15(Schwab et al.2005;Wu and Poethig 2006;Gandikota et al.2007;Addo-Quaye et al.2008).These miR156/157-regulated AtSPLs play divergent and redundant roles in the morphology and development of Arabidopsis.For instance,AtSPL2,AtSPL10,and AtSPL11 are closely related members of the SPL gene family.They redundantly control lateral organ development in the reproductive phase(Shikata et al.2009).AtSPL3,AtSPL4,and AtSPL5 regulate floral transition(Cardon et al.1997;Jung et al.2011;Yu et al.2012).AtSPL6 is able to activate the defense transcriptome and is a positive regulator in the TIR-NB-LRR receptor-mediated plant innate immunity(Padmanabhan et al.2013).AtSPL9 and AtSPL15 control shoot maturation(Schwarz et al.2008).In addition,AtSPL9 is also involved in the production and distribution of trichomes and the biosynthesis of anthocyanin(Yu et al.2010;Gou et al.2011).

Salvia miltiorrhiza,known as Danshen,has been widely used in Traditional Chinese medicine(TCM)for the treatment of dysmenorrhea,amenorrhea,and cardiovascular diseases(Cheng 2006).It belongs to the Labiatae family and is an emerging model plant for TCM studies(Ma et al.2012).Transcription factors and their regulation roles in S.miltiorrhiza are poorly understood.In order to elucidate the regulatory networks of SPLs in DanShen growth and development,we performed a genome-wide analysis and molecular dissection of the SmSPL gene family.It resulted in identification of the first set of SmSPLs,providing a great example for understanding transcription factors and their regulatory roles in S.miltiorrhiza.

RESULTS

Genome-wide identification,molecular cloning and sequence feature analysis of SmSPL genes

In order to identify SPL genes in S.miltiorrhiza,we performed a genome-wide prediction of SmSPLs by BLAST analysis of 16 AtSPLs against the working draft of the S.miltiorrhiza genome(Chen S.et al.unpubl.data 2010)using the tBLASTn algorithm(Altschul et al.1997).It resulted in the identification of 15 SmSPL genes,which were named SmSPL1–SmSPL15,respectively.The number of SPL genes in S.miltiorrhiza is comparable with that in A.thaliana(Cardon et al.1999),rice(Xie et al.2006),and P.trichocarpa(Lu et al.2011),which contain 16,19,and at least 17 SPLs,respectively.It suggests that the number of duplication events of SPLs occurring in these plant species is similar.The length of SmSPL genes varies from 757(SmSPL14)to 5,201 bp(SmSPL13)(Table 1).The wide size range of SmSPL genes is consistent with that of AtSPL genes,varying from 984(AtSPL5)to 4,798 bp(AtSPL14)(Table 1).We further predicted gene models of 15 SmSPLs using Genscan(http://genes.mit.edu/GENSCAN.html),and manually corrected the predicted models by comparing with known plant SPLs using the BLASTx algorithm(http://www.ncbi.nlm.nih.gov/BLAST).All of the deduced SmSPL proteins contain the conserved SBP domain,implying they are authentic SPLs.

To further verify the results from computational prediction of SmSPL gene models,we amplified,cloned,and sequenced the coding region of all 15 SmSPL cDNAs using polymerase chain reaction(PCR)technology.All cloned SmSPL cDNAs have been submitted to GenBank.The accession numbers in GenBank are shown in Table 1.Analysis of the experimentally confirmed cDNA sequence of SmSPLs revealed that the length of SmSPL cDNAs varied between 402(SmSPL15)and 3,216 bp(SmSPL1),which is consistent with the length of AtSPL cDNAs widely ranging from 396(AtSPL3)to 3,108 bp(AtSPL14)(Figure 1,Table 1).Moreover,the number of introns varies between 1 and 10 in both SmSPL and AtSPL genes(Figure 1,Table 1).These results suggest the diverse of SPL gene structures in a plant species.

Comparative analysis of conserved domains and motifs in SmSPLs and AtSPLs

BLAST analysis of SmSPLs against the National Center for Biotechnology Information(NCBI)conserved domain database(http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi)showed that all SmSPLs contained an SBP domain,which was located in a region close to the N-terminus and was encoded by the first two exons of SmSPL genes(Figure 1).The location of SBP domains in SmSPLs is similar to that of Arabidopsis SPLs(Figure 1),suggesting the conservation of SBP domain in two plant species.Sequence alignment of the SBP domain in SmSPLs showed many highly conserved amino acids(Figure S1),most of which were located in two zinc-binding sites,Zn1 and Zn2(Figure 2).Zn1 contains the CX4CX16CX2H(CCCH)or CX4CX16CX2C(CCCC)signature sequence,whereas Zn2 contains the CX2CX3HX11C(CCHC)signature(Figure 2).It is consistent with the results from Arabidopsis(Yamasaki et al.2004),indicating high conservation of zinc-binding sites in the SBP domain in plants.In addition to zinc-binding sites,a NLS overlapping with Zn2 was also found in the SBP domain(Figure 2).It is located in the region close to the C terminus of the SBP domain,and is a bipartite nuclear targeting sequence with the consensus sequence of KRX11RRRK(Figure 2)(Robbins et al.1991).Similarly,many SPLs in Arabidopsis and rice also contain the NLS with KRX11RRRK consensus sequence(Birkenbihl et al.2005;Xie et al.2006).The conservation of SBP domain location,zinc-binding sites and the NLS appear to be important for specific recognition and binding to cis-elements in the promoter of nuclear genes(Birkenbihl et al.2005).

Four S.miltiorrhiza SPLs(SmSPL1,SmSPL9,SmSPL10,and SmSPL13)and four A.thaliana SPLs(AtSPL1,AtSPL12,AtSPL14,and AtSPL16)contain an ANK or Ank-2 domain with four or three ankyrin repeats(Table 1),each of which usually contain about 33 amino acid residues forming two antiparallel helices separated by a beta-hairpin(Michaely et al.2002).Ankyrin repeats mediate protein–protein interactions(Michaely and Bennett 1992),indicating these SPLs may interact with other proteins for functions in plant cells.Although the ANK or Ank-2 domain of S.miltiorrhiza and A.thaliana SPLs is less conserved compared with the SBP domain(Figures S1,S2),there are several highly conserved amino acid residues among SmSPLs and AtSPLs(Figure 3).The significance of these conserved amino acid residues is currently unknown and need to be further investigated(Michaely et al.2002).

In addition to the conserved SBP domain and ankyrin repeats,other conserved motifs were found in rice and Arabidopsis SPLs(Xie et al.2006;Guo et al.2008).Although the actual function of these motifs is currently unknown,they may be structural units and play important roles in plants.Thus,we performed a search of motifs in S.miltiorrhiza and A.thaliana SPLs using the motif discovery tool MEME version 3.0.14(Bailey and Elkan 1994).It resulted in the identification of 20 conserved motifs(Figure 4,Table 2).Motif 1 is actually the SBP domain.Motif 2 and motif 8 correspond to the ANK domain.Functions of the other motifs need to be further investigated.

Phylogenetic analysis of SPLs in S.miltiorrhiza and A.thaliana

In order to analyze the evolutionary relationship of 15 SmSPLs and 16 AtSPLs,we constructed an unrooted neighbor-joining tree using MEGA4(Tamura et al.2007).SmSPLs and AtSPLs were clustered into six groups,G1–G6(Figure 5).G1–G3 and G5 to include short SPLs with no more than 465 amino acid residues,whereas the members of G4 and G6 are longer and vary from 774 to 1,040 amino acids.G1 is the largest group including four SmSPLs and six AtSPLs(Figure 5,Table 1).Based on the phylogenetic tree,it may be further divided into two subgroups,G1a(SmSPL2,SmSPL5,AtSPL2,AtSPL10,and AtSPL11)and G1b(SmSPL6,SmSPL11,AtSPL6,AtSPL9,and AtSPL15)(Figure 5,Table 1).Except AtSPL6,all of SPLs in G1 contain motifs 1 and 12,whereas G1a and G1b SPLs have an additional motif 14 and motif 19,respectively.It suggests the significance of these motifs and implies functional conservation of SPLs within a subgroup.G1a includes two highly related SmSPLs,SmSPL2,and SmSPL5,and three Arabidopsis homologs,AtSPL2,AtSPL10,and AtSPL11,all of which contain three introns with intron phase 2,1,and 1,respectively(Figure 1).AtSPL10 and AtSPL11 are tandem repeats in the Arabidopsis genome(Yang et al.2008),whereas the arrangement of SmSPL2 and SmSPL5 genes in the S.miltiorrhiza genome is currently unknown.Similarly,four of the five members of G1b,including SmSPL6,AtSPL6,AtSPL9,and AtSPL15,have the same number of intron and patterns of intron phase(Figure 1).It suggests the close evolutionary relationship of SPLs in a subgroup.

Based on the phylogenetic relationship,eight members of G4 were further grouped into G4a and G4b,each of which contains two SmSPLs and two AtSPLs(Figure 5,Table 1).Except SmSPL13 having 10 introns,the other seven SPLs in G4 contain nine introns and share the same intron phase patterns(Figure 1).G4 SPLs commonly share 10 motifs,which include the ankyrin repeats(motifs 2 and 8)involved in protein–protein interactions(Michaely and Bennett 1992),suggesting it is important to interact with other proteins for the function of SPLs in this group.In addition to the common motifs in G4,there are another three(motifs 5,17,and 20)commonly found in G4a and four(motifs 9,13,15,and 18)in G4b(Figure 4).The existence of group-common motifs suggests functional redundancy of G4 SPLs in some cellular processes,whereas subgroup-specific motifs indicate the specificity of G4a and G4b SPLs in the other plant cellular processes.It provides useful clues for elucidating the function of SPLs in G4.

All G5 members,including SmSPL3,SmSPL8,SmSPL15,and AtSPL3–AtSPL5,are short SPLs with only one conserved motif(motif 1)(Figure 4).Furthermore,all of them contain an intron with the intron phase 2(Figure 1).The results suggest the conserved evolutionary relationship among G5 SPLs and indicate functional conservation of these SPLs in plants.Consistently,in Arabidopsis,AtSPL4 and AtSPL5 are located in the duplicated genomic regions(Bowers et al.2003)and AtSPL3,AtSPL4,and AtSPL5 redundantly regulate flowering time and phase change(Cardon et al.1997;Jung et al.2011;Yu et al.2012).

There are two SPLs in G2(SmSPL14 and AtSPL13)and G3(SmSPL12 and AtSPL8),three(SmSPL4,SmSPL7,and AtSPL7)in G6(Figure 5).The number of SPLs is relatively small compared with G1,G4,and G5,suggesting less duplication events occurred for G2,G3,and G6 SPLs.G3 members contain motif 12 conserved among 9 of 10 G1 SPLs(Figure 5).Consistently,AtSPL8 plays redundant roles in male fertility with AtSPL2,AtSPL9,and AtSPL15,which belong to G1(Xing et al.2010).The members of G6 belong to long SPLs with multiple conserved motifs(Figure 4).AtSPL7 can directly bind to the conserved Cu-responsive element(CuRE)containing a core sequence of GTAC and is a significant regulator of Cu homeostasis in Arabidopsis(L?nnenp?? et al.2004;Yamasaki et al.2009).It indicates SmSPL4 and SmSPL7 may be the key regulators for Cu homeostasis in S.miltiorrhiza.

Figure 1.Gene structures of SPLs in Salvia miltiorrhiza and ArabidopsisExons,introns,SQUAMOSA promoter binding protein(SPB)domains,ANK/ANK-2 domains,and intron phases are shown.

Figure 2.Sequence logo of the SQUAMOSA promoter binding protein(SPB)-domain in SmSPLsBits represent the conservation of sequence at a position.Two zinc finger structures(Zn-1 and Zn-2)and the nuclear localization signal(NSL)are shown.

miR156/157-mediated posttranscriptional regulation of SPL genes in S.miltiorrhiza

A subset of SPL genes in Arabidopsis has been confirmed to be targets of miR156/157(Schwab et al.2005;Wu and Poethig 2006;Gandikota et al.2007;Addo-Quaye et al.2008).They belong to G1,G2,and G5,all of which are short SPLs(Figure 4,Table 1).With the online plant target prediction tool,known as psRNATarget(Dai and Zhao 2011),we screened SPL transcripts targeted by miR156/157 in S.miltiorrhiza.A total of eight SmSPLs were predicted to be targets of miR156/157(Figure 6).The complementary sites of miR156/157 are in coding regions for five SmSPLs(SmSPL2,SmSPL5,SmSLP6,SmSPL11,and SmSPL14)belonging to G1 and G2,and one(SmSPL8)belonging to G5,whereas it locates in the 3′UTR for the other two(SmSPL3 and SmSPL15)belonging to G5(Figure 6).It is consistent with the results from Arabidopsis(Rhoade et al.2002;Yang et al.2008).Using the modified 5′-rapid amplification of cDNA ends(Lu et al.2005),we experimentally validated all eight SmSPLs to be authentic targets of miR156/157(Figure 6).Similar to Arabidopsis SPLs,the eight SmSPLs regulated by miR156/157 belong to G1,G2,and G5 and all members in these groups are targets of miR156/157,suggesting the conservation of miR156/157-mediated regulation of SPLs in plants.

Figure 3.Sequence logo of the ANK/ANK-2 domain in SmSPLs and AtSPLsIt includes SmSPL1,SmSPL9,SmSPL10,SmSPL13,AtSPL1,AtSPL12,AtSPL14,and AtSPL16.Bits represent the conservation of sequence at a position.

Expression patterns of SmSPLs in S.miltiorrhiza at different developmental stages

SPLs play significant regulatory roles in various developmental processes of Arabidopsis.In order to obtain preliminary information on the functions of SPLs in S.miltiorrhiza,we investigated the expression levels of SmSPLs in roots,stems and leaves of S.miltiorrhiza at different developmental stages,including 1-and 3-month-old and flowering(Figure 7).All miR156/157-targeted SmSPLs,including SmSPL2(Figure 7B),SmSPL5(Figure 7E),SmSPL6(Figure 7F),and SmSPL11(Figure 7K)belonging to G1,SmSPL14(Figure 7N)belonging to G2,and SmSPL3(Figure 7C),SmSPL8(Figure 7H),and SmSPL15(Figure 7O)belonging to G5,showed the increased expression levels with the maturation of S.miltiorrhiza plants.It is particularly true for the expression in leaves.Moreover,the expression of G5 SmSPLs showed the highest expression level in flowers(Figure 7C,H,O).These results are consistent with those for miR156/157-regulated AtSPLs(Wu and Poethig 2006;Wang et al.2009;Yu et al.2010).The other short SmSPL,SmSPL12,which belongs to G3,is also highly expressed in flowers(Figure 7L).It is consistent with the role of its Arabidopsis homolog,AtSPL8,in the development of flowers(Unte et al.2003;Zhang et al.2007;Xing et al.2010).However,we observed a high expression of SmSPL12 in 1-month-old young stems(Figure 7L),indicating the function of SmSPL12 in the development of organs other than flowers.SmSPL1(Figure 7A),SmSPL9(Figure 7I),SmSPL10(Figure 7J),and SmSPL13(Figure 7M),long SPLs belonging to G4,showed more complicated expression patterns compared with miR156/157-regulated SmSPLs.All of them showed increased expression in leaves with the maturation of S.miltiorrhiza plants,whereas the expression in stems was decreased.Moreover,the expression of SmSPL1(Figure 7A)and SmSPL10(Figure 7J)were increased with the maturation of roots,whereas SmSPL9(Figure 7I)and SmSPL13(Figure 7M)were decreased.The expression of SmSPL4 and SmSPL7 in the other long SPL group,G6,were more constant in all the tissues analyzed compared with G4 SmSPLs,except the expression of SmSPL4 in 1-monthold roots and SmSPL7 in 1-month-old stems(Figure 7D,G).These results provide useful information for further elucidating the functions of SPLs in the development of S.miltiorrhiza plants.

Negative correlation of miR156/157 and miR172 expression in S.miltiorrhiza

In Arabidopsis,a subset of miR156/157-regulated AtSPLs,such as AtSPL9 and AtSPL10,activate the expression of miR172(Wu et al.2009).In order to know the expression patterns of miR156/157 and miR172 in the development of S.miltiorrhiza,the levels of two mature miR156/157,miR156a and miR156b,and two mature miR172,miR172a,and miR172b,were analyzed using the miRNA-specific qRT-PCR method(Shi and Chiang 2005).The levels of miR156a and miR156b decreased in roots,stems and leaves with the development of S.miltiorrhiza plants.Conversely,the levels of miR172a and miR172b increased(Figure 8).Negative correlation of the expression of miR156 and miR172 was also observed in Arabidopsis(Wu et al.2009),suggesting that miR156-and miR172-mediated regulation of developmental timing is conserved between Arabidopsis and S.miltiorrhiza.

DISCUSSION

Salvia is a large plant genus widely distributed in the world.It includes about 900 species,many of which have great economic and medicinal value.For instance,S.miltiorrhiza Bunge,known as Danshen in Chinese,has been widely used in Traditional Chinese medicine(TCM)for more than 1,700 years(Cheng 2006).It is also the first Chinese medicinal material entering the international market.Because of the relatively small genome size(～600 Mb),short life cycle,undemanding growth requirements and significant medicinal value,S.miltiorrhiza is being developed to be a model plant for TCM studies(Ma et al.2012).A working draft of the S.miltiorrhiza genome has been recently obtained(Chen S.et al.unpubl.data 2010).However,the progress of biological studies on S.miltiorrhiza is significantly less compared with other model plants,such as Arabidopsis and rice.The function of transcription factors is poorly understood in S.miltiorrhiza.Through a genome-wide identification and subsequent molecular cloning,we obtained the first set of SmSPLs(Table 1),showing the existence of at least 15 SmSPLs in S.miltiorrhiza.It provides a great example for understanding transcription factors and their regulatory roles in the growth and development of S.miltiorrhiza.

Figure 4.Conserved motifs predicted by MEME

Phylogenetic tree analysis showed that SmSPLs are clustered into six groups(Figure 5).Many sequence features are conserved between SmSPLs and their Arabidopsis homologs in the same group or subgroup(Figure 1),suggesting the close evolutionary relationship between SmSPLs and AtSPLs.Additionally,using the motif discovery tool,we identified a total of 20 conserved motifs in SmSPLs and AtSPLs(Figure 4).Although most of them are functionally unknown,many commonly exist in a group or subgroup of SPLs,implying functional redundancy of SPLs in a group or subgroup.Consistently,AtSPL2,AtSPL10,and AtSPL11,which belong to G1a,redundantly control proper development of lateral organs in association with shoot maturation in the reproductive phase(Shikata et al.2009).G5 AtSPLs,including AtSPL3,AtSPL4,and AtSPL5,activate the expression of LFY,FUL,and AP1 genes(Wang et al.2009)and play redundant roles in reproductive transition(Cardon et al.1997;Gandikota et al.2007;Wang et al.2009;Yamaguchi et al.2009;Jung et al.2012;Porri et al.2012;Yu et al.2012).Moreover,SmSPLs probably play similar biological functions as their Arabidopsis counterparts in the same group or subgroup because of high sequence homology and the presence of conserved motifs.

Figure 5.Phylogeny relationship of SmSPLs and AtSPLsThe unrooted neighbor-joining tree was constructed using MEGA4(Tamura et al.2007).G1–G6 indicate the six groups identified.

Figure 6.Experimental validation of miR156-directed cleavage of SmSPLsThe cleavage sites of SmSPLs(A–H)were determined by the modified 5′RNA ligase-mediated rapid amplification of cDNA ends(RACE).Heavy grey lines represent open reading frames(ORFs).The lines flanking ORFs represent nontranslated regions.SQUAMOSA promoter binding protein(SPB)domains are indicated in blue.MiRNA complementary sites with the nucleotide positions of SmSPL cDNAs are indicated in green.The RNA sequence of each complementary site from 5′to 3′and the predicted miRNA sequence from 3′to 5′are shown in the expanded regions.Watson-Crick pairing is indicated by vertical dashes.Vertical arrows indicate the 5′termini of miRNA-guided cleavage products,as identified by 5′-RACE,with the frequency of clones shown.

AtSPL7 belonging to G6,activates the expression of various genes associated with Cu homeostasis in Arabidopsis.It directly binds to the promoter regions known as Cu-responsive elements(CuREs),and is considered to be a central regulator for copper homeostasis(L?nnenp?? et al.2004;Yamasaki et al.2009;Lu et al.2011).Only one homolog of AtSPL7 was found in rice(Yang et al.2008),tomato,grape,and Physcomitrella patens(Li et al.2013).In this study,we identified two SmSPLs(SmSPL4 and SmSPL7)clustering with AtSPL7(Figure 5).These SPLs commonly contain motifs 1,3,4,9,and 15.However,SmSPL4 and AtSPL7 contain an additional motif 6,while SmSPL7 has an additional motif 2(Figure 4).Consistently,the expression patterns of SmSPL4 and SmSPL7 were similar in most tissues analyzed,while the levels of SmSPL4 in 1-monthold roots and SmSPL7 in 1-month-old stems were relatively high(Figure 7).It implies the redundant and specific roles of SmSPL4 and SmSPL7 in the development of S.miltiorrhiza plants.

Although SmSPL4,SmSPL7,AtSPL7,and members of the other long SPL group,G4,are not regulated by miR156/157,almost all short SPLs except AtSPL8 and SmSPL12 were confirmed to be targets of miR156/157(Figure 6),suggesting the existence of distinct regulatory mechanisms for SPLs.AtSPL8 regulates microsporangia development,megasporagenesis,trichome formation on sepals,stamen filament elongation(Unte et al.2003).It also acts as a local regulator in a subset of GA-dependent developmental processes(Zhang et al.2007).The regulatory roles of AtSPL8 in the development of anther and gynoecium are functionally redundant with AtSPL2,AtSPL9,and AtSPL15,which belong to G1 and target by miR156/157(Xing et al.2010,2013).SmSPL12 is the homolog of AtSPL8 in S.miltiorrhiza(Figure 5),indicating it probably regulates the development of reproductive organs in S.miltiorrhiza with miR156/157-targeted SPLs belonging to G1.Functional redundancy of miR156/157-targeted and nontargeted SPLs suggests the complexity and significance of SPL-related regulatory network.

All of the members of G1,G2 and G5 are regulated by miR156/157(Figure 6).In Arabidopsis,G5 SPLs,including AtSPL3,AtSPL4,and AtSPL5,redundantly regulate flowering time and phase change through direct activation of a subset of transcription factor genes,such as LEAFY(LFY),FRUITFULL(FUL),and APETALA1(AP1)(Yamaguchi et al.2009),whereas various G1 SPLs,such as AtSPL9,AtSPL10,and probably AtSPL11 and AtSPL15,act redundantly in controlling flowering time and phase change by directly activating the transcription of MIR172 genes,which further promote plant development and flowering(Wu et al.2009).Due to the conservation of AtSPLs and SmSPLs this regulatory mechanism may also exist in S.miltiorrhiza.Consistently,the expression of miR156/157-regulated SmSPLs increased with the maturation of S.miltiorrhiza plants,and the expression of miR156/157 was negatively correlated with miR172(Figures 7,8).Further analysis of transgenic S.miltiorrhiza plants with up-or downregulated SPLs,miR156/157 or miR172 will help us understand the regulatory network of SmSPLs.

Figure 7.Differential expression of SmSPLs in Salvia miltiorrhizaRelative expression of SmSPL1–SmSPL15(A–O)was quantified in total RNA isolated from roots(Rf),stems(Sf),leaves(Lf)and flowers(F)of field nursery-grown plants with flowers and roots(R1 and R3),stems(S1 and S3)and leaves(L1 and L3)of 1-and 3-month-old plants cultivated in vitro by quantitative real-time reverse transcription-polymerase chain reaction(RT-PCR)and normalized to the level of SmUBQ10 in the sample.Fold changes of transcript level are shown.Error bars represent the standard deviations of three technical replicates.Normalized mRNA levels in flowers were arbitrarily set to 1.

Figure 8.Negative correlation of miR156 and miR172 expression in Salvia miltiorrhizaRelative expression of miR156a(A),miR156b(B),miR172a(C)and miR172b(D)was quantified in total RNA isolated from roots(Rf),stems(Sf),leaves(Lf)of field nursery-grown plants with flowers and roots(R1 and R3),stems(S1 and S3)and leaves(L1 and L3)of 1-and 3-month-old plants cultivated in vitro by quantitative real-time reverse transcription-polymerase chain reaction(RT-PCR)and normalized to the level of 5.8S rRNA in the sample.Fold changes of transcript level are shown.Error bars represent the standard deviations of three technical replicates.Normalized mRNA levels in R1 were arbitrarily set to 1.

MATERIALS AND METHODS

Plant materials

Salvia miltiorrhiza Bunge(line 993)was grown in a field nursery of the Institute of Medicinal Plant Development.Roots,stems,leaves,and flowers were collected from 1-year-old S.miltiorrhiza in August when the plants were blooming.The tissues were named Rf(roots from plants with flowers),Sf(stems from plants with flowers),Lf(leaves from plants with flowers),and F(flowers),respectively,and then stored in liquid nitrogen until use.Roots,stems and leaves were also collected from 1-and 3-month-old S.miltiorrhiza plants cultivated in vitro and named R1,S1,and L1,respectively,for 1-month-old plants,and R3,S3,and L3,respectively,for 3-month-old plants.

Database search and gene prediction

The nucleotide and amino acid sequence information of Arabidopsis AtSPLs were obtained from the Arabidopsis Information Resource(TAIR,http://www.arabidopsis.org/).SmSPL genes were identified by tBLASTn analysis(Altschul et al.1997)of AtSPL protein sequences against the current assembly of the S.miltiorrhiza genome(Chen S.et al.unpubl.data 2010).Gene models of SmSPLs were predicted on the Genscan web server(http://genes.mit.edu/GENSCAN.html)(Burge and Karlin 1998).The predicted models were manually corrected by comparison with known plant SPLs using the BLASTx algorithm(http://www.ncbi.nlm.nih.gov/BLAST).

Analysis of gene structures,conserved domains and motifs

Gene structures were analyzed using the Gene Structure Display Sever(http://gsds.cbi.pku.edu.cn/index.php).Conserved domains were analyzed by BLAST analysis of SmSPL protein sequences against the Conserved Domain Database(CDD,http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi)with the expected E-value threshold of 0.01 and the maximum size of hits to be 500 amino acids(Marchler-Bauer et al.2011).Sequence logos were created using WebLogo(http://weblogo.berkeley.edu/).Protein alignment of SBP and ANK domains were carried out using DNAMAN version 6(Lynnon BioSoft).Conserved motifs were predicted using MEME version 3.0.14(Bailey and Elkan 1994,Bailey et al.2009).

Phylogenetic analysis

A phylogenetic tree was constructed with the full-length SmSPL and AtSPL protein sequences using MEGA version 4.0 by the neighbor-joining method with 1,000 bootstrap replicates(Tamura et al.2007).

RNA isolation

Total RNA was extracted from roots,stems,leaves and flowers of S.miltiorrhiza using the EASYspin Plant microRNA Extract kit(Aidlab Biotechnologies,Beijing,China).The quality and quantity of total RNA was analyzed with agarose gel electrophoresis and nanodrop 2000 spectrophotometer(Thermo Scientific,Wilmington,DE,USA).Genomic DNA was removed by treating total RNA with RNase-free DNase(Promega,Madison,WI,USA).

Molecular cloning of SmSPLs

The 5′and 3′regions of SmSPL8 and SmSPL14,showing low sequence homology with known plant SPLs,were amplified by the 5′and 3′rapid amplification of cDNA ends(RACE)method with the GeneRacer kit(Invitrogen,Carlsbad,CA,USA).Plant mRNA was isolated using the Oligotex mRNA Mini kit(Qiagen,Hilden,Germany).Total RNA was reverse-transcribed into cDNA using Superscript III reverse transcriptase(Invitrogen).5′and 3′RACE was performed according to the instruction of GeneRacer kit using gene-specific nesting and nested primers listed in Table S1.The full-length coding regions of SmSPLs were amplified by PCR using the gene-specific forward and reverse primers listed in Table S1.PCR products were gel-purified,cloned,and then sequenced.

Prediction and experimental validation of SmSPLs targeted by miR156

SmSPLs targeted by miR156 were predicted using psRNATarget(Dai and Zhao 2011,http://plantgrn.noble.org/psRNATarget/?function=3)with the maximum expectation of 3 and the target accessibility(UPE)of 50.For experimental validation,total RNA was extracted from S.miltiorrhiza tissues using the EASYspin Plant microRNA Extract kit(Aidlab Biotechnologies).mRNA was isolated using the Oligotex mRNA mini kit(Qiagen).The modified RNA ligase-mediated rapid amplification of 5′cDNAs method(5′RLM-RACE)was performed using the GeneRacer kit(Invitrogen)as described previously(Lu et al.2005).Genespecific primers used are listed in Table S2.

Quantitative real-time PCR

RNase-free DNase-treated total RNA were reverse-transcribed into cDNA using Superscript III reverse transcriptase(Invitrogen).Quantitative real-time PCR(qRT-PCR)of SmSPLs was performed as described previously(Ma et al.2012).Genespecific primers were designed using the IDT online tools(http://www.idtdna.com/site)and listed in Table S3.The expressions of miR156 and miR172 were analyzed using the miRNA-specific qRT-PCR method as described previously(Shi and Chiang 2005).Primers are shown in Table S3.

ACKNOWLEDGEMENTS

We thank Dr Shilin Chen and the sequencing group in our institute for kindly providing the Salvia miltiorrhiza genome sequence.We appreciate Professor Xian’en Li for providing S.miltiorrhiza plants.This work was supported by grants from the Beijing Natural Science Foundation(Grant No.5112026 to S.L.),the Major Scientific and Technological Special Project for Significant New Drugs Creation(Grant No.2012ZX09301002-001-031 to S.L.),the Research Fund for the Doctoral Program of Higher Education of China(20111106110033 to S.L.),the Program for Changjiang Scholars and Innovative Research Team in University(PCSIRT,Grant No.IRT1150),and the Program for Xiehe Scholars in Chinese Academy of Medical Sciences&Peking Union Medical College(to S.L.).

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SUPPORTING INFORMATION

Additional supporting information can be found in the online version of this article:

Figure S1.Protein sequence alignment of the SBP domain identified in 15 SmSPLs.

Figure S2.Protein sequence alignment of the ANK domain identified in SmSPL1,SmSPL9,SmSPL10,SmSPL13,AtSPL1,AtSPL12,AtSPL14,and AtSPL16.

Table S1.Gene-specific primers used for PCR amplification of SmSPLs.

Table S2.Primers used for 5′RLM-RACE validation of SmSPLs targeted by miR156.

Table S3.Gene-specific primers used for quantitative real-time PCR of SmSPLs,miR156a/b and miR172a/b.