- Research article
- Open Access
miRNA164-directed cleavage of ZmNAC1 confers lateral root development in maize (Zea maysL.)
- Jing Li†1, 2,
- Guanghui Guo†1, 2,
- Weiwei Guo1, 2,
- Ganggang Guo1, 2,
- Dan Tong1, 2,
- Zhongfu Ni1, 2,
- Qixin Sun1, 2 and
- Yingyin Yao1, 2, 3Email author
© Li et al.; licensee BioMed Central Ltd. 2012
Received: 15 August 2012
Accepted: 20 November 2012
Published: 21 November 2012
MicroRNAs are a class of small, non-coding RNAs that regulate gene expression by binding target mRNA, which leads to cleavage or translational inhibition. The NAC proteins, which include NAM, ATAF, and CUC, are a plant-specific transcription factor family with diverse roles in development and stress regulation. It has been reported that miR164 negatively regulates NAC1 expression, which in turn affects lateral root development in Arabidopsis; however, little is known about the involvement of the maize NAC family and miR164 in lateral root development.
We collected 175 maize transcripts with NAC domains. Of these, 7 ZmNACs were putative targets for regulation by miR164. We isolated one gene, called TC258020 (designated ZmNAC1) from 2 maize inbred lines, 87-1 and Zong3. ZmNAC1 had a high expression level in roots and showed higher abundance (1.8 fold) in Zong3 relative to 87-1, which had less lateral roots than Zong3. There was a significant correlation between the expression level of ZmNAC1 and the lateral root density in the recombinant inbred line (RIL) population. Transgenic Arabidopsis that overexpressed ZmNAC1 had increased lateral roots in comparison to the wild type. These findings suggest that ZmNAC1 played a significant role in lateral root development. An allelic expression assay showed that trans-regulatory elements were the dominant mediators of ZmNAC1 differential expression in 87-1 and Zong3, and further analysis revealed that miR164 was a trans-element that guided the cleavage of endogenous ZmNAC1 mRNA. Both mature miR164 and miR164 precursors had higher expression in 87-1 than Zong3, which was the opposite of the expression pattern of ZmNAC1. Additionally, the allelic assay showed that the cis-regulatory element most likely affected Zm-miR164b's expression pattern. A β-glucuronidase (GUS) assay showed that the Zm-miR164b promoter had higher GUS activity in 87-1 than in Zong3. In addition, we detected miR164b expression in the RIL population, and the results indicated that miR164b had a higher expression level in the RILs containing 87-1 promoter than those containing Zong3 promoter.
Our results indicate one possible pathway in maize by which differences in miR164b promoter activity resulted in a different expression pattern for mature miR164 which negatively regulates ZmNAC1 expression in 87-1 and Zong3, thereby contributing to a significantly different lateral root phenotype.
NAC genes that encode plant-specific transcription factors influence a diverse set of developmental processes. The NAC family proteins contain a consensus sequence known as the NAC domain (petunia NAM and Arabidopsis ATAF1, ATAF2, and CUC2) [1, 2], which is located in the N-terminal region, but their C-terminal sequences are divergent in both length and amino acid sequence . NAC proteins make up a large family, with a total of 109 and 140 predicted proteins identified in Arabidopsis and rice, respectively [4, 5]. Members of the NAC family seem to play different roles in plant development. For example, NAM from petunia  and CUC2 of Arabidopsis (the first reported NAC genes) are involved in shoot apical meristem (SAM) development. By contrast, the Arabidopsis NAC gene CUC3 has been reported to contribute to the establishment of the cotyledon boundary and the shoot meristem . Another Arabidopsis NAC gene called NAP (NAC-LIKE, activated by AP3/PI) has been characterized as the target of two MADS box genes, APETAL3 and PISTILLATA, which control cell division and cell expansion in stamens and petals . AtNAC1 has been characterized as an intermediary in the auxin-signaling pathway that activates genes encoding molecules involved in the specification of lateral root initiation . In addition, NAC proteins have been implicated in defense and abiotic stress responses. Some ATAF subfamily genes, including StNAC from tomato and AtAF1-2 from Arabidopsis, have been shown to be induced by pathogen attack and wounding . More recently, some NAC genes such as BnNAC from Brassica, AtNAC072 (RD26), AtNAC019 and AtNAC055 from Arabidopsis, and SNAC1 and SNAC2 from rice were found to be involved in the plant's response to various environmental stresses, including drought, salinity, and/or low temperature.
MicroRNAs (miRNAs) are a class of small, non-coding RNAs that regulate gene expression by guiding target mRNA cleavage or translational inhibition [14–16]. In Arabidopsis, miR164 can target five NAC domain-encoding mRNAs, including the NAC1, CUC1, CUC2, At5g07680, and At5g61430 mRNAs . NAC1 is able to transmit auxin signals that promote lateral root emergence, and miR164 guides the cleavage of NAC1 mRNA, which is followed by a mechanism to cleave NAC1 mRNA and downregulate auxin signals [8, 18]. Other groups have independently reported evidence for the miR164-mediated regulation of CUC1 and CUC2[19, 20], which have been implicated in meristem development and the separation of aerial organs.
The process of lateral root (LR) development has been extensively studied in flowering plants and gymnosperms, and LRs begin in a specialized cell layer (called the pericycle) in the primary root (PR) [21, 22]. In Arabidopsis and most other dicots, LRs are only formed from the pericycle cells that overlay the developing xylem tissue (the xylem pole pericycle). In other species, particularly cereals such as maize, rice and wheat, LRs arise specifically from the phloem pole pericycle, with additional contributions from the endodermis [23, 24]. In Arabidopsis, a large body of evidence has indicated that auxin plays a pivotal role in lateral root development. Lateral root initiation begins with auxin-induced IAA14 degradation. This step allows the activation of ARF7 and ARF19 transcription factors, which then activate the expression of LBD/ASL genes. The LBD/ASL proteins in turn activate cell cycle genes and cell patterning genes, allowing the formation of a new lateral root primordium (LRP) [25, 26]. In addition, auxin activates the transcription of NAC1, which upregulates the expression of two downstream auxin-responsive genes including DBP and AIR3 to stimulate LR initiation . The post-transcriptional and post-translational regulation of NAC1 was also reported as follows: the role of miR164 expression in late auxin response was intended to clear NAC1 mRNA, which would attenuate the auxin signaling that inhibits lateral root development . A RING-finger ubiquitin E3 ligase called SINAT5 promoted NAC1 ubiquitination and its subsequent degradation in order to attenuate the auxin response .
In this study, a miR164-targeted NAC domain gene that was designated ZmNAC1 was isolated from maize. It was demonstrated that ZmNAC1 overexpression in Arabidopsis leads to increased number of lateral roots. Further study showed that miR164 is one of the trans-acting factors that negatively regulates ZmNAC1, resulting in a different ZmNAC1 expression pattern between the two inbred lines 87-1 and Zong3, contributing to a significant difference in the lateral root phenotype between these two lines. Moreover, our analysis also indicated that the promoter variation of maize pri-miR164b in 87-1 and Zong3 might be one in which the dominant cis-element affects the expression of pri-miR164b, leading to differences in mature miR164 expression.
Identification of putative miR164-regulated NAC genes in maize
Putative NAC proteins in maize were identified using the conserved NAC domain sequences in a query that was performed by TBLASTN search. Predicted amino acid sequences of the full-length maize cDNA (PlantGDB) were used for the analysis. In this study, a total of 175 putative maize NAC proteins were obtained, and 105 NACs from Arabidopsis were used to construct the polygenetic tree. These NAC proteins could be classified into 3 groups and 14 subgroups on the basis of their predicted NAC domain amino acid sequences (Additional file 1). It has been reported that miR164 directs the regulation of 5 target NAC-domain transcription factor mRNAs in Arabidopsis. To obtain putative miRNA-regulated NAC genes from maize, we searched the reverse complementary site for the mature miR164 in the 175 ZmNACs. Maize miR164 sequences were identified in http://www.mirbase.org/. Seven ZmNAC genes, TC258020, Zm390255026, Zm029753, Zm020717, Zm020987, Zm4253255028 and Zm017452, were found to be putative miR164 target genes (Additional file 2), and are shown in pink dots in supplemental Figure 1 (Additional file 1). Among these genes, the amino acid sequence corresponding to three of them, Zm020987, TC258020 and Zm017452, showed high homology to Arabidopsis NAC1 protein (Additional file 1).
Expression of putative miR164-regulated ZmNACgenes
The ZmNAC1gene and its expression in maize roots
Arabidopsis NAC1 has been shown to play an important role in lateral root development. To determine the putative biological function of ZmNAC1 in maize root development, the inbred lines 87-1 and Zong3 along with a set of RIL (recombination inbred lines derived from the cross between Zong3/87-1) were used, and in this population, either two inbreeds or the RILs showed a significant difference in their lateral root density. The lateral root density of 87-1 and Zong3 were 4.13 and 6.82 respectively (Figure 2B p < 0.01) and a large amplitude of variation in lateral root density (from 2.3 to 12.6) was observed among the RILs (Additional file 4).
The accumulation of ZmNAC1 transcripts in roots among the inbred lines Zong3, 87-1 and their RILs was determined by real-time PCR, which revealed that ZmNAC1 showed 1.8-fold (P < 0.05) higher expression in Zong3 than in 87-1 (Figure 2C) and had different expression levels among different RIL lines with a maximum of 1.41-fold higher than Zong3 and 3.04-fold lower than 87-1 (Additional file 5). To determine whether the lateral root phenotypes in RIL populations were associated with ZmNAC1 expression level, the correlation coefficient between the lateral root density and the expression levels of ZmNAC1 was calculated. These results showed that the correlation coefficient value reached 0.41 (P < 0.01), suggesting that differential ZmNAC1 expression contributed 16.8% to the lateral root phenotype. Based on these results, we argued that ZmNAC1 played important roles in maize lateral root development and contributes to the lateral root number difference between the inbred lines Zong3 and 87-1.
Overexpression of ZmNAC1 in Arabidopsispositively regulated lateral root development
ZmNAC1allelic expression between maize hybrids and parents
Allele-specific transcript ratio of ZmNAC1 in hybrids and parents (cDNA-RT PCR)
P Allelic ratio 87-1: Zong3
H Allelic ratio 87-1: Zong3
Allele-specific transcript ratio of Zm-miR164b in hybrids and parents (cDNA-RT PCR)
P Allelic ratio 87-1: Zong3
H Allelic ratio 87-1: Zong3
miR164 as the trans-acting factor that regulates expression of ZmNAC1
We then investigated the expression of mature miR164 to determine whether miR164, which can act as the trans-acting regulator of ZmNAC1, also showed differential expression between 87-1 and Zong3. Northern blot analysis showed that the mature miR164 had higher expression levels in 87-1 than in Zong3 (Figure 5C), which was the opposite of the ZmNAC1 expression pattern in the two inbred lines.
MiR164 was potentially transcribed from 8 loci, specifically from miR164a to miR164h, in maize. The mature miR164 from these loci differed by one or two nucleotides at the 3' end. We designed specific primers to amplify the 8 precursors, and only miR164b and miR164d had a higher expression level in the roots than in other tissues. Because the oligonucleotide probe used in the RNA gel blot cannot discriminate among these eight transcripts, we used gene-specific RT-PCR to analyze pri-miR164 expression. The results revealed that pri-miR164b (primary miRNA) and pri-miR164d showed 2.4-fold and 3.6-fold higher expression levels in 87-1 than in Zong3 (P < 0.01), respectively (Figure 5D), suggesting that a higher expression of miR164 precursors may contribute to the higher expression of mature miR164s in 87-1.
ZmmiR164ballelic expression between maize hybrids and parents
Promoters of Zm-miR164bfrom inbred line 87-1 showed higher activity
To provide evidence for the cis-regulation of the miR164 precursor, we isolated a 2.6 kb region upstream of the miR164b transcription start site and then determined the activity of the promoters from the two inbred lines 87-1 and Zong3. Sequence analysis revealed that the promoters from these two lines exhibited 71.07% nucleotide identity for miR164b. Based on the sequence polymorphism, specific primers were designed to differentiate the promoters from the two inbred lines.
To further investigate whether the ZmmiR164b promoter from 87-1 showed higher activity in vitro than that from Zong3, we designed a promoter: GUS reporter assay, including 87p-ZmmiR164b: GUS (the promoter of miR164b from 87-1) and Zong3p-ZmmiR164b: GUS (the promoter of miR164b from Zong3). Tobacco leaves were infiltrated with Agrobacterium that harbored these constructs, and GUS activity expression was analyzed by spectrophotometric assays. We also transformed tobacco leaves with a GFP vector to normalize the transformation efficiency. Our results showed that GUS activity that was driven by the promoter from 87-1was significantly higher than that driven by the promoter from Zong3 and by the background (Figure 7B), which strongly suggested that the different promoter activities of pri-miR164 resulted in the diversity of pri-mir164 transcript levels that was found between the two inbred lines, which may have led to the difference in mature miR164 expression between 87-1 and Zong3. In addition, the correlation coefficient between the expression levels of ZmmiR164b and the lateral root numbers among the RILs was also calculated, but no significant correlation was observed (Additional file 7).
ZmNAC1played an important role in lateral root development
We isolated and characterized ZmNAC1, which is a member of the NAC-domain gene family and the first NAC gene shown to be involved in maize lateral root development. We demonstrated that ZmNAC1has high amino acid similarity to a homologous gene, NAC1, in Arabidopsis, which has also been reported to play an important role in lateral root development. In contrast to the other members of its family, ZmNAC1 showed higher expression levels in the roots than in other tissues. Moreover, ZmNAC1 was more highly expressed in the roots of inbred Zong3 than inbred 87-1, and inbred Zong3 had more lateral roots than inbred 87-1. The RIL populations derived from the Zong3/87-1 hybrid showed significant differences in lateral root numbers and in ZmNAC1 expression. There was a significant correlation (correlation coefficient value reached 0.41, P < 0.01) between the expression levels of ZmNAC1 and the lateral root number phenotype of the RILs, illustrating that ZmNAC1 is important for maize lateral root development. Overexpression of ZmNAC1 in Arabidopsis resulted in a higher number of lateral roots than in the wild type, whereas no other dramatic phenotype change was detected in transgenic plants. However, once the lateral roots initiated in the transgenic plants, no further alterations in root development occurred. These results suggest that ZmNAC1 is specifically involved in the control of lateral root initiation. The phenotype of transgenic plants that overexpress ZmNAC1 was consistent with NAC1 having its main function in the roots, as only lateral root development was affected.
Nonetheless, we cannot exclude the possibility that ZmNAC1 plays roles in other developmental pathways. ZmNAC1 also showed a higher expression level in the ears, stems and male spikes than in the leaves, so we speculated that ZmNAC1 might also be involved in the branching development of ears and male spikes.
miR164-mediated ZmNAC1mRNA cleavage in vivo
Plant miRNAs have been implicated in the control of various developmental processes, including leaf development [30–32], flower development  and lateral root development . In Arabidopsis, miR164 has been predicted to target 5 NAC-domain transcripts: NAC1, CUC1, CUC2, At5g07680 and At5g61430. Among these targets, CUC1 and CUC2 contribute to embryonic and floral development, and NAC1 plays important roles in lateral root development. Here we provide evidence showing that miR164 directs ZmNAC1 mRNA cleavage in vivo at the 11th nucleotide of the complementary miR164 binding site, and this cleavage was conserved between maize and Arabidopsis. In addition, ZmmiR164 showed higher expression levels in inbred 87-1 than in Zong3, which was the opposite of the expression pattern found for ZmNAC1, suggesting that miR164 negatively regulates ZmNAC1. These findings confirmed that this conserved miRNA can regulate a target gene in a conserved pathway throughout evolution.
miR164 as a trans-acting factor contributed to the differential expression of ZmNAC1between 87-1 and Zong3
The results of the allelic variation analysis for ZmNAC1 in 87-1, Zong3, and hybrid Zong3/87-1 maize showed that the trans-acting factor, and not the cis-element, was most likely the major factor underlying the differential expression of ZmNAC1 between 87-1 and Zong3. We further isolated and compared the 1 kb ZmNAC1 promoter sequences between the inbred lines 87-1 and Zong3, and no sequence difference was found except for one SNP (Additional file 8). Based on the ZmNAC1 mRNA cleavage that was directed by miR164, we consider miR164 to be one of the post-transcriptional trans-acting factors that regulates ZmNAC1 expression. However, no significant negative correlation was detected between the expression levels of ZmmiR164b and ZmNAC1 in the RIL population, suggesting that the expression of ZmNAC1 is also regulated by other genes, as observed in Arabidopsis. For example, Xie reported that the F-box protein TIR1 is likely involved in ubiquitin-mediated proteolysis of regulatory proteins that are required for an auxin response and can induce the expression of NAC1 through auxin signals .
Further study revealed that both the mature miR164 and the precursor pri-miR164b were expressed at higher levels in 87-1 than in Zong3 and that the promoter of ZmmiR164b from 87-1 showed higher activity in vivo and in vitro than that of Zong3, which might lead to the higher expression level of miR164 in 87-1 than in Zong3. It has been reported that Arabidopsis miR164 negatively regulates lateral root development, showing a strict inverse correlation between changes in the miR164 level and the NAC1 mRNA levels. Mutant plants that were defective in miRNA biogenesis showed a higher level of NAC1 mRNA and more lateral roots. Conditional overexpression of miRNA164 decreased NAC1 mRNA and lateral root numbers. Based on our results, it is possible to improve the root development in maize by altering the miR164 pathway.
Arabidopsis NAC1 has been shown to be a transcriptional activator in auxin-induced lateral root initiation. This study shows that ZmNAC1 also plays an important role in maize lateral root development and that ZmNAC1 expression is regulated at the post-transcriptional level by miR164. Overall, our data suggest that in 87-1 maize (which has fewer lateral roots than Zong3), the miR164b promoter has higher activity than in Zong3, leading to a higher expression level of mature miR164, which then downregulates ZmNAC1 expression at the post-transcriptional level. This pathway might contribute to the smaller lateral root numbers in 87-1. By contrast, both pri-miR164b and mature miR164 had a lower expression level in inbred Zong3 maize than in 87-1 maize, leading to a higher expression of ZmNAC1, thus contributing to greater numbers of lateral roots. It should be noted that no significant negative correlation was detected between the expression levels of ZmmiR164b and lateral root numbers in the RIL population, which revealed the complexity of ZmmiR164 and its regulation of lateral root development in maize.
Arabidopsis NAC1 is a transcriptional activator in auxin-induced lateral root initiation. Our study shows that the maize homologue, ZmNAC1, also plays an important role in lateral root development in maize. Our study then extends the research by showing that ZmNAC1 expression is regulated at the post-transcriptional level by maize miRNA164. Overall, our data suggest that the miR164b promoter showed higher activity in inbred 87-1 maize than in Zong3 maize, leading to higher expression of mature miR164, which down-regulated ZmNAC1 expression at the post-transcriptional level. This pathway might contribute to 87-1 having fewer lateral root numbers than Zong3.
Plant materials and growth condition
The maize (Zea mays L) inbred lines 87-1 and Zong3, the hybrid line Zong3/87-1, and 40 recombinant inbred lines (RIL) that were derived from the hybrid Zong3/87-1 were used for this study. For phenotypic analysis of lateral root numbers, seed germination was initiated by soaking in distilled water in a Petri dish for 12h at room temperature. Seeds were then placed between layers of moist paper towels at 30°C for 24 h and transplanted into pots. Seeds were cultivated in vermiculite with no additional fertilizer and grown in a growth chamber at a relative humidity of 40-60% and a 26/24°C day and night temperature under a 16-h-light/8-h-dark photoperiod. Eight individuals were planted for each genotype, and half of them were collected for gene expression analysis (the rest were used for the investigation of lateral root density). All of the root samples were collected at 8 days after germination between 8:00 a.m. and 9:00 a.m., and root samples were collected carefully and cleaned with water without causing any injury. Lateral root numbers in primary root were counted manually, and the number was divided by the length of primary root to calculate the parameter for lateral root density . Three biological replicates were performed for both gene expression and phenotypic analysis of lateral roots. For tissue expression pattern analysis, the root, leaf, sheath in the V3 stage, spike in the V12 stage, and stem in the V9 stage were collected for real-time PCR and Northern blot analysis.
The Arabidopsis thaliana Col-0 ecotype was used for transformation. Seeds were germinated after 3 d of vernalization on Murashige and Skoog (MS) medium at 4°C. Plants that were intended for transformation were grown under continuous light (150 mE/m2per s) at 22°C in a greenhouse.
Gene expression analysis
For Northern blot hybridization, total RNA was isolated using Trizol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. Low molecular weight RNA was enriched with 0.5 M NaCl and 10% PEG 8000 precipitation. Ten μg of low molecular weight RNA was loaded per lane, resolved on a denaturing 15% polyacrylamide gel, and electrophoretically transferred to Hybond-N+ membranes (Amersham Biosciences, Buckinghamshire, UK). Membranes were UV-crosslinked and baked for 2 h at 80°C. DNA oligonucleotides that were complementary to miR164 were end-labeled with γ-32P-ATP using T4 polynucleotide kinase (TaKaRa, Dalian, China). Membranes were pre-hybridized for more than 8 h and then hybridized overnight using Church buffer at 38°C. Blots were washed three times (twice with 2 × SSC + 1% SDS and once with 1 × SSC + 0.5% SDS) at 50°C. The membranes were briefly air-dried and then exposed to X-ray films for autoradiography at −80°C.
For the real-time PCR analysis, total RNA was isolated by using Trizol (Invitrogen, USA) according to the manufacturer's instructions and treated with RNase-free DNase I (Promega, Madison, USA). Two μg of total RNA from each sample was used for first-strand cDNA synthesis in 20 μL reactions containing 50 mM Tris–HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM DTT, 50 μM dNTPs, 200 U M-MLV reverse transcriptase (Promega, Madison, USA) and 50 pmol T15 oligonucleotides. Reverse transcription was performed at 37°C for 60 min with a final denaturation at 95°C for 5 min. Gene-specific RT-qPCR primers for 7 putative miR164 targets were designed according to the EST sequences. A 250 bp β-actin gene fragment was amplified as a positive control using the primer pair 5'-TGGCATTGTCAACAACTGG-3' and 5'-TCATTAGGTGGTCGGTGAGG-3'. PCR reactions were performed in a volume of 20 μL containing 10 pmol primers, 10 mmol/L Tris–HCl (pH8.5), 50 mmol/L KCl, 2 mmol/L MgCl2, 0.4 μL DMSO, 200 mmol/L dNTPs, 1 U Taq DNA Polymerase (TaKaRa, Dalian), and 0.5 μL SYBR GREEN I. The following PCR amplification protocol was used: 95°C (3 min) and 40 cycles of amplification cycle (95°C (30 s), 55°C (30 s),and 72°C (1 min)) using an Opticon PTC200 system (MJ Research, Waltham, MA, USA). All reactions were run in triplicate, and no template and no reverse transcription controls were included. Quantification results were expressed in terms of the cycle threshold (CT) values according to the baseline, which was adjusted to 0.04. The comparative CT method (PE Applied Biosystems, Foster City, CA, USA) was used to quantify gene expression in comparison with actin. In brief, the CT values were averaged for each triplicate. The differences between the mean CT value of a specific gene and that of β-actin was calculated as ΔCT sample = CTgene-CTβ-actin. In the final results, the sample's relative expression level was determined by 2-ΔCt method. Statistical significance was tested using Student’s t-test (P < 0.05).
Mapping of the miRNA-guided cleavage site
The GeneRacer Kit (Invitrogen USA) was used for the RACE assay according to the manufacturer’s instructions. Total RNA was extracted from the roots of 87-1 seedlings, and Poly(A)mRNA was purified and ligated to the RLM-RACE 5'RACE RNA Oligo adaptor (5'-CGACUGGAGCACGAGGACACUGACAUGGACUGAAGGAGUAGAAA-3'). The oligo (dT) (15-mer) primer was used for cDNA synthesis with reverse transcriptase, and the resultant cDNA was used for the first round of nested PCR using the 5' RACE primer 5'-CGACTGGAGCACGAGGACACTGA-3' together with ZmNAC1 gene-specific primer 5’-ACCCAAGCCTCTTGTAGCACTCATC-3'. The 5' RACE nested primer 5'-GGACACTGACATGGACTGAAGGAGAT-3' and the gene-specific nested primer 5'-GTCGAGGCATTTCGATCCGCATC-3' were used for the second round of nested PCR. Gel-purified PCR products were cloned into the pGEM-T Easy Vector (Promega) and sequenced.
Transformation vectors and construction of transgenic plants
For the transgenic Arabidopsis plants, constructs were made in the binary vector pCAMBIASuper1300. For the plasmid super-p: ZmNAC1, the forward primer (5'-GCTCTAGACGCAGAAGTTGACCACGTAC-3') (the underlined sequence is the XbaI site) and the reverse primer (5'-GGGGTACCATCCATCCTGTTATCGTCGAG-3') (the underlined sequence is the KpnI site) were designed to introduce XbaI and KpnI sites, and Zong3 root cDNA samples were used to amplify a 1463 bp cDNA from ZmNAC1. The construct was transformed into Agrobacterium GV3101. Six-week-old Arabidopsis plants were transformed via Agrobacterium-mediated transformation by the floral-dip method . Transgenic plants were selected on 1/2 MS medium (Gibco BRL, Grand Island, NY, USA) containing hygromycin B. T3 generation was used for further experiments.
Agrobacterium-mediated transient expression assay and GUS activity assay
The miR164b promoters from the inbred lines 87-1 and Zong3 were ligated into the Gateway pDONR 211Vector (Invitrogen USA), and then the vector miR164b-87-1promoter:: GUS and miR164b-Zong3promoter::GUS construct made according to the manufacturer’s instructions. Two primers in different orientations (5'-CAAAAAAGCAGGCTGTGATTGACGACAACATGAACAAATC-3',5'-ACAAGAAAGCTGGGTCGCAATTCTCGAATTCACCTTC-3'; the underlined sequence is the attB1 and attB2 site) were used to amplify the promoter sequences from 87-1 and Zong3 in separate DNA samples. Agrobacterium GV3101 that harbored each of the promoter:GUS constructs was grown on yeast extract peptone medium (10 g yeast extract, 10 g Bacto peptone, 5 g NaCl, and 15 g agar/l) supplemented with rifampicin (60 μg/mL), kanamycin (50 μg/mL) and spectinomycin (100μg/mL). Agrobacterium was cultured at 28°C and harvested by centrifugation for 15 min at 6,000 g, re-suspended in infiltration media (10 mM MgCl2 and 10 mM MES containing 150 mM acetosyringone) for 2 h at room temperature to induce T-DNA transfer functions , and then adjusted to an OD600 of 0.8. After the infiltration of the Agrobacterium suspension into the abaxial surfaces of tobacco leaves from a syringe without a needle , the tobacco plants were maintained in a moist chamber at 26°C for 48 h for GUS activity analysis.
GUS activity in these tobacco leaves was measured as described by Jefferson . Tobacco leaf tissues were homogenized in 1 ml of extraction buffer (50 mM NaH2PO4, pH 7.0, 10 mM EDTA, 0.1% Triton X-100, 0.1% (w/v) sodium laurylsarcosine, 10 mM β-mercaptoethanol, and 6 mM L-ascorbic acid). After centrifugation for 10 min at 12,000 rpm (4°C), the supernatant was transferred to a fresh microtube. The spectrophotometric reaction was carried out in a 1 mL volume with 1 mM PNPG (P-nitrophenyl-β-D-glucuronide) in the extraction buffer supplemented with protein extract supernatants. GUS activity was normalized to the protein concentration in each of the crude extracts and was expressed as nmol of P-nitrophenol min/mg protein. The total protein in the sample extract was quantified using bovine serum albumin as a standard, according to the Bradford method . The GUS measurement was repeated at least three times.
RT-PCR analysis using the WAVE dHPLC system
Total RNA was isolated using Trizol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. We used gene-specific primers (5'-CAGCTCCACACCTGTACGT-3' and 5'-CCATGCTCAGCGACTTGATG-3' for ZmNAC1, and 5'-ACGTGCATTACCATCCAATGC-3', 5'-CTGCATGACGAGGTATGTACG-3' for ZmmiR164b) to obtain the corresponding cDNA from each inbred line by RT-PCR. The PCR products were then sequenced to identify allele-specific sequence polymorphisms between the inbred lines 87-1 and Zong3 that would allow separation of the two parental alleles on the WAVE dHPLC system (Transgenomic, Omaha, NE). The gene-specific primers were designed to amplify the regions that are conserved between alleles and that encompass a sequence polymorphism indel to minimize the amplification preference of either allele and to optimize the amplicon for WAVE analysis. The β-actin gene fragment was amplified as a positive control using 26 cycles. Three PCR replicates were performed for each RNA sample. The RT-PCR products were then separated and quantified by the WAVE dHPLC system, and detailed WAVE dHPLC analysis was performed according to the manufacturer’s instructions .
This work was financially supported by the National Basic Research Program of China (2012CB910900), 863 Project of China (2012AA10A309) and the National Natural Science Foundation of China (30930058, 30600392, 30871529).
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