However, to date, knowledge concerning their initiation or early development at the molecular level remains poor, and only the RTCS gene encoding a LOB domain protein was suggested to be
Trang 1maize brace root via Solexa sequencing
Yan-Jie Li*, Ya-Ru Fu*, Jin-Guang Huang, Chang-Ai Wu and Cheng-Chao Zheng
State Key Laboratory of Crop Biology, College of Life Sciences, Shandong Agricultural University, China
Introduction
Maize (Zea mays L.) develops a complex root
architec-ture, including embryonic primary roots, seminal
roots, lateral roots and shoot borne roots, which form
at different developmental stages and have distinct
physiological functions [1,2] The shoot borne roots,
which represent the major portion of the
postembryon-ic roots, include crown roots formed underground and
brace roots born on the stem nodes of successive basal
phytomers In cereal crops, the brace roots (also termed nodal adventitious roots), which are specifically developed in maize and sorghum, differ from the main roots in that they are mostly very short, lose their mer-istem and rapidly become determinate [3] They con-tribute enormously to lodging resistance, water and nutrient uptake in the late growth and development of the maize plants [4,5] Most importantly, they have a
Keywords
brace root; differential expression; maize;
Solexa sequencing; tag
Correspondence
C.-C Zheng or C.-A Wu,
College of Life Sciences, Shandong
Agricultural University, Taian, Shandong
271018, China
Fax: +86 538 8226399
Tel: +86 538 8242894; +86 538 8246678
E-mail: cczheng@sdau.edu.cn;
cawu@sdau.edu.cn
*These authors contributed equally to this
work
(Received 16 September 2010, revised 19
October 2010, accepted 26 October 2010)
doi:10.1111/j.1742-4658.2010.07941.x
Currently, the molecular regulation mechanisms involved in the early development of maize brace root are poorly known To gain insight into the transcriptome dynamics that are associated with its development, genome-wide gene expression profiling was conducted by Solexa sequencing (Illumina Inc., San Diego, CA, USA) More than five million tags were generated from the stem node tissues without and with just-emerged brace roots, including 149 524 and 178 131 clean tags in the two libraries, respec-tively Of these, 82 864 (55.4%) and 91 678 (51.5%) tags were matched to the reference genes The most differentially expressed tags with a log2ratio
> 2 or <)2 (P < 0.001) were analyzed further, representing 143 up-regu-lated and 152 down-reguup-regu-lated genes, except for unknown transcripts, which were classified into 11 functional categories The most enriched categories were those of metabolism, signal transduction and cellular transport Many genes or biological pathways were found to be commonly shared between brace root and lateral or adventitious root development, such as genes par-ticipating in cell wall degradation and synthesis, auxin transport and signal-ing, ethylene signalsignal-ing, etc Next, the expression patterns of 20 genes were assessed by quantitative real-time PCR, and the results obtained showed general agreement with the Solexa analysis Furthermore, a comparison of the brace root transcriptome with that of maize primary root revealed sub-stantial differences in the categories and abundances of expressed tran-scripts In conclusion, we first reveal the complex changes in the transcriptome during the early development of maize brace root and pro-vide a comprehensive set of data that are essential for understanding its molecular regulation
Abbreviations
DGE, Illumina ⁄ Solexa digital gene expression; EST, expressed sequence tag; N, node tissues; NR, node tissues with just-emerged brace roots; qRT-PCR, quantitative real-time PCR; TPM, transcript per million.
Trang 2substantial influence on grain yield under soil flooding
and water-limited conditions [6]
In the past decades, research on the maize brace
roots has been focused mainly at the morphological
and physiological levels It has been well described that
the primordia of brace roots develop from
dedifferenti-ated cells of the stem parenchyma, just behind the stem
cortex and below the intercalary meristem of the
over-laying internodes [7] Previous studies have
demon-strated that many nutritional or environmental factors
could affect brace root formation Demotes-Mainard
and Pellerin [8] reported that the number of brace
roots emerging from the upper phytomers was lower
when the carbohydrate nutrition of plants was reduced
by shading or low light as a result of large plant
densi-ties Nutrient deficiencies such as phosphorus and
nitrogen could decrease the rate of emergence or the
number of brace roots [9,10] Soil ridging is also an
important factor that could increase the number of
functional brace roots, and later ridging tends to result
in shorter internodes and more functional nodal roots,
leading to better lodging resistance [11,12] However,
to date, knowledge concerning their initiation or early
development at the molecular level remains poor, and
only the RTCS gene encoding a LOB domain protein
was suggested to be a potential regulator of the maize
crown and brace root initiation [13–15] Recently,
sev-eral genes involved in regulating the development of
other root types of maize have been identified The
SLR1 and SLR2 genes were reported to be required
for lateral root elongation [16] Ten ZmGSL members
in the gibberellic acid stimulated-like gene family were
found to be involved in modulating the lateral root
development [17] The rum1 and lrt1 mutants failed to
develop lateral roots [18] In addition, the rth1 mutant,
which is deficient in encoding a subunit of the exocyst
complex, exhibited a reduction in root hairs [19] Some
possible regulatory genes involved in the lateral or
adventitious root formation of Arabidopsis, rice and
Pinus contorta were also proposed [20–23] Therefore,
it is interesting to determine whether the maize brace
root formation shares the same regulatory mechanisms
with these underground roots To obtain a
comprehen-sive and unbiased transcript profile during maize brace
root formation, we performed deep sequencing analysis
using the Illumina⁄ Solexa digital gene expression
(DGE) system The DGE system is an improved
tag-based method that can sequence in parallel millions of
DNA molecules that are derived directly from mRNA
[24] The development of the DGE 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 functional complexity of transcriptome can be studied
In the present study, an overall impression of gene profiles during the early development of the maize brace root was acquired by deep sequencing For the first time, we have comprehensively characterized the molecular basis of the physiological processes during maize brace root formation and provide useful infor-mation for further research
Results
Characterization of the sequenced Solexa libraries
To identify genes involved in brace root initiation, two maize Solexa libraries were constructed from tissues of node and node with just-emerged brace roots Sequencing depths of 4 172 448 and 5 713 648 tags were achieved in the two libraries, including 328 043 and 432 881 distinct tags, respectively To make the libraries meaningful, tags recorded only once were first wiped off as a result of their unreliability; leaving
149 524 and 178 131 distinct tags in each library that were detected multiple times (clean tags) The fre-quency of these tags is shown in Table 1, which lists the copy numbers in the range 2–100 or higher, in which the majority of clean tags (68% from each) were present at low copy numbers (< 10 copies), and approximately 26% tags from each library were counted between 11 and 100 times Only approxi-mately 5% tags were detected more than 100 times
To identify the genes corresponding to the 149 524 and 178 131 meaningful tags in each library, an essential dataset containing 163 919 reference genes expressed in the maize genome from the MaizeSequence database (http://maizesequence.org/index.html) was prepared by
Table 1 Distribution of the experimental tags sequenced from the two solexa libraries.
Total number of distinct tags 328 043 432 881 Tag copy number > 1 (clean tag) 149 524 178 131
Trang 3expressed sequence tag (EST) analysis Altogether,
144 768 genes (88.3%) have the CATG sites, resulting in
a total number of 1 417 555 reference tags By assigning
the experimental Solexa tags to the virtual reference
ones (Table 2), we observed that 68 639 (45.9%) and
71 250 (40%) tags were perfectly matched to the
refer-ence genes in node tissues (N) and node tissues with
just-emerged brace roots (NR) libraries respectively
Out of the tags matched to reference genes,
approxi-mately 13% were mapped to multiple locations,
includ-ing low complexity tags with poly(A) tails and tags
derived from repetitive sequences Further sequences
analysis revealed that some of them were mapped to
highly conserved domains shared by different genes For
example, CATGGACAAGTTCGGCGGCGT could be
matched to AC194430.3_FG026 and AC194430.3_
FG036 sharing a 792 bp sequence encoding a fatty acid
hydroxylase domain In addition, approximately 14%
tags in two libraries were mapped to the antisense
strands, demonstrating that those regions might be
bidi-rectionally transcribed However, for the discrepancy
between the reference tags and experimental tags [25],
10% of 1 bp mismatched tags were present in the two
libraries
Altogether, there are 82 864 (55.4%) tags in the N
library and 91 678 (51.5%) tags in the NR library
matched to the reference genes The unmatched tags
were then blasted against the maize genome, and
approximately 31% tags were matched to the genomic
sequences in the two libraries These might represent
non-annotated genes or noncoding transcripts that
derived from intergenic regions As a result of the
sig-nificant sequencing depth of Solexa technology and
incomplete annotation of the maize genome, however,
13.7% and 15.7% unmatched tags in each library were
observed
Identification of differentially expressed transcripts
By comparing our two Solexa libraries, a great number
of differentially expressed transcripts were identified The distribution of fold-changes in tag number between the two libraries is shown in Fig S1 The great major-ity of transcripts were expressed at similar levels in the two libraries: approximately 98.6% tags showed a < 5-fold difference in expression Tags with expressional changes in the range 5–200-fold accounted for 1.39%, and only 0.01% tags showed > 200-fold changes in expression level At a statistically significant value (P < 0.01), 7239 differentially expressed tags exhibiting substantial changes were detected, including 3720 anno-tated genes (51%) Scatter plot analysis also presented
a broader scope of differentially expressed tags than annotated genes, demonstrating that a great number of unknown transcripts were revealed (Fig S2) To study
a subset of genes that were associated with brace root development and to assess the molecular basis of brace root development, we analyzed the most differentially regulated tags with a log2 ratio > 2 or <)2 using a greater statistically significant value (P < 0.001) as well
as false discovery rates (FDR < 0.01), representing
307 up-regulated and 372 down-regulated transcripts Apart from the unknown transcripts (55%), predicted
or known genes were categorized according to their functions Altogether, 143 up- and 152 down-regulated genes were listed and classified into 11 categories (Tables 3 and S1) Of these, the most enriched func-tional categories are those of metabolism (19.6%), sig-naling pathway (12.8%) and cellular transport (12.6%) Gene categories showed an obvious increase in tran-script abundance involved in protein binding and cell wall metabolism By contrast, transcriptional abun-dance for genes participating in transcript regulation, cell cycle and DNA replication pathways were reduced
At the cellular level, brace root initiation involves two major steps similar to that of lateral or adventi-tious roots: the degradation of overlaying cells and the reorganization of new root cells, which are regulated
by auxin associated networks [23] In total, 25 differen-tially expressed genes involved in cell wall metabolism and cell morphogenesis were listed (Table S1) Nine transcripts encoding endoglucanase, pectin lyase and chitinase genes, which were involved in cell wall degra-dation, were greatly up-regulated On the other hand, another 10 genes directly participating in cell wall bio-synthesis and cell morphogenesis were obviously induced as well, such as xyloglucan fucosyltransferase, cellulose synthase, COBRA and expansin Interestingly, many auxin associated genes were also differentially
Table 2 Summary of Solexa distinct tag-to-gene mapping data.
Tag mapping
Distinct tags
Sense
Perfect match 59 802 (40%) 62 276 (35%)
Antisense
All tags mapping to gene 82 864 (55.4%) 91 678 (51.5%)
Tags mapping to genome 46 247 (30.9%) 58 430 (32.8%)
No matched tags 20 413 (13.7%) 28 023 (15.7%)
Total distinct tags
(clean tags)
Trang 4regulated in the present study, such as AUX, ARF,
H+ pyrophosphatase, P450 CYP81A and ABC
trans-porters (Table S1) In accordance with previous studies
[22,26], the AUX gene was sharply induced and two
ARF proteins were greatly decreased in the present
analysis, which could positively regulate brace root
development
In addition, it was observed that many gene families
were over-represented in the data of the present study
(Table S1) For example, six transcripts encoding
members of major facilitator super family were
classi-fied into cellular transport categories, and five of them
are down-regulated in the NR library The most
abun-dant gene family present is the protein kinase family;
16 genes out of the total 24 members were significantly
induced in the NR library, implying the importance of
signal transduction during the early brace root
devel-opment The over-representation of the same family
members strongly suggests their regulating roles in this
developmental transition processes
Furthermore, with the benefit of Solexa sequencing
(i.e meaning that the entire transcriptome is surveyed),
numerous novel transcripts with an unclear function
were also detected (Table S2)
Quantitative quantitative real-time PCR
(qRT-PCR) confirmation
To evaluate the validity of Solexa analysis and to
further assess the patterns of differential gene
expres-sion, 20 candidate genes were selected and detected
by qRT-PCR, including two unknown transcripts
AC211140.2_FG010 and AC209357.3_FG031 (Table S2)
As shown in Table 4, the expression patterns showed
general agreement with the Solexa sequencing Because
of the apparent discrepancies with respect to ratio, it
should be attributed to the essentially different algo-rithms determined by the two techniques [27] In the analysis of gene profiles, the deep sequencing method generates absolute rather than relative expression mea-surements As expected, transcripts from highly abun-dant Solexa tags appeared at the expected lower cycle numbers in the quantitative PCR analyses Addition-ally, high-fold changes were observed for genes that showed low copy numbers in the N library but high abundances in the NR library For example, PI12 showed no expression in the N library, whereas it was detected 230 times in the NR library It was signifi-cantly up-regulated by 166.7-fold in the RT-PCR analysis Similarly, a b-1,3-glucanase-like gene was induced by 62-fold These results basically confirmed the reliability of our transcriptome analysis
To further investigate the expression profiles of these genes, stem node tissues were harvested from separate successive phytomers of the V6 stage maize, representing four different developmental phases of brace roots: pre-initiation, initiation, emergence and post-emergence (Fig 1A) From the qRT-PCR results (Fig 1B), altered expression was observed for all the candidate genes, indicating that they were involved in the regulatory networks during brace root formation GRP (glycine-rich cell wall structural protein), PI 12 (proteinase inhibitor I 12), PI 13 (proteinase inhibitor
I 13), BGL (B-1,3-glucanase like), Chn1 (Chitinase) and ABC transporter gene were up-regulated, whereas E3, AC209357.3_FG031 and AC211140.2_FG010 genes were down-regulated as the brace root devel-ops, indicating that these genes play positive or negative roles during both brace root initiation and later development Other genes such as expansin, XF (xyloglucan fucosyltransferase), CesA (cellulose syn-thase), NOI (nitrogen transporter) and CYP 81A
Table 3 Functional classification of genes differentially expressed during the early development of the maize brace root.
Functional category
Number (proportion)
Trang 5were first induced to a high expression level and then
decreased, indicating that they might play major roles
in specific developmental stages Moreover, the
expression of ARF, two DNA methyltransferases and
HMG fluctuated during brace root development,
demonstrating that these genes might be regulated in
a temporal manner
Comparison of the maize brace root and primary
root transcript profiles
To determine the differentially expressed transcripts
involved in the maize brace root and primary root
development, we compared the 80 most highly
abun-dant brace root transcripts in the NR library
(Table S3) and the primary root transcripts reported
by Poroyko et al [28] The results obtained revealed
that the selected tags in brace root and primary root
showed little overlap in functional category or
tran-script identity Functional analysis of the 80 abundant
brace root transcripts revealed that a large proportion
of them are enzymes involved in the metabolic and
energy processes, such as glutathione S-transferase,
glycoside hydrolase and dTDP-glucose dehydratase,
indicating that these metabolic processes are more
active in the early development of brace roots
More-over, as shown in Table 5, comparison of gene
catego-ries showed that, except for the categories of
chromatin structure and energy production, substantial differences were observed For example, in the maize primary root tissue, the most abundant genes were involved in translation and ribosome structure, which accounted for 24% of genes, whereas only 11.3% of genes were classified into this category in the brace root tissue For the rest of the categories, the tran-script abundances in the brace root were higher than that in the primary root In the 55 annotated tags in the primary root reported by Poroyko et al [28], 19 tags showed no match to any transcript in our NR libraries, suggesting that these genes were not expressed in brace root tissue Thirty-one tags, as a result of being only 14 nucleotides in length, were mapped to more than one brace root tag (21 nucleo-tides) Only five transcripts were present in both libraries, encoding a initiation factor 5A, elongation factor 1-b, 60S ribosomal protein L5, ribosomal pro-tein S10 and calmodulin, respectively (Table 6) The obvious discrepancies between the two root expression profiles imply that different regulation mechanisms are involved in maize brace root and primary root early development
Discussion
The major goal of the present study is to preliminarily explore transcripts involved in the early development
Table 4 Confirmation of the expression profiles of selected genes by qRT-PCR.
Solexa
qRT-PCR NR ⁄ N b
a To avoid division by 0, we used a tag value of 1 for any tag that was not detected in any sample b Ratio of relative concentrations.
Trang 6of the maize brace root, as well as to provide
ground-work for investigating their regulating mechanisms To
our knowledge, this is the first report that
comprehen-sively shows the transcriptional changes during the
onset of brace root branching We used the
Illu-mina⁄ Solexa DGE system, which is essentially a serial
analysis of gene expression-based tag profiling
approach Several previous studies for the plant
pri-mary and lateral root transcript profiles using deep
sequencing have been reported [25,27–29] Fizames
et al [25] investigated root transcriptome responses to
2,4,6-trinitrotoluene exposure in Arabidopsis Poroyko
et al [28] defined the number and abundance of tran-scripts in the root tip of the maize seedlings In the present study, a sequencing depth of more than five million tags was finally achieved, which was increased
by approximately 25-fold compared to maize primary root transcript profiling [28] Recently, Wang et al [29] revealed the epigenetic modifications in maize shoots and roots by Solexa sequencing, demonstrating that Solexa sequencing analysis has emerged as an efficient and economical method for sampling transcript
P1 P2 P3 P4
XF P1
P2 P3 P4
GRP
B A
P1 P2 P3 P4
P1 P2 P3 P4 AC209357.3_FG031
P1 P2 P3 P4
P1 P2 P3 P4
P1 P2 P3 P4
P1 P2 P3 P4
PI12
P1 P2 P3 P4
E3
P1 P2 P3 P4
Expansin
P1 P2 P3 P4 P1
P2
P3
P4
Chn1
LysM P1
P2 P3 P4
ABC transporter
MET
P1 P2 P3 P4
NOI
CesA
P1 P2 P3 P4 AC211140.2_FG010
Endochitinase A
P1 P2 P3 P4
P1 P2 P3 P4
ARF
Sugar transporter
P1 P2 P3 P4
PI13
P1 P2 P3 P4
BGL
P1 P2 P3 P4
CYP81A
P1 P2 P3 P4
MCM
Fig 1 The expression profiles of 20 selected genes in different developmental stages of the maize brace roots (A) Schematic drawings illustrating different developmental stages of the maize brace root in different phytomers P1, P2, P3 and P4 represent the first, second, third and fourth phytomer, respectively (B) Relative expression of the 20 genes detected by qRT-PCR in different phytomers The transcript levels were normalized to that of elongation factor 1-a, and the level of each gene in the first phytomer (P1) was set at 1.0 Error bars repre-sent the SE for three independent experiments GRP (glycine-rich cell wall structural protein), endochitinase A, CesA (cellulose synthesis), Chn1 (chitinase chem5), XF (xyloglucan fucosyltransferase), BGL (b1,3-glucanase like), expansin, LysM (peptidoglycan-binding LysM), E3 (ubiquitin-conjugating enzyme E3), CYP 81A (cytochrome P450 81A), ARF (auxin response factor), ABC transporter, NOI (nitrate-induced NOI, correlated to nitrate transport), MCM (MCM protein 5), sugar transporter, MET (DNA cytosine methyltransferase MET2a), PI13 (protein-ase inhibitor I13), PI12 (protein(protein-ase inhibitor I12) (Table S1), AC211140.2_FG010, AC209357.3_FG031 (Table S2).
Trang 7profiles under specific experimental conditions
Gener-ally, a significant sequencing depth would greatly help
to explore clean tags that were detected more than
once In the present study, the clean tags and the
sin-gletons accounted for approximately 43% and 57% in
the two Solexa libraries However, a total of 40 599
different tags were sequenced in the maize primary
root tissues, of which 63.5% tags were singletons [28]
Furthermore, 84% tags in the wheat grain tissues were
singletons [30] These data suggest that, as the
sequenc-ing depth increases, the proportion of clean tags will
also increase
A crucial step in deep sequencing studies is the
annotation of the tags In the present study, we
gener-ated a 21 bp tag for mapping to the existing
tran-scripts data, which is more specific than the 14 or
19 bp tags emploted in previous serial analyses of gene
expression [25,27,31] In total, we could map
approxi-mately 85.3% of tags to unique or non-unique
posi-tions, which is the same proportion as that described
by Wang et al [29], demonstrating that Illumina⁄
So-lexa sequencing and tag mapping are feasible with
great accuracy in large and repeat-rich maize genomes
However, these approaches still suffer limitations in
the reference gene databases In the present study, only
88.3% of reference genes from the maize EST library
have the CATG sites, leaving 19 151 genes unanalyzed,
which means that a number of the related genes
involved in brace root initiation would be neglected
For example, because of the absence of CATG in the
EST sequence, we did not detect tags representing the
RTCSgene, which was reported to be involved in
con-trolling brace root development [15]
Solexa sequencing could provide a comprehensive
and unbiased dataset in the global analysis of gene
expression Recently, Zenoni et al [32] profiled the
Table 5 Comparison of the functional categories of the 80 most
abundant tags in the maize brace root and primary root tissues.
Functional category
% maize brace root
% maize primary root Translation, ribosome structure 11.3 24
Energy production and conversion 3.8 4
Carbohydrate transport and
metabolism
Lipid transport and metabolism 3.8 1.6
Cell wall ⁄ membrane ⁄ envelope
biogenesis
Copy number Abundance (%)
Copy number Abundance (%)
Trang 8patterns of gene expression during plant developmental
transition in Vitis vinifera, and a wide range of
tran-scriptional responses associated with berry
develop-ment were investigated, demonstrating that the plant
developmental transition is a complex process that
requires the regulation of many transcripts [32,33] In
the present study, 7239 differentially expressed
anno-tated and novel transcripts (P < 0.01) were explored
Except for unknown transcripts, the most differentially
expressed genes with a log2 ratio > 2 or <)2 (P <
0.001) also participate in various biological pathways,
such as cell wall metabolism, signal transduction,
envi-ronmental response and transcription regulation
Inter-estingly, many transcripts detected at very low copy
numbers in the control library (N) were significantly
up-regulated in the NR library (Table S1), implying
that these genes might be specifically expressed in
brace root tissues For example, the presence of sugar
transporter and potassium uptake channel genes in the
NR library rather than the N library indicates that the
emerged brace roots begin to take on new functions
with respect to ion uptake or nutrient transport
Previous studies have reported the transcript profiles
of P contorta adventitious root development and
Ara-bidopsislateral root development by microarray
analy-sis [20,21] A large number of differentially expressed
genes involved in critical pathways during the lateral
or adventitious root development were also found in
the present study, such as genes participating in cell
wall degradation and synthesis, auxin transport and
signaling In Arabidopsis, P450 CYP83A1, CYP83B1,
CYP79B2 and CYP79B3 were previously reported to
act as key enzymes in the auxin biosynthesis pathway
[34,35] We proposed that the sharp increase of
tran-script encoding the P450 CYP81A might be required
for auxin biosynthesis during maize brace root
pattern-ing, indicating that auxin represents a regulator in
both brace root and lateral or adventitious root
devel-opment in higher plants [20,23,36,37] In addition,
blocking ethylene responses by etr1 (ethylene triple
response1) or ein2 (ethylene insensitive 2) mutation
was reported to increase lateral root formation in
Ara-bidopsis [38] We observed that an EIN3
(ethylene-insensitive 3) gene was markedly decreased in the NR
library (Table S1), implying that ethylene signaling was
also negatively involved in brace root development In
addition, a large number of transcripts involved in
wound, pathogen or disease defenses were significantly
induced, including proteinase inhibitors, disease
resis-tance proteins and pathogenesis-related genes, etc.,
which is also observed in P contorta adventitious root
development [20] We thus propose that these
tran-scripts might be also induced by the break up of
tissues during the emergence of the brace root from the stem node, which in turn contributes to a defensive barrier against extrinsic biotic intrusion Taken together, some regulating mechanisms appear to be commonly shared with respect to maize brace root and plant lateral or adventitious root formation
Although numerous nomatched or unknown tags were detected, the value of this tag collection will increase as more maize genomic sequences become available Further functional analysis of the differen-tially expressed genes will provide deeper insight into the regulation of maize brace root development
Materials and methods
Plant material and RNA extraction
The maize (Zea mays) inbred line H5468 was used in the present study Seeds of H5468 were surface-sterilized with 3% sodium hypochlorite for 10 min and rinsed in distilled water Sterilized seeds were pre-germinated on moistened filter papers in a plant growth chamber at 60% humidity and 28C, under a 16 : 8 h light ⁄ dark cycle for 3 days Then, the seedlings were transferred into the field in a greenhouse and cultivated at a mean temperature of 28C with both natural light and an additional 16 : 8 h light⁄ dark cycle For solexa analysis and qRT-PCR verification, plants were harvested when they are at the V4 (four-leaf stage) or V6 (six-leaf stage) stages (http://www.exten sion.iastate.edu/hancock/info/corn.htm) Each sample was derived from at least five independent plants and the tissues were mixed together The transverse section of stem node tissues at the first aboveground phytomer (from the bottom
to the top) of the V4 stage maize with no brace root initia-tion were harvested as the control (N), and the same loca-tion 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) For analysis of the different developmental phases, the transverse sections of the stem node tissues at the first, second, third and fourth above-ground phytomers were harvested from at least five inde-pendent plants of V6 stage All samples were immediately frozen in liquid nitrogen Then, total RNA were prepared from 0.1 g tissues and extracted with Trizol Reagent (Invi-trogen, Carlsbad, CA, USA) in accordance with the manu-facturer’s instructions
Tag library construction and sequencing
For Solexa tag preparation and sequencing, 1 lg of total RNA was incubated with oligo(dT) beads to capture the mRNA with a poly(A) tail First- and second-strand cDNA was synthesized and bead-bound cDNA was subsequently digested with NlaIII The GEX adapters 1 containing a
Trang 9restriction site for MmeI were ligated to the free 5¢ ends of
the digested bound cDNA fragments Then, the
bead-bond fragments were digested with MmeI, which could cut
17–18 bp downstream of the CATG site (NlaIII site),
releasing 21–22 bp tags starting with the NlaIII recognition
sequence A second adaptor (GEX adaptor 2) was ligated
at the site of MmeI cleavage, and the adapter-ligated cDNA
tags were enriched using PCR primers that anneal to the
adaptor ends The resulting PCR fragments were purified
from a 6% acrylamide gel and subjected to the
Illu-mina⁄ Solexa sequencing system (Illumina Inc., San Diego,
CA, USA) using a sequencing-by-synthesis method in
accordance with the manufacturer’s instructions
Tag-to-gene assignment and functional
categorization
Sequencing quality evaluation and data summarization were
performed using Illumina⁄ Solexa pipeline software after
sequencing For tag-to-gene assignment, soap (Short
Oligo-nucleotide Alignment Program) was used, allowing no more
than one base mismatch [39] The schematic overview of the
procedure is given in Fig S3 Reference ESTs or cDNA
sequences represented by bacteria artificial chromosomes
were obtained from the MaizeSequence database, release
3b.50 (http://maizesequence.org/index.html) The
experimen-tal tags were first filtered to eliminate unauthentic ones and
to leave the clean tags for mapping to the reference tags
derived from cDNA or EST sequences, and the unmatched
ones were then blasted against the maize genome sequences
For those sequenced tags, their expressional abundances in
each library were shown by copy numbers in the library For
differential expression analysis, the fold changes were
assessed by the log2 ratio (TPM-NR⁄ TPM-N) after the
expressional abundances in each library were normalized to
transcript per million (TPM), which is accordance with the
study of Audic and Claverie [40] After tag-to-gene
assign-ment, genes were grouped into functional categories based
on the MIPS Functional Catalogue Database (http://mips
gsf.de), which was developed for the functional description
of proteins The presence of identified functional domains by
blastxsearch in the examined sequence was accepted for the
assignment of functions A criterion of sequence similarity
E-value < 1· 10)5 was used for the significant hits
Func-tional categorization was carried out in the same way and
included a blast search against the Arabidopsis protein
database at TAIR (http://www.arabidopsis.org/index.jsp)
and the rice protein database (http://www.tigr.org/) The
search results were linked to the MIPS Functional Catalogue
Database for further functional categorization
Real-time PCR
Reverse transcription reactions were performed using 5 lg
of RNA by M-MLV reverse transcriptase (Takara Bio Inc.,
Otsu, Japan) in accordance with the manufacturer’s instruc-tions after incubation with RNase-free DNase I For real-time PCR, selected gene sequences were obtained from the MaizeSequence database Primers were designed using bea-con designer 7.0 software (Premier Biosoft International, Palo Alto, CA, USA) Elongation factor 1-a was used as the inner control The PCR primers are shown in Table S4 Real-time PCR reactions were performed with a Bio-Rad real-time thermal cycling system (Bio-Rad, Hercules, CA, USA) using SYBR-Green to detect gene expression abun-dances The reaction mixture (25 lL) contained 0.5 lm of each primer and the appropriate amounts of enzymes, cDNA and fluorescent dyes All runs used a negative con-trol without adding target cDNA, resulting in no detectable fluorescence signal from the reaction A range of five dilu-tions of the total cDNA was tested under the same condi-tions as the samples Amplification reaccondi-tions were initiated with a pre-denaturing step at 95C for 10 s and followed
by denaturing (95C for 5 s), annealing (60 C for 10 s) and extension (72C for 15 s) steps for 49 cycles during the second stage, and a final stage of 55C to 95 C to deter-mine dissociation curves of the amplified products All reac-tions were performed with at least three replicates Data were analyzed using Bio-Rad cfx manager software
Acknowledgements
This work was supported by the National Natural Science Foundation (grant numbers 30970225 and 30970230) and the Genetically modified organisms breeding major projects (2009ZX08009-092B) in China
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