Transcript profiling of microRNAs during the early development of the maize brace root via Solexa sequencing

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Transcript pro

ﬁling of microRNAs during the early development of the maize brace

root via Solexa sequencing

Peng Liu

1

, Kang Yan

1

, Yun-xue Lei, Rui Xu, Yue-min Zhang, Guo-dong Yang, Jin-guang Huang,

Chang-Ai Wu

⁎

, Cheng-Chao Zheng

⁎⁎

State Key Laboratory of Crop Biology, College of Life Sciences, Shandong Agricultural University, Taian, Shandong 271018, PR China

a b s t r a c t

a r t i c l e i n f o

Article history: Received 16 June 2012 Accepted 2 November 2012 Available online 10 November 2012 Keywords:

Deep sequencing Zea mays Brace roots Auxins

Small non-coding RNAs

To characterize the microRNAs that contribute to the development of brace root, Solexa high-throughput sequencing of three libraries derived from tissues of node (N), nodes with just-emerged brace roots (NR), and nodes with just-emerged brace roots after IAA treatment (NRI) was performed. Total 650,793, 957,303 and 1,082,948 genome-matched unique reads were obtained in N, NR and NRI libraries, respectively. Further analysis confirmed the authenticity of 137 known miRNAs and the discovery of 159 novel miRNAs in maize. 14 conserved and 16 novel miRNAs differentially expressed in brace root, as well as 15 target genes, were identified and validated by qRT-PCR during maize brace root development. Moreover, we identified 9 miRNA precursor-matched novel sRNAs that may form miRNA clusters, as well as 24 nt siRNAs in the three libraries. In addition, we suggest that auxin represent a regulator in brace root development and can be reg-ulated at the posttranscriptional level by miRNAs.

1. Introduction

Maize (Zea mays L.) displays a complex root structure comprising several root types. The underground portion includes the primary root, lateral root, seminal root, and crown root, whereas the above-ground portion includes brace roots emerging from the stem nodes of successive basal phytomers [1,2]. The brace root (also termed nodal adventitious root) is a type of aerial root that contributes enor-mously to lodging resistance and water and nutrient uptake during the later growth and development stage of maize plants[3,4]. Most importantly, brace roots have a substantial inﬂuence on grain yield under soil_{ﬂooding and water-limited conditions}[5].

In the past decades, research on the maize brace roots has been focused mainly at the morphological and physiological levels. The primordia of brace roots have been well described as developing from dedifferentiated cells of the stem parenchyma, just behind the stem cortex and below the intercalary meristem of the overlaying internodes[6]. Previous studies have demonstrated that many nutri-tional or environmental factors could affect brace root formation. Nu-trient deﬁciencies, such as those of phosphorus and nitrogen, could decrease the rate of emergence or the number of brace roots[7,8]. The number of brace roots emerging from upper nodes is lower when the carbohydrate nutrition of plants is reduced by shading or

low light as a result of large plant densities[9]. Soil ridging is also an important factor that could increase the number of functional brace roots. Later ridging tends to result in shorter internodes and more func-tional nodal roots, leading to better lodging resistance[10]. However, to date, knowledge concerning their initiation or early development at the molecular level remains poor, and only two maize mutants deficient in shoot-borne root formation have been isolated. The mutant rt1 is thefirst root formation mutant isolated and shows a reduced number of shoot-borne roots at higher nodes (nodes 7 and 8), whereas only a slight difference is observed in the number of crown roots at thefirst two nodes[11]. The rt1 mutation is inherited as a monogenic recessive trait and mapped on chromosome 3[6]. In contrast, the rtcs mutation

[12]is completely devoid of all shoot-borne roots and embryonic sem-inal roots because of inhibition of the initiation of the affected root-types. The RTCS encodes a LOB domain protein[12,13]. We performed deep sequencing analysis and impressed the complex changes in the transcriptomes during the early development of maize brace root[14]. However, no other reports on genes regulating brace root development have yet been found.

In recent years, small regulatory RNAs have attracted attention be-cause of their important roles in posttranscriptional or translational gene regulation[15–17]. MicroRNAs (miRNAs) and small interfering RNAs (siRNAs) are the two major groups of small RNAs in plants that serve as negative regulators of gene expression. miRNAs are viewed as endogenous and purposefully expressed products of an organism's own genome and processed from hairpin precursors by the ribonucle-ase III-like enzyme Dicer Dicer-like1 (DCL1) or DCL4. Unlike miRNAs, siRNAs are thought to be primarily exogenous in origin, derived directly

⁎ Corresponding author.

⁎⁎ Corresponding author. Fax: +86 538 8226399.

E-mail addresses:[email protected](C.-A. Wu),[email protected](C.-C. Zheng).

1

These authors contributed equally to this work.

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Genomics

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from the virus, transposon, or transgene trigger, and are generated from perfectly complementary, long, double-stranded RNAs by RNA– dependent RNA polymerase 6 (RDR6)/RDR2[18,19].

Studies indicate that miRNA-regulated auxin homeostasis is an important regulator of plant root development: MiR160 targets AUXIN RESPONSE FACTOR17 (ARF17) to regulate adventitious rooting in Arabidopsis[20]; MiR164-directed cleavage of NAC1 mRNA affects auxin regulation in lateral root development in Arabidopsis [21]; MiR165 and miR166 target HD-ZIP III transcription factor mRNAs in the endodermis and stele periphery and further determine root cell fates in a dosage-dependent manner[22]; MiR390 and ARFs form an auxin-responsive regulatory network controlling lateral root growth in Arabidopsis[23]. Brace root initiation is similar to that of lateral or adventitious roots[24]. Therefore, it is interesting to determine whether the maize brace root formation shares the common miRNA regulatory mechanisms with these underground roots. To obtain a com-prehensive and unbiased miRNA transcript proﬁle during maize brace root formation together with auxin treatment, we performed deep se-quencing analysis using the Illumina_{⁄ Solexa digital gene expression} (DGE) system because that this system enables the sequencing of total cDNA for the derivation of an accurate measure of gene expression, both individually and comprehensively, and the discovery of novel regions of transcription, dramatically changing the way that the func-tional complexity of transcriptome can be studied.

In the present study, an overall impression of miRNA proﬁles dur-ing the early development of the maize brace root was acquired by deep sequencing. For theﬁrst time, we have comprehensively charac-terized the miRNA regulation basis of the physiological processes during maize brace root formation and provide useful information for further research.

2. Results and discussion

2.1. High-throughput sequencing of maize small RNAs

To identify microRNAs involved in brace root development and further auxin-dependent brace root development, we performed Solexa sequencing of three small RNA libraries derived from maize node tissues (N), nodes with just-emerged brace roots (NR), and nodes with ust-emerged brace roots after IAA treatment (NRI) (Fig. S1). The sequenc-ing effort resulted in over 11,357,201, 12,728,574, and 11,949,415 raw reads from the N, NR, and NRI datasets, respectively (Table S1). After discarding low-quality sequences and adapter sequences, 8,558,568 (75.36%), 9,723,311 (79.39%), and 10,605,379 (88.75%)

clean reads ranging from 18 to 31 nucleotides were generated from the N, NR, and NRI libraries, respectively. The overall size distribution of all the sequenced reads from three sequencing efforts were very similar, with the 24 nt class being the most abundant, followed by the 21 nt class (Fig. 1). Such a size distribution suggests that sRNAs were speci_{ﬁcally enriched between 21 and 24 nt in maize. The length of} miRNA is ~21 nt, as well as siRNA ~24 nt, thus, the peaks of abundance in maize reveals that miRNAs and siRNAs may play important roles in the nodes.

Generating a reference set of annotations is essential to exploring the small RNA categories. All identical Solexa reads in each library were sorted into unique sequence tags for further analysis. The unique reads out of the clean reads for N, NR, and NRI libraries were 650,793 (7.6%), 957,303 (9.8%), and 1,082,948 (10.2%), respectively (Table S1), which were perfectly mapped to maize B73 RefGen_v2 (release 5a.59 in November 2010). Genome-matched sequences were classi_{ﬁed into} sev-eral groups, including known miRNAs, rRNAs, tRNAs, snRNAs, snoRNAs, and others (Table 1). Known miRNAs accounted for 0.08%, 0.06%, and 0.04% of the small RNA libraries for N, NR, and NRI, respectively. The highest proportion of genome-matched sequences included un-annotated small RNA sequences, which may include novel miRNA candidates. However, these results indicate that maize brace root con-tains a large and diverse small RNA population at very early stages of development.

Small interfering RNA (siRNA), another important class of non-coding small RNA, is a 21–24 nt long double-strand RNA, each strand of which is 2 nt longer than the other on the 3′ end. According to this structural feature, we aligned tags from clean reads to each other toﬁnd sRNAs meeting this criterion as potential siRNA candi-dates. We analyzed 24nt siRNA in the three libraries (Fig. 2A). Among these, 3347 were commonly predicted in the N and NR libraries; 3957 were commonly predicted in the N and NRI libraries; 7298 were commonly predicted in the NR and NRI libraries; and 2732 were commonly predicted in all three libraries. In addition, 427 down-regulated and 68 up-down-regulated 24nt siRNAs in barce roots were identi-ﬁed (Fig. 2B). The disparity of classes and numbers suggest that 24nt siRNAs may also play roles in the early development of brace roots. 2.2. Analysis of differentially expressed conserved miRNAs

All small RNA sequences were Blastn-searched against known ma-ture maize miRNAs and their precursors in the miRNA database miRBase (http://www.mirbase.org/, released 18 November 2011) to identify conserved miRNAs in the three libraries. Blastn searches and

10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 0 10 20 30 40 50 N NR NRI

Proportion of small RNA (%)

Length(nt)

Fig. 1. Length distribution and abundance of the sequence. Histogram presentation of sequence-length distribution for clean reads in three libraries. The x-axis indicates sequence sizes from 10 nt to 31 nt. The y-axis indicates the percent of reads for every given size. The histogram shows there are two peaks in small RNAs proﬁle in maize.

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further sequence analysis showed that 137 known miRNAs belong-ing to 26 miRNA families were identiﬁed in the three libraries (Table S2). The identiﬁed miRNA families are conserved in a variety of plant species. For example, miR156, miR159, miR166, and miR169 have been found in 51, 45, 41, and 40 plant species, respectively[25,26]. The diversity of maize miRNAs could also be found in the number of members they contained (Fig. S2). The largest miRNA family sequenced was miR166, consisting of 14 members, followed by

miR171, miR156, miR169, miR395, and miR167 with 13, 12, 11, 11, and 10 members, respectively. Other miRNA families, such as miR162, miR529, miR827, miR1432, and miR2118, had only one member detected in each this period. The size of miRNA families may be indicative of their functional complexity.

Maize miRNA families displayed signi_{ficantly varied abundances} with one another (Table S2). This variation in abundance of the miRNA families suggests that miRNA genes would be differentially transcribed during brace root development. For example, maize miR168 families were sequenced more than 550,000 times, while miR166 families were sequenced more than 20,000 times. In contrast, miR395 families were detected less than 10 times; miR169 families were detected less than 50 times. Two known miRNA families, miR397 and miR482, were not successfully detected in our datasets. Different family members also displayed drastically different abundance levels (Table S2). For instance, the abundance of the miR156 family varied from 8 (zma-miR156j) to 11,538 reads (zma-miR156d) during deep sequencing. The same results were obtained for some other miRNA families, such as zma-miR159 (6 to 37,748 reads) and zma-miR166 (5100 to 353,290 reads). Abundance comparison among different members within a miRNA family may provide valuable information on the role that the miRNAs play in that plant-specific development stage. These may suggest tissue-, development-, and species-specific expression profiles of miRNAs.

We identified differentially expressed miRNAs in brace root by comparing normalized expression profiles of 137 known miRNAs (Fig. S3). Overall, 19 miRNAs forming 11 mature miRNAs were differ-entially expressed with a fold change (FC, Log2Ratio) >1.5 orb−1.5 and a p-valueb0.001 in the N and NR libraries (Table 2). However, only three miRNAs, including zma-miR156j and zma-miR169a/b, were downregulated. The most significant upregulation was observed for zma-miR171g, with an FC > 9 in the NR library compared with the N library, followed by zma-miR164e and zma-miR171h/k with FCs >5 and >4, respectively.

Using the same selection criterion, 21 known miRNAs were differ-entially expressed in NRI library compared with NR library (data not shown). Thus, a larger number of miRNAs were regulated by auxin. Six of 21 miRNAs regulated by IAA were differentially expressed in the N and NR libraries simultaneously (Table 2, marked with a star). These expression proﬁles strongly indicate that auxin may contribute to the early development of maize brace root.

Nineteen miRNAs differently expressed in N vs. NR were validated by detecting the mature miRNA by qRT-PCR to validate the data. Ex-pressions of miR156j and miR169a/b were downregulated (Figs. 3G and1H) while those of others miRNAs were upregulated in the NR library compared with the N library (Figs. 3A to1F), in agreement with the sequencing datasets. However, mature miRNA levels of zma-miR171a, zma-miR399a/c/h, and zma-miR399d showed almost

Table 1

Total genome matching reads from three libraries.

Locus Unique reads in N Unique reads in NR Unique reads in NRI Exon_antisense 14,020 (2.15%) 20,564 (2.15%) 22,010 (2.03%) Exon_sense 23,072 (3.55%) 32,682 (3.41%) 34,418 (3.18%) Intron_antisense 24,595 (3.78%) 36,314 (3.79%) 40,447 (3.73%) Intron_sense 60,530 (9.30%) 84,593 (8.84%) 89,031 (8.22%) rRNA 57,726 (8.87%) 43,291 (4.52%) 50,695 (4.68%) siRNA 11,418 (1.75%) 31,761 (3.32%) 27,073 (2.50%) tRNA 4288 (0.66%) 3975 (0.42%) 4735 (0.44%) snRNA 858 (0.13%) 995 (0.10%) 835 (0.08%) snoRNA 302 (0.05%) 353 (0.04%) 409 (0.04%) miRNA 511 (0.08%) 617 (0.06%) 462 (0.04%) Unannotated 453,473 (69.68%) 702,158 (73.35%) 812,833 (75.06%) Total 650,793 957,303 1,082,948

A

B

up regulated down regulated Number of 24nt siRNA 600 400 200 0 200 NR/N 7916 NRI 13686 N 2572 NR 2732 615 4566 1225

Fig. 2. Number of 24 nt siRNA in three libraries. (A) Classes of 24nt siRNA sequenced in the three libraries. 7154, 15,829 and 21,209 were obtained in N, NR and NRI, respectively. (B) Number of differentially expressed 24 nt siRNAs in barce roots; down-regulated, grey; up-regulated, black. The x-axis indicates NR library compared with N library. The y-axis indicates the number of 24nt siRNAs.

Table 2

Differentially expressed conserved miRNAs.

Name Sequence (5'- 3') L (nt) Fold change

Log2 (NR/N) Log2 (NRI/NR)

Up-regulated zma-miR171g GAGGUGAGCCGAGCCAAUAUC 21 9.03 −1.55⁎ zma-miR164e UGGAGAAGCAGGACACGUGAG 21 5.96 −0.29 zma-miR171h/k GUGAGCCGAACCAAUAUCACU 21 4.06 −0.30 zma-miR167a/b/c/d UGAAGCUGCCAGCAUGAUCUA 21 2.35 −0.61 zma-miR390a/b AAGCUCAGGAGGGAUAGCGCC 21 2.17 −0.52 zma-miR399d UGCCAAAGGAGAGCUGCCCUG 21 2.17 −2.63⁎ zma-miR399a/c/h UGCCAAAGGAGAAUUGCCCUG 21 1.80 −2.47⁎ zma-miR171a UGAUUGAGCCGCGCCAAUAU 20 1.75 −3.93⁎ zma-miR393b UCCAAAGGGAUCGCAUUGAUCC 22 1.74 −0.50 Down-regulated zma-miR156j UGACAGAAGAGAGAGAGCACA 21 −2.75 −0.71 zma-miR169a/b CAGCCAAGGAUGACUUGCCGA 21 −1.84 −0.07

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no change (Fig. 3I), which was not consistent with the results of the sequencing data; for these miRNAs, FCs of only 1.75, 1.8, and 2.17 FC, respectively, were obtained (Table 2). Zma-miR396a/b, which showed no discrepancies in the three libraries, was selected as control (Fig. 3I). Therefore, 14 conserved miRNAs were conﬁrmed to be differentially expressed during brace root development.

Nitrogen deﬁciencies could decrease the rate of emergence or num-ber of brace roots[7,8]. The expressions of miR156j, miR164e, miR171g,

and miR393b have also been reported to be upregulated by nitrogen de-ﬁciency in maize root[27]. In the present study, we demonstrated that miR156j, miR164e, miR171g, and miR393b are regulated by the emer-gence of brace roots. Therefore, the emeremer-gence of brace roots may re-quire high levels of nitrogen nutrition, which could be regulated at the posttranscriptional level by miRNAs. Moreover, miR171g, as well as miR167a/b/c/d, were responsive to salt stress in maize root[28]. In Arabidopsis, miR169 was reported to target the NFYA transcription factor

0 2 8 10 12 0.0 .5 1.0 1.5 2.0 2.5 0.0 .5 1.0 1.5 2.0 2.5 3.0 3.5 0.0 .5 1.0 1.5 2.0 2.5 3.0 0 2 4 6 8 10

E

D

A

B

C

G

0.0 .5 1.0 1.5 2.0 2.5 3.0 3.5 0.0 .2 .4 .6 .8 1.0 1.2 1.4

H

I

F

0.0 .5 1.0 1.5 2.0 2.5

Relative expression level

Relative expression level 0.0 .2 .4 .6 .8 1.0 1.2 1.4 1.6

N

NR

NRI

Fig. 3. Expression patterns of mature conserved miRNAs and their predicted target genes related to the brace root. (A–H) Differentially expressed conserved miRNAs and their target genes: (A) zma-miR164e, zma-miR396a/b is a control sequenced with an unchanged level, (B) zma-miR171g, (C) zma-miR171h/k, (D) zma-miR167a/b/c/d, (E) zma-miR390a/b, and (F) zma-miR393b are up-expressed; (G) zma-miR156j and (H) zma-miR156j are down-expressed; error bars represent SE for three independent experiments. N: black; NR: dark-grey; NRI: French grey. Real-time RT-PCR quantiﬁcations were normalized to the expression of U6 RNA (for mature miRNAs) or EF1-α (for target genes). The x-axis indicates name of genes. The y-axis indicates the Relative expression level.

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to promote drought resistance[29]. In the present study, miR171g and miR167a/b/c/d were upregulated whereas miR169a/b was downregulated by the emergence of the brace root (Fig. 3). These ﬁnd-ings suggest the function of the brace root to keep the plant upright, provide a surface for nutrient and water uptake and gas exchange, and help plants adapt well to environmental stresses.

2.3. Target prediction and validation of conserved maize miRNAs differentially expressed in brace root

Investigation of the target mRNAs of the miRNAs identified can as-sist us in understanding their biological roles. As most plant miRNAs exhibit near-perfect complementation to their targets, we utilized the psRNA Target program (http://plantgrn.noble.org/psRNATarget/) to predict the mRNA targets of the 19 differentially expressed and conserved miRNAs related to brace root. Fifty-four targets were iden-ti_{fied (Table S3). We also performed qRT-PCR to detect the predicted} target mRNA level (Figs. 3A to 1H). Some genes were differentially expressed in the N, NR, and NRI libraries, consistent with the expression of miRNAs; thesefindings indicate that the miRNAs may truly act on their predicted targets through mRNA cleavage. For example, zma-miR164e was induced about 10-fold in the brace root, whereas its target, GRMZM2G163975, coding a protein with HLH DNA-binding domain, was downregulated by about 3.5-fold. Similarly, zma-miR171h/k was induced more than 7-fold in the brace root, while its targets GRMZM2G026218 and GRMZM2G021299 were downregulated by about 8-fold and 2-fold, respectively. Three targets (GRMZM2G061734, GRMZM2G307588, and GRMZM5G806833) of miR156j and one target (GRMZM2G038303) of miR169a/b, which encode three SBP-box tran-scription factors and a NF-YA trantran-scription factor, respectively, showed expression patterns similar to those of miR156j and miR169a/b. This result confirms that the four predicted genes were not targets of zma-miR156j or miR169a/b but they may participate in the early develop-ment of maize brace root. Fifteen of 24 target mRNAs showed expres-sions opposite to those of the miRNAs (Table S3, genes marked with

stars) and were thus considered the target genes of miRNAs related to maize brace root.

Then we performed GO analysis to these 15 target genes. They were predicted to have 7 different molecular functions (Fig. 4A) relat-ed to DNA binding, ATP binding, and Auxin binding; they were locatrelat-ed in 5 cellular components (Fig. 4B) including the nucleus, the membrane and the cytoplasm; they were involved in 9 different biological process-es (Fig. 4C), such as auxin mediated signaling pathway, protein phos-phorylation, regulation of transcription, and protein transport.

Four target genes appeared to be auxin-related genes, including two AUXIN RESPONSE FACTOR genes (GRMZM2G078274 and GRMZM2G035405, targets of zma-miR167a/b/c/d), one TRANSPORT INHIBITOR RESPONSE gene (GRMZM2G135978, target of zma-miR393b/c), and one AFB (GRMZM2G137451, target of zma-miR393b/c). These ex-pression proﬁles strongly indicate that auxin may contribute to the early development of maize brace root. In Arabidopsis, intracellular auxin is detected by TIR1/AUXIN SIGNALING F-BOX PROTEIN1-3 (TIR1/AFB1-3) receptors, thereby activating ARF transcription factors, which regulate the expression of auxin-responsive genes[30]. TIR1/AFP has been prov-en to be the target of miR393 in Arabidopsis[31]. MiR390 downregulates ARFs to control lateral root growth in Arabidopsis[23]. These data are in agreement with the report that brace root initiation involves two major steps regulated by auxin-associated networks, similar to those of lateral or adventitious roots at the cellular level[24], and with our previous data, which showed that auxin positively regulates brace root develop-ment[14]. Thus, auxin represents a regulator in both brace root and lat-eral or adventitious root development in higher plants[24,32–35].

2.4. Prediction and invalidation of novel miRNAs differentially expressed in brace root

Deep sequencing of the small RNA transcriptomes yielded data that would help determine known miRNAs and successfully explore novel miRNAs with high accuracy and efﬁciency. We used the Mireap (http://sourceforge.net/projects/mireap/) software to predict novel

Cellular components Unknown 21.43% Nucleus 42.86% Integral to membrane 14.29% Plasma membrane 14.29% Cytoplasm 7.14%

Molecular functions Unknown 21.43% DNA binding 21.43% ATP binding 21.43% Auxin binding 14.29%

G-protein coupled receptor activity 7.14% Ubiquitin-protein ligase activity 7.14% Receptor activity 7.14%

Biological process

Unknown 21.43%

Response to auxin stimulus 14.29% Auxin mediated signaling pathway 14.29% Protein phosphorylation 14.29% Response to light stimulus 7.14% Regulation of transcription 7.14% Protein ubiquitination nucleus 7.14% Microtubule-based movement 7.14% Protein transport 7.14%

A

C

B

Fig. 4. GO annotation of 15 miRNA target gene proteins. Molecular functions (A), cellular components (B), biological processes (C). Fifteen proteins were predicted to have 7 dif-ferent molecular functions, were located in 5 cellular components, and involved in 9 difdif-ferent biological processes.

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miRNAs and their stem-loop structures. To better distinguish miRNAs from other RNAs, we calculated minimal folding free energy index (MFEI), which should be greater than 0.85[36,37]. A total of 497 novel miRNA candidates were obtained from the three libraries, and then we selected only 159 candidate novel miRNAs (belonging to 88 families) commonly found in two or three of the libraries for fur-ther analysis (Table S4). The length of the newly identiﬁed miRNAs ranged from 20 bp to 23 bp in length, and the negative folding free en-ergies varied from−171.5 kcal mol−1_to_{−22.1 kcal mol}−1_{, with an} average of−67.4 kcal mol−1_{. These results are similar to the free} ener-gy values of Arabidopsis miRNA precursors (−57 kcal mol−1_{) and much} lower than the folding free energies of tRNA (−27.5 kcal mol−1_{) or} rRNA (−33 kcal mol−1_{) obtained in a previous report}_[38]_.

Novel miRNAs differentially expressed in brace root were identi-ﬁed. Overall, 25 miRNA were differentially expressed with an FC Log2Ratio >1.5 or_{b−1.5 at a p-valueb0.001 in the N and NR} librar-ies, which includes 12 upregulated and 13 downregulated miRNAs (Table 3). miRNAs were validated by mature miRNA qRT-PCR (Fig. S4). Zma-nmiR21, zma-nmiR38 zma-nmiR70, and zma-nmiR76a/b were more upregulated in the NR library compared in the N one, whereas zma-nmiR9a/b/c, zma-nmiR11a/b, zma-nmiR45a/b/c, zma-nmiR81, zma-nmiR85, and zma-nmiR86 were downregulated. The results were in agreement with the sequencing datasets. The nine other novel miRNA candidates were detected at almost unchanged or opposite levels in qRT-PCR compared with the results observed during sequenc-ing. However, results of the mature miRNA qRT-PCR of the nine novel miRNA candidates were at least invalidated as true miRNAs.

2.5. miRNA precursor-matched novel sRNAs

Total of 511, 617 and 462 sRNAs were matched to the known miRNA precursors in N, NR and NRI libraries, respectively, including mature miRNA, miRNA*, and novel sRNAs. Nine novel sRNAs with high expression level in all three libraries came from zma-miR159, 169 and 319 families (Fig. 5B). These sRNAs were located between the mature miRNA and miRNA* (Fig. 5A, Table S5), thus probably gen-erated in the miRNA processing. In addition, sequences of some sRNAs differed for deriving from varied precursors, such as sRNA319-5′ac (AGCTGCCGACTCATCCATTCA) and sRNA319-5′bd (AGCTGCCGACTCA TTCACCCA), however, which came from the same location of precursor sequences relatively. These novel sRNAs may exist in the form of miRNA clusters with known maize miRNAs, but in need of further examination.

In conclusion, we have sequenced three independent small RNA li-braries derived from N, NR, and NRI through Solexa sequencing. Our data con_{firmed the authenticity of 137 known miRNAs in maize. We} found 159 miRNAs that had not been reported in other species. 14 conserved and 16 novel miRNAs differentially expressed in brace root, as well as 15 target genes, were identi_{fied and validated by} qRT-PCR during maize brace root development. Moreover, we identi-fied 9 miRNA precursor-matched novel sRNAs that may form miRNA clusters, as well as 24nt siRNAs in the three libraries. In addition, auxin may regulate maize brace root development through miRNAs. This work extends insights into miRNA-mediated gene expression regulations in brace root and provides a useful resource for future studies on maize small non-coding RNAs. Further functional analysis of the differentially expressed siRNA, miRNAs and their target genes will provide deeper insight into the regulation of maize brace root development.

3. Materials and methods 3.1. Plant material

The maize (Zea mays) inbred line H5468 was used in the present study. Seeds of H5468 were surface-sterilized with 3% sodium hypochlo-rite for 10 min and rinsed in distilled water. Sterilized seeds were pre-germinated on moistenedﬁlter papers in a plant growth chamber at 60% humidity and 28°C, under a 16: 8 h light⁄ dark cycle for 3 days. Then, the seedlings were transferred into theﬁeld in a greenhouse and cultivated at a mean temperature of 28°C with both natural light and an additional 16: 8 h light_{⁄dark cycle.}

3.2. Library construction and sequencing

Plants were harvested when they are at the V4 (four-leaf stage) or V6 (six-leaf stage) stages (http://www.extension.iastate.edu/hancock/info/ corn.htm). Each sample was derived from at leastﬁve independent plants and the tissues were mixed together. The transverse section of stem node tissues at theﬁrst aboveground phytomer (from the bottom to the top) of the V4 stage maize with no brace root initiation were harvested as the control (N), the same location stem node tissues of the V6 stage where the brace roots just emerge were sampled as NR (i.e. node tissue with just-emerged brace roots), and the same location stem node tissues of the V6 stage where the brace roots just emerge under IAA treatment (10μM, sprayed, from V4 to V6) were sampled

Table 3

Differentially expressed novel miRNAs.

Name Sequence (5′–3′) L (nt) Fold change

Log2 (NR/N) Log2 (NRI/NR)

Up-regulated zma-nmiR66 CAAGCAAACUCAUCAACCUCAA 22 8.22 −0.90 zma-nmiR76a/b UUAUUUUUAGUUGUCGCUGGAUA 23 8.17 −0.76 zma-nmiR59 AAAUGAGACUAAAAUAGAGGG 21 8.01 −0.25 zma-nmiR71 UACAAGCCUUAUUGACCUGCCU 22 7.95 −0.90 zma-nmiR73a/b/c UGAAGGGGAUUGAGGGGGCUA 21 7.27 −0.86 zma-nmiR70 GCAUCCAUUCUUGGCUAAGUG 21 6.82 −0.58 zma-nmiR61 ACUGUUUUGUAUCGUUUUGU 20 6.82 −0.41 zma-nmiR21 UCUGGAUCAAAGAAGGUUGGA 21 1.69 −0.34 zma-nmiR38 UUGAUAUGGAUUGAGAGGGAUU 22 1.53 −1.02 Down-regulated zma-nmiR45a/b/c CAGGGCGGCUUUAAAUUUUGUG 22 −1.71 −6.36 zma-nmiR9a/b/c AUUGGAGGGGAUUGAGGAGGCU 22 −1.92 0.10 zma-nmiR11a/b CGGAGGGGAUUGGAGAGGCUA 21 −2.23 −0.67 zma-nmiR33 UUAGGCUCGGGGACUACGGUG 21 −2.83 −1.27 zma-nmiR81 AAAUAACGGGAGGUGGUAGACC 22 −7.01 6.56 zma-nmiR86 UGAGUUGGCUAGGUGGUUUGA 21 7.35 6.04 zma-nmiR84 UCCUUAAGGGUUUGUUCGGUUA 22 −9.10 9.56 zma-nmiR85 UGAAAGGCGGAUGGCUCUCUA 21 −9.45 8.75

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as NRI (i.e. node tissue with just-emerged brace roots under IAA treatment).

Total RNA was isolated from different maize tissues with TRIZOL reagent (Invitrogen, Carlsbad, CA, USA); Small RNAs of 16–28 nt were gel-puriﬁed and then sequentially ligated to RNA/DNA chimeric oligo-nucleotide adapters, reversely transcribed, and ampliﬁed through PCR (polymerase chain reaction). Finally, Solexa sequencing technology was employed to sequence the sRNAs from the three samples.

3.3. Sequencing data mapping and analysis

Raw sequence reads were processed. During this procedure all low quality reads, including 3′ adapter reads and 5′ adapter contaminants were removed. The remaining high quality sequences were trimmed of their adapter sequences and sequences larger than 30 nt and smaller than 18 nt were discarded. All high quality sequences, even those with only a single unique read, were considered as signiﬁcant and further analyzed. Unique small RNA sequences were mapped to maize genome (B73 RefGen_v2, release 5a.59 in November 2010) refer-ence sequrefer-ences by SOAP[39]. Small RNAs derived from rRNAs, tRNAs, snRNAs and snoRNAs deposited at the Rfam and NCBI GenBank data-baseshttp://www.ncbi.nlm.nih.gov/Ftp/were identiﬁed by NCBI blast. In order to determine conserved miRNAs, unique sequences were aligned with known maize miRNAs from miRBase (Released 18 November 2011) with a maximum of two mismatches, where gaps count as mismatches.

3.4. Identiﬁcation of conserved and novel miRNAs

The unique small RNA sequences were Blastn searched against the known maize mature miRNAs and their precursors in the miRNA data-base miRBase (Release 18, November 2011,http://www.mirbase.org/). Only those small RNAs whose mature and precursor sequences perfectly

matched known maize miRNAs in miRBase were considered to be con-served miRNAs.

To discover potential novel miRNAs, we used the software Mireap (http://sourceforge.net/projects/mireap/) to predict the precursor se-quences and their secondary structures. Then in order to distinguish miRNAs from other non-coding and coding sRNAs, the MFEI was cal-culated and potential novel maize miRNA candidates were further screened. Previous study indicated that more than 90% of miRNA pre-cursors had an MFEI greater than 0.85, and no other RNAs had MFEI higher than 0.85 (MFEI= [(MFE/length of the RNA sequence) × 100]/ (G+ C)%)[37].

3.5. siRNAs identiﬁcation

Small interfering RNA (siRNA) is a 21–24 nt long double-strand RNA, each strand of which is 2 nt longer than the other on the 3′ end. According to this structural feature, we aligned tags from clean reads to each other toﬁnd sRNAs meeting this criteria. These tags might be potential siRNA candidates. Program and Parameters: Software devel-oped by BGI-tag2siRNA.

3.6. Differential expression of miRNA

Compare the miRNA expression between two samples to deter-mine the differentially expressed miRNA. The procedures are shown as below: (1) normalize the expression of miRNA in two samples (control and treatment) to get the expression of transcript per million (TPM). Normalization forum: normalized expression = Actual miRNA count/ Total count of clean reads *1,000,000; (2) calculate fold-change and P-value from the normalized expression according the Bayesian method developed by Audic and Claverie[40]. Then generate the log2 ratio plot and scatter plot.

TCGATGCTTTGGGTTTGAAGCGGAGCTCCTATCATTCCAATGAAGGGTCGTTCCGAAGGGCTGGTTCCGCTGCTCGTTCA TGGTTCCCACTATCCTATCTCATCATGTGTATATATGTAATCCATGGGGGAGGGTTTCTCTCGTCTTTGAGATAGGCTTGTGG TTTGCATGACCGAGGAGCTGCACCGCCCCCTTGCTGGCCGCTCTTTGGATTGAAGGGAGCTCTGCATCCTGATCCACCCCTCC GAGCTCCTATCATTCCAATGA TTTGGATTGAAGGGAGCTCTG AGGGTCGTTCCGAAGGGCTGGTTC TTTGCATGACCGAGGAGCTGC miR159a* miR159a sRNA159-5 a sRNA159-3 5 3 1 80 81 163 164 246

Name sequence Length(nt) matched precursor zma-MIR159 sRNA159- TTTGCATGACCGAGGAGCTGC 21 zma-MIR159a/b/f/g/i/j/k

sRNA159- AGGGTCGTTCCGAAGGGCTGGTTC 24 zma-MIR159a sRNA159- CGCTGCTCGTTCATGGTTCC 20 zma-MIR159f zma-MIR169 sRNA169- ATGGATGAAATGTGGATGATGG 22 zma-MIR169m zma-MIR319 sRNA319- AGCTGCCGACTCATCCATTCA 21 zma-MIR319a/c

sRNA319- AGTGGGTGGCGCGGGAGCTAA 21 zma-MIR319a/c sRNA319- AGCTGCCGACTCATTCACCCA 21 zma-MIR319b/d sRNA319- AGTGAATGAAGCGGGAGGTAA 21 zma-MIR319b/d sRNA319-3'bd2 AAGCTTCGATCTCGCACCGTCTTT 24 zma-MIR319b/d

A

B

Fig. 5. SRNAs matched in the zma-MIRNA precursors. (A) Sequence analysis of sRNAs located on the zma-MIR159a precursor as an example. Precursor sequence (246 nt), black; miR159 (21 nt) and miR159* (21 nt), blue; sequenced novel sRNAs sRNA159-5′a (24nt) and sRNA159-3′ (21nt), red. (B) List of miRNA precursor-matched novel sRNAs sequenced in all three libraries.

(8)

3.7. Mature miRNA qRT-PCR analysis

Small RNA was extracted using miRcute miRNA Isolation kit (TIANGEN) from different tissues of maize. Ploy (A) modification and first strand cDNA synthesis were performed with miRcute miRNA first strand cDNA synthesis kit. Mature miRNA qRT-PCR analysis were performed with miRcute miRNA qRCR detection kit. The experiments had been carried out at least three times under identical conditions using U6 RNA as an internal control. Details of primers were listed in Table S5.

3.8. QRT-PCR analysis

Total RNA was extracted using TRIZOL reagent (Invitrogen, Carlsbad, CA, USA) from different tissues of maize. Contaminated DNA was re-moved with RNase-free DNase I. First strand cDNA synthesis was performed with Prime Script RT reagent kit using oligo (dT) primer. The qRT-PCR experiment had been carried out at least three times under identical conditions using EF1-α as an internal control. Primers for amplifying genes were designed according to the sequences down-loaded from the maize sequence database (http://maizesequence.org/ index.html). Details of primers were listed in Table S6.

3.9. Prediction of target genes

The potential targets of the differentially expressed miRNAs were predicted using psRNATarget (http://plantgrn.noble.org/psRNATarget/). Information about maize Genome was acquired from the maize se-quence database (http://maizesequence.org/index.html).

3.10. Gene Ontology (GO) analysis

Gene Ontology (GO) (http://www.geneontology.org) analyses were performed with the PartiGene program (http://www.nematodes.org/ bioinformatics/annot8r/index.shtml). GO (protein) terms based on BLASTX similarity (E-value_{b10e-5) and known GO annotations. Results} for GO were summarized in three independent categories (Biological Process, Cellular Component, and Molecular Function).

Supplementary data to this article can be found online athttp://dx. doi.org/10.1016/j.ygeno.2012.11.004.

Acknowledgments

The current work was supported by the National Natural Science Foundation (grant nos. 30970225 and 31070240).

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