# tasiRNA-ARF Pathway Moderates Floral Architecture inArabidopsis Plants Subjected to Drought Stress

Authors: Akihiro Matsui; Kayoko Mizunashi; Maho Tanaka; Eli Kaminuma; Anh Hai Nguyen; Maiko Nakajima; Jong-Myong Kim; Dong Van Nguyen; Tetsuro Toyoda; Motoaki Seki
Journal: BioMed Research International
 (2014)

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## Abstract

In plants, miRNAs and siRNAs, such as transacting siRNAs (ta-siRNAs), affect their targets through distinct regulatory mechanisms. In this study, the expression profiles of small RNAs (smRNAs) inArabidopsis plants subjected to drought, cold, and high-salinity stress were analyzed using 454 DNA sequencing technology. Expression of three groups of ta-siRNAs (TAS1, TAS2, and TAS3) and their precursors was downregulated in Arabidopsis plants subjected to drought and high-salinity stress. Analysis of ta-siRNA synthesis mutants and mutated ARF3-overexpressing plants that escape the tasiRNA-ARF target indicated that self-pollination was hampered by short stamens in plants under drought and high-salinity stress. Microarray analysis of flower buds of rdr6 and wild-type plants under drought stress and nonstressed conditions revealed that expression of floral development- and auxin response-related genes was affected by drought stress and by the RDR6 mutation. The overall results of the present study indicated that tasiRNA-ARF is involved in maintaining the normal morphogenesis of flowers in plants under stress conditions through fine-tuning expression changes of floral development-related and auxin response-related genes.


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## Body

## 1. Introduction

In order to adapt and survive the exposure to biotic and abiotic stress, plants have evolved various molecular responses for fine-tuning the control of adaptive responses that involve posttranscriptional regulatory mechanisms, as well as epigenetic and posttranslational modifications [1, 2]. Recent genome-wide transcriptome analyses using tiling arrays and next generation sequencing (NGS) have revealed a large number of stress-responsive noncoding RNAs (ncRNAs) [3–5].Several small RNAs (smRNAs), such as miRNAs and siRNAs, were shown to function in development and stress responses in plants [6–9]. In plants, smRNAs exhibit a high level of complexity in their biogenesis and function. At the moment, smRNAs are classified into microRNAs (miRNAs) and three classes of small interfering RNAs (siRNAs) [6–9]. Transacting siRNAs (ta-siRNAs) are derived fromTAS ncRNAs that are targeted by miR173 or miR390 [10–14]. Double-stranded RNAs (dsRNAs) are generated from cleaved ncRNAs by RNA-dependent RNA polymerase 6 (RDR6) and dsRNAs are processed into 21nt ta-siRNAs.ARF2,ARF3 (ETT), andARF4 were demonstrated to be targets ofTAS3ta-siRNA (tasiRNA-ARF) [10–14].In the present study,Arabidopsis deep smRNA sequencing was used to identify novel roles for smRNAs in abiotic stress response and it was discovered that ta-siRNAs and their precursors (TAS1, TAS2,andTAS3) were downregulated by drought and high-salinity stress treatments. Analysis of ta-siRNA synthesis mutants subjected to drought and high-salinity stresses revealed a short stamen phenotype and changes in the expression of floral development-related and auxin response-related genes, which was enhanced by theRDR6mutation. These results demonstrate that the tasiRNA-ARF pathway functions in maintaining normal flower morphogenesis under environmental stress.


## 2. Materials and Methods

### 2.1.454 Sequencing of Small RNAs

Two-week-old wild-type plants (Arabidopsis thaliana ecotype Columbia), grown on MS medium [3], were transferred to drought, cold (4°C), and high-salinity (250 mM NaCl) stress　as previously reported [3]. The treated plants were harvested hourly from 1 to 10 hrs after the treatment was initiated. The 1–5 hr stress-treated samples and the 6–10 hr stress-treated samples were pooled into two groups. Total RNAs were prepared using an ISOGEN kit (Nippon Gene) and precipitated with 2 M LiCl and equal volume of ethanol. RNAs were resuspended in RNase-free water at 65°C and extracted twice with an equal volume of phenol for 30 min on ice. The RNAs were then precipitated with 2 M LiCl and equal volume of ethanol. They were resuspended in RNase-free water at 65°C and extracted twice with an equal volume of phenol for 30 min on ice and precipitated again by adding 1/10 volume of 3 M sodium acetate and 3 volume of ethanol. Subsequently, 17–30 nt smRNAs were extracted using flashPAGE (Life Technologies). A cDNA library was then constructed using a small RNA Cloning Kit (Takara). First, smRNAs were ligated with a 5′ adapter F: and a 3′ adapter to generate cDNAs. The cDNAs were electrophoresed in 8 M urea and 7.5% acrylamide gel, and 60–80 nt cDNAs were recovered. The cDNAs were amplified by 12–15 cycles of PCR and subjected to 454 DNA sequencing according to the manufacturer’s instructions. To eliminate RNA degradation fragments, the number of RNA sequences was normalized against the total number of miRNA sequences obtained. The normalized number of smRNAs was then subjected to data analysis. Data sets of 454 sequencing are available in DDBJ (http://www.ddbj.nig.ac.jp/index-e.html) under the accession number AB948670-AB967973.


### 2.2. Stress Treatments Applied to tasiRNA-ARF Pathway-Related Mutants

rdr6-15(SAIL_617), ago7(SALK_095997), sgs3(SALK_039005), dcl4-2(GK-160G05), ARF3pro;ARF3[15], ARF3pro:ARF3mut[15],arf3 (ett-15) [10],arf4-2 (SALK_070506), and wild-typeArabidopsis plants were grown for two weeks in pots containing 30 g of vermiculite soil. The drought stress treatment consisted of subjecting the two-week-old plants to water depletion for one week. After the one-week period, the watering of plants was then reinitiated. The high-salinity stress treatment consisted of watering three-week-old plants that started to bolt, with 100 mM NaCl-containing water for five days.


### 2.3. RNA Extraction

Total RNA was extracted with a Plant RNA Isolation Reagent (Life Technologies) and treated with DNase I (Life Technologies). The RNAs were then subjected to RT-quantitative PCR (RT-qPCR), microarray, and Northern analyses.


### 2.4. RT-qPCR Analysis

cDNAs were prepared from 1μg of total RNA using Superscript III (Life Technologies). The target RNA concentration was obtained by measuring 1/10 of the cDNA using an ABI Prism 3100 (Life Technologies) and SYBR Premix Ex Taq II kit (Takara). For detecting small RNAs, a SYBR Advantage qPCR Kit (Takara) was used. The relative expression was calculated using the delta-delta CT method. U2 andACT2for smRNA and mRNA, respectively, were used as a reference gene for normalization. Three independent biological replicates were used in all of the RT-qPCR analyses.


### 2.5. Microarray Analysis

Two-week-oldrdr6mutants and wild-type plants were subjected to a drought stress treatment consisting of withholding water for one week. Microarray experiments using flower buds subjected to a drought stress or nonstressed treatment were carried out according to the manufacturer’s (Agilent) preferred protocol using three biological replicates [16]. Fluorescent-labeled cRNAs were prepared from each total RNA sample using a Low Input Quick Amp Labeling Kit and were then hybridized to an AgilentArabidopsis V4 microarray. The microarrays were scanned using an Agilent DNA Microarray Scanner G2539A ver. C. 75 percentile normalization was performed for the signals generated by the microarray probes according to the Agilent data analysis protocol. For microarray analysis, R program ver. 2.12.1 was used. Significant differentially expressed genes were identified by 2-way ANOVA analysis (FDR < 0.075) [17, 18]. The data set derived from the microarray analysis is available in GEO (http://www.ncbi.nlm.nih.gov/geo/info/linking.html) under the accession number GSE57174.


## 2.1.454 Sequencing of Small RNAs

Two-week-old wild-type plants (Arabidopsis thaliana ecotype Columbia), grown on MS medium [3], were transferred to drought, cold (4°C), and high-salinity (250 mM NaCl) stress　as previously reported [3]. The treated plants were harvested hourly from 1 to 10 hrs after the treatment was initiated. The 1–5 hr stress-treated samples and the 6–10 hr stress-treated samples were pooled into two groups. Total RNAs were prepared using an ISOGEN kit (Nippon Gene) and precipitated with 2 M LiCl and equal volume of ethanol. RNAs were resuspended in RNase-free water at 65°C and extracted twice with an equal volume of phenol for 30 min on ice. The RNAs were then precipitated with 2 M LiCl and equal volume of ethanol. They were resuspended in RNase-free water at 65°C and extracted twice with an equal volume of phenol for 30 min on ice and precipitated again by adding 1/10 volume of 3 M sodium acetate and 3 volume of ethanol. Subsequently, 17–30 nt smRNAs were extracted using flashPAGE (Life Technologies). A cDNA library was then constructed using a small RNA Cloning Kit (Takara). First, smRNAs were ligated with a 5′ adapter F: and a 3′ adapter to generate cDNAs. The cDNAs were electrophoresed in 8 M urea and 7.5% acrylamide gel, and 60–80 nt cDNAs were recovered. The cDNAs were amplified by 12–15 cycles of PCR and subjected to 454 DNA sequencing according to the manufacturer’s instructions. To eliminate RNA degradation fragments, the number of RNA sequences was normalized against the total number of miRNA sequences obtained. The normalized number of smRNAs was then subjected to data analysis. Data sets of 454 sequencing are available in DDBJ (http://www.ddbj.nig.ac.jp/index-e.html) under the accession number AB948670-AB967973.


## 2.2. Stress Treatments Applied to tasiRNA-ARF Pathway-Related Mutants

rdr6-15(SAIL_617), ago7(SALK_095997), sgs3(SALK_039005), dcl4-2(GK-160G05), ARF3pro;ARF3[15], ARF3pro:ARF3mut[15],arf3 (ett-15) [10],arf4-2 (SALK_070506), and wild-typeArabidopsis plants were grown for two weeks in pots containing 30 g of vermiculite soil. The drought stress treatment consisted of subjecting the two-week-old plants to water depletion for one week. After the one-week period, the watering of plants was then reinitiated. The high-salinity stress treatment consisted of watering three-week-old plants that started to bolt, with 100 mM NaCl-containing water for five days.


## 2.3. RNA Extraction

Total RNA was extracted with a Plant RNA Isolation Reagent (Life Technologies) and treated with DNase I (Life Technologies). The RNAs were then subjected to RT-quantitative PCR (RT-qPCR), microarray, and Northern analyses.


## 2.4. RT-qPCR Analysis

cDNAs were prepared from 1μg of total RNA using Superscript III (Life Technologies). The target RNA concentration was obtained by measuring 1/10 of the cDNA using an ABI Prism 3100 (Life Technologies) and SYBR Premix Ex Taq II kit (Takara). For detecting small RNAs, a SYBR Advantage qPCR Kit (Takara) was used. The relative expression was calculated using the delta-delta CT method. U2 andACT2for smRNA and mRNA, respectively, were used as a reference gene for normalization. Three independent biological replicates were used in all of the RT-qPCR analyses.


## 2.5. Microarray Analysis

Two-week-oldrdr6mutants and wild-type plants were subjected to a drought stress treatment consisting of withholding water for one week. Microarray experiments using flower buds subjected to a drought stress or nonstressed treatment were carried out according to the manufacturer’s (Agilent) preferred protocol using three biological replicates [16]. Fluorescent-labeled cRNAs were prepared from each total RNA sample using a Low Input Quick Amp Labeling Kit and were then hybridized to an AgilentArabidopsis V4 microarray. The microarrays were scanned using an Agilent DNA Microarray Scanner G2539A ver. C. 75 percentile normalization was performed for the signals generated by the microarray probes according to the Agilent data analysis protocol. For microarray analysis, R program ver. 2.12.1 was used. Significant differentially expressed genes were identified by 2-way ANOVA analysis (FDR < 0.075) [17, 18]. The data set derived from the microarray analysis is available in GEO (http://www.ncbi.nlm.nih.gov/geo/info/linking.html) under the accession number GSE57174.


## 3. Results

### 3.1. RNA Sequencing ofArabidopsis Small RNAs in Plants under Abiotic Stress

Two-week-old wild typeArabidopsis plants were subjected to drought, cold, and high-salinity stress as described in Section 2. Six smRNA libraries were prepared from pooled samples of stress-treated plants and one pooled sample from nonstressed control plants using 454 DNA sequencing technology as described in Section 2. A total of 480,343 reads were obtained from these seven libraries. The smRNA sequences (17–30 nt) were used for further analysis. After the smRNA sequence data were assembled to unique sequences in each library and they were mapped to theArabidopsisgenome, resulting in 59,284 sequences that were perfectly matched to at least one locus (Table 1). The sequences represented 12,028 unique signatures in whole 7 libraries. Approximately 39% (4,681) of the unique signatures were represented by a single sequence. The smRNAs were classified based on their mapped genomic position. The composite profiles of the types of identified smRNAs were different in each stress treatment (Supplemental Figure 1; Supplementary Material available online at  http://dx.doi.org/10.1155/2014/303451). The percentile of smRNAs mapped to the sense strand of AGI code genes was higher in drought-stress and high-salinity-stress treated samples than in the nonstressed control sample, suggesting that mRNA degradation occurs preferentially under these stress conditions.Table 1
Number of smRNA-seqs and smRNA loci in each treatment.
Treatment
Number of sequencesa
Number of locib
Drought
1–5 h
17,758
4,574
6–10 h
10,367
3,138
Cold
1–5 h
2,104
1,595
6–10 h
2,869
2,018
High salinity
1–5 h
10,247
3,645
6–10 h
6,148
2,417
No treatment
9,791
4,162
asmRNA sequences were mapped on Arabidopsis genome. Sequences of tRNAs, rRNAs snoRNAs, and snRNAs were eliminated. bsmRNAs mapped within less than 150 nt distance were grouped as the same loci.


### 3.2. Identification of Stress-Responsive miRNAs

Deep RNA sequencing analysis of smRNAs identified signatures of various miRNAs, including those previously reported to be stress　responsive miRNAs inArabidopsis (Supplemental Figure 2). Expression of miR169 was downregulated in response to drought stress (Supplemental Figure 2). Downregulation of miR169 by drought has also been previously reported and demonstrated to be required for the acquisition of drought stress tolerance [19]. Expression of miR156 and miR319 was upregulated by salinity stress and miR408 expression was upregulated by cold stress (Supplemental Figure 2). These results are also consistent with a previous report [20].


### 3.3. Accumulation of ta-siRNA Expression

A further analysis of smRNA sequences identified in the present study revealed that the number of ta-siRNAs was reduced in response to the stress treatments when compared to the number of ta-siRNAs identified in the nonstressed control (Figure1(a) and Supplemental Figure 2). The precursors of tasiRNA are transcribed as ncRNAs and processed into siRNAs after a miRNA cleavage event [10–14]. An analysis ofArabidopsis expression profiling data obtained from a previous study utilizing a tiling array [3] indicated that the expression of theTAS1/2/3 family was downregulated under drought and high-salinity stress (Figure 1(b)). Semiquantitative RT-PCR analysis, using primer sets that span the miRNA cleavage sites, showed that the expression ofTAS1/2/3precursors decreased under drought and high-salinity stress (Figure 1(c)). These results are consistent with the ta-siRNA expression profiling data obtained from smRNA sequencing. Collectively, the data indicate that abiotic stress signaling regulates ta-siRNA production through transcriptional regulation of their precursors.Downregulation of ta-siRNAs and TAS precursors in plants under drought and high-salinity stress. (a) Relative number of ta-siRNA sequences obtained by 454 DNA sequencing of cDNA libraries prepared from small RNAs. Orange, blue, and red boxes represent the relative number ofTAS1ta-siRNA,TAS2ta-siRNA,andTAS3ta-siRNA, respectively. (b) Relative expression ofTAS precursors was analyzed using previous data from a tiling array [3]. Two-week-oldArabidopsis plants were subjected to drought (2 hr, 10 hr), cold (2 hr, 10 hr), and high-salinity (2 hr, 10 hr) (see Section 2). (c)PCR primer sets (Supplemental Table 1) spanning miRNA cleavage site were designed to determine the expression profiles ofTAS precursors. RNA samples were isolated from drought (2 hr, 10 hr), cold (2 hr, 10 hr), and high-salinity (2 hr, 10 hr) treated wild-type plants and nonstress controls. Expression ofTAS precursors was analyzed by semiquantitative RT-PCR.
(a)
(b)
(c)It is known that ta-siRNA-ARF guides the cleavage ofARF3 andARF4mRNAs [10, 11, 14, 15]. Therefore, the accumulation ofTAS3 precursor, ta-siRNA-ARF,MIR390, ARF3, andARF4in wild-type plants and a ta-siRNA synthesis mutant,rdr6, in response to a five-hour-drought stress treatment was measured using RT-qPCR in order to better understand the function of ta-siRNA in abiotic stress response (Figure 2). Expression of theTAS3a precursor andRDR6 was downregulated under drought stress (Figure 2). The TAS3a expression data is consistent with the tiling array data (Figure 1(b)) [3] and results obtained by semiquantitative RT-PCR (Figure 1(c)). Expression ofARF3 andARF4 was downregulated under drought stress in wild-type plants but the level of downregulation inrdr6 mutants was much less (Figure 2). These results suggest that effective downregulation of ARF3/ARF4 mRNAs under drought stress occurs by degradation activity of tasiRNA-ARF (Figure 2) and transcriptional repression of ARF3/ARF4 mRNAs (Figure 2) under drought stress. Downregulation of tasiRNA-ARF might function in fine-tuning quantitative expression of ARF3/ARF4 under drought stress.Figure 2
Expression profiles of miR390,TAS3 precursor,ARF3, andARF4 in plants under drought stress. Expression profiles of tasiRNA-ARF pathway-related genes were analyzed inrdr6 mutants and wild-type plants subjected to a 5 hr drought stress and nonstressed conditions by RT-qPCR. The bar graphs indicate relative expression compared toACT2. Values represent the mean and standard deviation of three experiments.


### 3.4. ta-siRNA Generation-Related Mutants Fail to Self-Pollinate due to Modifications in Flower Architecture

In order to identify the biological function of ta-siRNA under environmental stress, a moderate drought stress was applied to a variety ofArabidopsis mutants that are deficient in ta-siRNA biosynthesis (rdr6, sgs3,dcl4, andago7). The moderate drought stress was applied to two-week-old plants grown in soil by withholding water for one week, resulting in an approximate 20% decrease in soil water content (Supplemental Figure 3(a)). Following the drought treatment, the plants were rewatered. After recovery, wild-type plants produced a number of seeds that was similar to the numbers produced in nonstressed control plants (Figures 3(a) and 3(b)). On the other hand, ta-siRNA mutants, such asrdr6, sgs3, dcl4, andago7,produced a lower number of seeds after recovery from the drought stress compared to the numbers produced in nonstressed ta-siRNA mutants (Figures 3(a) and 3(b) and Supplemental Figure 3(b)).Modification of flower architecture and reduction in seed number in the ta-siRNA biosynthesis mutant,rdr6,under drought stress. (a) Two-week-oldrdr6 and wild-type plants were subjected to drought stress treatment for one week and then rewatered. Seed number in the 1st through the 5th siliques was counted (n
=
12). Siliques were numbered starting at the basal end of the main shoot. (b) Siliques in plants subjected to drought stress followed by rewatering. (c) Floral architecture in drought-stress and nonstressed wild-type,rdr6,ARF3pro:ARF3,andARF3pro:ARF3mutplants. Flowers inrdr6 andARF3pro:ARF3plants have a slightly exerting stigma phenotype under nonstressed conditions. In contrast, flowers inrdr6 andARF3pro:ARF3mut plants under drought stress exhibit short stamens, as shown as white arrows. (d) Average length of the stigmas and stamens in drought-stressed and nonstressed WT,rdr6,ARF3pro:ARF3,andARF3pro:ARF3mutplants.
(a)
(b)
(c)
(d)The reproduction failure phenotype was further investigated and it was found that the reproduction failure ofrdr6 plants under drought stress could be rescued by artificial pollination (Supplemental Figure 3(c)). These data suggested that the reproduction failure under drought stress was due to insufficient contact between the anther and the stigma. Abnormal floral architecture was observed in stage 13 flowers ofrdr6 plants, which had shorter stamens relative to the length of the stigma (Figure 3(c)). The length of stigmas and stamens in stage 13 flowers was examined inrdr6 and wild-type plants under drought stress and nonstressed conditions. Stigma lengthinrdr6 and wild-type plants was similar in plants under drought stress and nonstressed conditions. In contrast, the length ofrdr6 stamens was shorter in plants under drought stress compared to the length of stamens in wild-type plants under drought stress (Figures 3(c) and 3(d)). Althoughrdr6 plants exhibited a tendency to produce slightly shorter stamens, relative to wild-type plants, even in nonstressed conditions, the difference was not statistically significant (Figures 3(c) and 3(d)). These data were consistent with a previous report [21].It has been demonstrated that tasiRNA-ARF negatively regulatesARF3 andARF4,both of which are genes that control flower organ identity [22–24]. The ability of tasiRNA-ARF to regulate correct stamen development under drought stress was examined usingARF3pro:ARF3mut. This transgenic genotype has a mutatedARF3 sequence that confers the ability ofARF3 to avoid tasiRNA-ARF targeted degradation and still translate ARF3 protein [15]. Similar tordr6plants, theARF3pro:ARF3mut exhibited a short stamen phenotype in plants subjected to drought stress (Figures 3(c) and 3(d)), suggesting that the tasiRNA-ARF pathway has an important role in regulating stamen length under drought stress. Additionally, reproductive failure and the short stamen phenotype were also observed inrdr6 mutants andARF3pro:ARF3mut plants that were subjected to high-salinity stress (Supplemental Figures 3(d), 3(e), and 3(f)). These results suggest that the tasiRNA-ARF pathway plays a critical role in the regulation of floral organ development under both drought and high-salinity stress.


### 3.5.RDR6 Functions in the Stabilization of Stress-Dependent Changes in the Expression of Floral Development-Related and Auxin-Related Genes in Plants under Drought Stress

Gene expression profiles in flower buds ofArabidopsis plants under moderate drought stress and nonstressed conditions were analyzed using a microarray in order to better understand the regulation of the ta-siRNA-mediated network in response to drought stress. Results identified 513 (30.3%) genes whose level of expression was significantly different inrdr6 plants compared to wild-type plants. The list of genes overlapped with drought stress-responsive genes (Figure 4(a), Supplemental Table 2). No correlation (
R
2
=
0.09
) was observed between the expression ratios of drought/control and those ofrdr6/wild-type for the GO category of water deprivation response-related genes (GO:0009414) (Figure 4(b)).rdr6and wild-type plants showed similar drought stress-responsive expression in the water deprivation response-related genes (Supplemental Figure 4(a)). These results suggest that a similar level of drought stress was applied tordr6 and wild-type plants.Microarray analysis of flower buds in plants under drought stress. (a) Venn diagram of differentially expressed genes inRDR6 plants compared to wild-type plants and drought stress-responsive genes. Statistically significant differentially expressed genes were identified based on the following criteria: FDR of 2-way ANOVA (rdr6 versus WT or drought-stressed versus nonstressed) < 0.075. ((b)–(d)) Scatter plot analysis of genes from different categories of GO terms. Horizontal axis represents log2 ratio of (rdr6 non-stressed +rdr6 drought)/(WT nonstressed + WT drought). Vertical axis represents log2 ratio of (WT drought+rdr6 drought)/(WT nonstressed +rdr6 non-stressed). (b) Scatter plot analysis of water deprivation response-related genes. (c) Scatter plot analysis of floral organ development-related genes. (d) Scatter plot analysis of auxin response-related genes. (e) RT-qPCR expression profiles of tasiRNA-ARF,ARF3,ARF4,GH3-3,YUCCA1,andYUCCA4 in flower buds ofrdr6mutant and wild-type plants subjected to drought stress or nonstressed control.
(a)
(b)
(c)
(d)
(e)Among the differentially expressed genes inrdr6 plants were a number of floral development-related genes (Supplemental Table 3). Expression levels of the C-class homeotic geneAGAMOUS  [25] andAGAMOUSdownstream genes that promote the development of stigma, style, and medial tissue of ovules, such asSHATTERPROOF 1 andSHATTERPROOF 2 [26], and the stigma and stamen identity gene,SUPERMAN [27], were upregulated under drought stress and their upregulation was affected inrdr6mutants (Supplemental Table 3). In contrast, expression of the E-class organ identity gene,SEPALLATA 4[28], and the petal identity genePETALLOSS[29] was downregulated in response to drought stress in both wild-type andrdr6 mutants; however, their expression was significantly lower inrdr6mutants relative to wild-type plants (Supplemental Table 3). These results indicate that theRDR6 mutation affects organ whorl identity genes and their downstream genes. It seems that tasiRNA-ARF regulation represses central floral organ development and enhances peripheral floral development under drought stress.To better understand the relationship between drought stress response and theRDR6 mutation on flower development-related genes (GO:0048437), the expression ratios of drought/control and those ofrdr6/wild-type were compared. A moderate positive correlation (
R
2
=
0.387
) was observed between the drought response andRDR6 mutation (Figure 4(c)). These data suggest that tasiRNA-ARF is involved in fine-tuning the expression of floral development-related genes in plants subjected to drought stress.


## 3.1. RNA Sequencing ofArabidopsis Small RNAs in Plants under Abiotic Stress

Two-week-old wild typeArabidopsis plants were subjected to drought, cold, and high-salinity stress as described in Section 2. Six smRNA libraries were prepared from pooled samples of stress-treated plants and one pooled sample from nonstressed control plants using 454 DNA sequencing technology as described in Section 2. A total of 480,343 reads were obtained from these seven libraries. The smRNA sequences (17–30 nt) were used for further analysis. After the smRNA sequence data were assembled to unique sequences in each library and they were mapped to theArabidopsisgenome, resulting in 59,284 sequences that were perfectly matched to at least one locus (Table 1). The sequences represented 12,028 unique signatures in whole 7 libraries. Approximately 39% (4,681) of the unique signatures were represented by a single sequence. The smRNAs were classified based on their mapped genomic position. The composite profiles of the types of identified smRNAs were different in each stress treatment (Supplemental Figure 1; Supplementary Material available online at  http://dx.doi.org/10.1155/2014/303451). The percentile of smRNAs mapped to the sense strand of AGI code genes was higher in drought-stress and high-salinity-stress treated samples than in the nonstressed control sample, suggesting that mRNA degradation occurs preferentially under these stress conditions.Table 1
Number of smRNA-seqs and smRNA loci in each treatment.
Treatment
Number of sequencesa
Number of locib
Drought
1–5 h
17,758
4,574
6–10 h
10,367
3,138
Cold
1–5 h
2,104
1,595
6–10 h
2,869
2,018
High salinity
1–5 h
10,247
3,645
6–10 h
6,148
2,417
No treatment
9,791
4,162
asmRNA sequences were mapped on Arabidopsis genome. Sequences of tRNAs, rRNAs snoRNAs, and snRNAs were eliminated. bsmRNAs mapped within less than 150 nt distance were grouped as the same loci.


## 3.2. Identification of Stress-Responsive miRNAs

Deep RNA sequencing analysis of smRNAs identified signatures of various miRNAs, including those previously reported to be stress　responsive miRNAs inArabidopsis (Supplemental Figure 2). Expression of miR169 was downregulated in response to drought stress (Supplemental Figure 2). Downregulation of miR169 by drought has also been previously reported and demonstrated to be required for the acquisition of drought stress tolerance [19]. Expression of miR156 and miR319 was upregulated by salinity stress and miR408 expression was upregulated by cold stress (Supplemental Figure 2). These results are also consistent with a previous report [20].


## 3.3. Accumulation of ta-siRNA Expression

A further analysis of smRNA sequences identified in the present study revealed that the number of ta-siRNAs was reduced in response to the stress treatments when compared to the number of ta-siRNAs identified in the nonstressed control (Figure1(a) and Supplemental Figure 2). The precursors of tasiRNA are transcribed as ncRNAs and processed into siRNAs after a miRNA cleavage event [10–14]. An analysis ofArabidopsis expression profiling data obtained from a previous study utilizing a tiling array [3] indicated that the expression of theTAS1/2/3 family was downregulated under drought and high-salinity stress (Figure 1(b)). Semiquantitative RT-PCR analysis, using primer sets that span the miRNA cleavage sites, showed that the expression ofTAS1/2/3precursors decreased under drought and high-salinity stress (Figure 1(c)). These results are consistent with the ta-siRNA expression profiling data obtained from smRNA sequencing. Collectively, the data indicate that abiotic stress signaling regulates ta-siRNA production through transcriptional regulation of their precursors.Downregulation of ta-siRNAs and TAS precursors in plants under drought and high-salinity stress. (a) Relative number of ta-siRNA sequences obtained by 454 DNA sequencing of cDNA libraries prepared from small RNAs. Orange, blue, and red boxes represent the relative number ofTAS1ta-siRNA,TAS2ta-siRNA,andTAS3ta-siRNA, respectively. (b) Relative expression ofTAS precursors was analyzed using previous data from a tiling array [3]. Two-week-oldArabidopsis plants were subjected to drought (2 hr, 10 hr), cold (2 hr, 10 hr), and high-salinity (2 hr, 10 hr) (see Section 2). (c)PCR primer sets (Supplemental Table 1) spanning miRNA cleavage site were designed to determine the expression profiles ofTAS precursors. RNA samples were isolated from drought (2 hr, 10 hr), cold (2 hr, 10 hr), and high-salinity (2 hr, 10 hr) treated wild-type plants and nonstress controls. Expression ofTAS precursors was analyzed by semiquantitative RT-PCR.
(a)
(b)
(c)It is known that ta-siRNA-ARF guides the cleavage ofARF3 andARF4mRNAs [10, 11, 14, 15]. Therefore, the accumulation ofTAS3 precursor, ta-siRNA-ARF,MIR390, ARF3, andARF4in wild-type plants and a ta-siRNA synthesis mutant,rdr6, in response to a five-hour-drought stress treatment was measured using RT-qPCR in order to better understand the function of ta-siRNA in abiotic stress response (Figure 2). Expression of theTAS3a precursor andRDR6 was downregulated under drought stress (Figure 2). The TAS3a expression data is consistent with the tiling array data (Figure 1(b)) [3] and results obtained by semiquantitative RT-PCR (Figure 1(c)). Expression ofARF3 andARF4 was downregulated under drought stress in wild-type plants but the level of downregulation inrdr6 mutants was much less (Figure 2). These results suggest that effective downregulation of ARF3/ARF4 mRNAs under drought stress occurs by degradation activity of tasiRNA-ARF (Figure 2) and transcriptional repression of ARF3/ARF4 mRNAs (Figure 2) under drought stress. Downregulation of tasiRNA-ARF might function in fine-tuning quantitative expression of ARF3/ARF4 under drought stress.Figure 2
Expression profiles of miR390,TAS3 precursor,ARF3, andARF4 in plants under drought stress. Expression profiles of tasiRNA-ARF pathway-related genes were analyzed inrdr6 mutants and wild-type plants subjected to a 5 hr drought stress and nonstressed conditions by RT-qPCR. The bar graphs indicate relative expression compared toACT2. Values represent the mean and standard deviation of three experiments.


## 3.4. ta-siRNA Generation-Related Mutants Fail to Self-Pollinate due to Modifications in Flower Architecture

In order to identify the biological function of ta-siRNA under environmental stress, a moderate drought stress was applied to a variety ofArabidopsis mutants that are deficient in ta-siRNA biosynthesis (rdr6, sgs3,dcl4, andago7). The moderate drought stress was applied to two-week-old plants grown in soil by withholding water for one week, resulting in an approximate 20% decrease in soil water content (Supplemental Figure 3(a)). Following the drought treatment, the plants were rewatered. After recovery, wild-type plants produced a number of seeds that was similar to the numbers produced in nonstressed control plants (Figures 3(a) and 3(b)). On the other hand, ta-siRNA mutants, such asrdr6, sgs3, dcl4, andago7,produced a lower number of seeds after recovery from the drought stress compared to the numbers produced in nonstressed ta-siRNA mutants (Figures 3(a) and 3(b) and Supplemental Figure 3(b)).Modification of flower architecture and reduction in seed number in the ta-siRNA biosynthesis mutant,rdr6,under drought stress. (a) Two-week-oldrdr6 and wild-type plants were subjected to drought stress treatment for one week and then rewatered. Seed number in the 1st through the 5th siliques was counted (n
=
12). Siliques were numbered starting at the basal end of the main shoot. (b) Siliques in plants subjected to drought stress followed by rewatering. (c) Floral architecture in drought-stress and nonstressed wild-type,rdr6,ARF3pro:ARF3,andARF3pro:ARF3mutplants. Flowers inrdr6 andARF3pro:ARF3plants have a slightly exerting stigma phenotype under nonstressed conditions. In contrast, flowers inrdr6 andARF3pro:ARF3mut plants under drought stress exhibit short stamens, as shown as white arrows. (d) Average length of the stigmas and stamens in drought-stressed and nonstressed WT,rdr6,ARF3pro:ARF3,andARF3pro:ARF3mutplants.
(a)
(b)
(c)
(d)The reproduction failure phenotype was further investigated and it was found that the reproduction failure ofrdr6 plants under drought stress could be rescued by artificial pollination (Supplemental Figure 3(c)). These data suggested that the reproduction failure under drought stress was due to insufficient contact between the anther and the stigma. Abnormal floral architecture was observed in stage 13 flowers ofrdr6 plants, which had shorter stamens relative to the length of the stigma (Figure 3(c)). The length of stigmas and stamens in stage 13 flowers was examined inrdr6 and wild-type plants under drought stress and nonstressed conditions. Stigma lengthinrdr6 and wild-type plants was similar in plants under drought stress and nonstressed conditions. In contrast, the length ofrdr6 stamens was shorter in plants under drought stress compared to the length of stamens in wild-type plants under drought stress (Figures 3(c) and 3(d)). Althoughrdr6 plants exhibited a tendency to produce slightly shorter stamens, relative to wild-type plants, even in nonstressed conditions, the difference was not statistically significant (Figures 3(c) and 3(d)). These data were consistent with a previous report [21].It has been demonstrated that tasiRNA-ARF negatively regulatesARF3 andARF4,both of which are genes that control flower organ identity [22–24]. The ability of tasiRNA-ARF to regulate correct stamen development under drought stress was examined usingARF3pro:ARF3mut. This transgenic genotype has a mutatedARF3 sequence that confers the ability ofARF3 to avoid tasiRNA-ARF targeted degradation and still translate ARF3 protein [15]. Similar tordr6plants, theARF3pro:ARF3mut exhibited a short stamen phenotype in plants subjected to drought stress (Figures 3(c) and 3(d)), suggesting that the tasiRNA-ARF pathway has an important role in regulating stamen length under drought stress. Additionally, reproductive failure and the short stamen phenotype were also observed inrdr6 mutants andARF3pro:ARF3mut plants that were subjected to high-salinity stress (Supplemental Figures 3(d), 3(e), and 3(f)). These results suggest that the tasiRNA-ARF pathway plays a critical role in the regulation of floral organ development under both drought and high-salinity stress.


## 3.5.RDR6 Functions in the Stabilization of Stress-Dependent Changes in the Expression of Floral Development-Related and Auxin-Related Genes in Plants under Drought Stress

Gene expression profiles in flower buds ofArabidopsis plants under moderate drought stress and nonstressed conditions were analyzed using a microarray in order to better understand the regulation of the ta-siRNA-mediated network in response to drought stress. Results identified 513 (30.3%) genes whose level of expression was significantly different inrdr6 plants compared to wild-type plants. The list of genes overlapped with drought stress-responsive genes (Figure 4(a), Supplemental Table 2). No correlation (
R
2
=
0.09
) was observed between the expression ratios of drought/control and those ofrdr6/wild-type for the GO category of water deprivation response-related genes (GO:0009414) (Figure 4(b)).rdr6and wild-type plants showed similar drought stress-responsive expression in the water deprivation response-related genes (Supplemental Figure 4(a)). These results suggest that a similar level of drought stress was applied tordr6 and wild-type plants.Microarray analysis of flower buds in plants under drought stress. (a) Venn diagram of differentially expressed genes inRDR6 plants compared to wild-type plants and drought stress-responsive genes. Statistically significant differentially expressed genes were identified based on the following criteria: FDR of 2-way ANOVA (rdr6 versus WT or drought-stressed versus nonstressed) < 0.075. ((b)–(d)) Scatter plot analysis of genes from different categories of GO terms. Horizontal axis represents log2 ratio of (rdr6 non-stressed +rdr6 drought)/(WT nonstressed + WT drought). Vertical axis represents log2 ratio of (WT drought+rdr6 drought)/(WT nonstressed +rdr6 non-stressed). (b) Scatter plot analysis of water deprivation response-related genes. (c) Scatter plot analysis of floral organ development-related genes. (d) Scatter plot analysis of auxin response-related genes. (e) RT-qPCR expression profiles of tasiRNA-ARF,ARF3,ARF4,GH3-3,YUCCA1,andYUCCA4 in flower buds ofrdr6mutant and wild-type plants subjected to drought stress or nonstressed control.
(a)
(b)
(c)
(d)
(e)Among the differentially expressed genes inrdr6 plants were a number of floral development-related genes (Supplemental Table 3). Expression levels of the C-class homeotic geneAGAMOUS  [25] andAGAMOUSdownstream genes that promote the development of stigma, style, and medial tissue of ovules, such asSHATTERPROOF 1 andSHATTERPROOF 2 [26], and the stigma and stamen identity gene,SUPERMAN [27], were upregulated under drought stress and their upregulation was affected inrdr6mutants (Supplemental Table 3). In contrast, expression of the E-class organ identity gene,SEPALLATA 4[28], and the petal identity genePETALLOSS[29] was downregulated in response to drought stress in both wild-type andrdr6 mutants; however, their expression was significantly lower inrdr6mutants relative to wild-type plants (Supplemental Table 3). These results indicate that theRDR6 mutation affects organ whorl identity genes and their downstream genes. It seems that tasiRNA-ARF regulation represses central floral organ development and enhances peripheral floral development under drought stress.To better understand the relationship between drought stress response and theRDR6 mutation on flower development-related genes (GO:0048437), the expression ratios of drought/control and those ofrdr6/wild-type were compared. A moderate positive correlation (
R
2
=
0.387
) was observed between the drought response andRDR6 mutation (Figure 4(c)). These data suggest that tasiRNA-ARF is involved in fine-tuning the expression of floral development-related genes in plants subjected to drought stress.


## 4. Discussion

The results of the present study demonstrate that tasiRNA-ARF functions in keeping correct flower architecture which is critical to self-pollination under drought and high-salinity stress. Various abiotic stresses, such as heat, high-salinity, drought, and cold, induce reproductive failure in plants [30–35]. This failure is the result of morphological abnormalities that arise during various stages of floral development. Molecular mechanisms responsible for the abortion of pollen development have been well-characterized [30–35]. Although defects in stamen development in plants under drought, high-salinity, and heat stress have been reported [32, 34, 35], the molecular mechanisms that protect floral development from the adverse effects of abiotic stress, however, remain unclear.tasiRNA-ARF is required for the normal development of lateral organs, such as leaves, lateral roots, and flowers [14, 21, 36, 37]. For example,rdr6 mutant exhibits altered stamen and pistil elongation that results in variable seed production and supports the premise that reproduction inrdr6plants is sensitive to growth conditions [22]. Interestingly, a mutation ofRDR6 enhanced self-incompatibility in a transgenic, self-incompatible,Arabidopsis thalianasystem [38]. These results suggest that tasiRNA-ARF functions as a key mediator for maintaining the correct pattern of flower architecture, as well as their development.ARF3 andARF4 function in central organ identity in flowers and apical-basal patterning defects in the gynoecium [22–24].ARFs regulate the expression of auxin-responsive genes by binding specifically to auxin response elements (AuxRE) [39]. Regarding auxin response-related genes (GO:0009733), a moderate positive correlation was observed between drought stress response and the effect of theRDR6 mutation (Figure 4(d)). The genes downregulated by both the drought stress treatment and theRDR6 mutation included the auxin-induced conjugating enzymes,GH3-2,GH3-3,GH3-6,GH3-10, and anauxin-responsive GH3-like protein (AT1G48660) (Supplemental Table 2). In addition, microarray analysis also identified that an expression of auxin biosynthesis-related gene,YUCCA 4, was significantly downregulated by drought stress and theRDR6 mutation (Supplemental Table 2). After conducting a closer study of 4YUCCA genes that are mainly expressed in flowers [40],YUCCA 1 andYUCCA 4 were downregulated by drought stress andRDR6 mutation andYUCCA 2 andYUCCA 6 were downregulated byRDR6 mutation (Supplemental Figure 4(b)). The regulation of tasiRNA-ARF and mRNAs ofARF3, GH3-3,YUCCA 1, andYUCCA 4 in flower buds was confirmed by RT-qPCR (Figure 4(e)). The previous report showed that expression of an auxin reporter, DR5:GUS, was decreased inARF3pro:ARF3mut plants [41]. These results suggest that the auxin biosynthesis and auxin response were attenuated in floral development by drought stress and loss of tasiRNA-ARF regulation. It was known that auxin signaling was important for floral development. The pin-shaped flower ofyucca1/4 was similar to the flowers produced inARF3- orARF4-overexpressing plants [40]. Experiments utilizing an auxin transport inhibitor indicate thatARF3 functions as a modulator of auxin response during floral development [42]. There is a possible hypothesis that tasiRNA-ARF controls floral development by maintaining the proper level of auxin signaling under drought stress (Figure 5).Figure 5
Proposed model for the fine-tuning of whorl architecture in flowers subjected to environmental stress via tasiRNA-ARF. tasiRNA-ARF is required for normal morphogenesis of floral whorl organs in plants subjected to drought stress. tasiRNA-ARF acts by modulating the expression of floral development-related genes in plants subjected to drought stress. Expression ofYUCCA4 is downregulated by drought stress and in theRDR6 mutation, suggesting that auxin biosynthesis is modulated by tasiRNA-ARF.Both tasiRNA-ARF andARF3/4 were downregulated under drought stress, suggesting that tasiRNA-ARF are required for quantitative adjustment ofARF3/4 expression. Positive feedback regulation of auxin signaling might also function in the regulation of these genes (Figure 5). It is known that initiation of lateral roots modulated by positive and negative feedback regulation between tasiRNA-ARF andARF2/3/4 through auxin signaling [38, 39].ARF3 expression was also induced by increased auxin biogenesis through upregulation ofYUCCA 4 in shoot initiation [43]. These previous reports invoke that drought stress affects tasiRNA-ARF regulatory network, but it remained unclear.To search candidate genes connecting abiotic stress and tasiRNA-ARF regulatory network, microarray coexpression analysis of ARF3 (P
<
0.01) and ARF4 (P
<
0.01) was performed. 155 genes coexpressed withARF3andARF4involved twenty-five abiotic stress response-related genes, such asDREB2C[44],DWD (DDB1-binding WD40 protein) [45],ascorbate peroxidase 1 [46],glutathione S-transferase[47], andRD21B [48] (Supplemental Table 4). Thegenes also included ta-siRNA pathway-related genes, such asRDR6,TAS3, otherTAS genes, and ta-siRNA target genes [49]. Sixty-four of the 155 genes possessed an ARF binding motif (tgtctc) [50] in the promoter region within 1kb upstream of the start codon (Supplemental Table 4). These genes may represent candidates that connect tasiRNA-ARF regulatory network and drought stress signaling pathway.In conclusion, this study demonstrates that tasiRNA-ARF acts as a central modifier, negatively regulating changes in the expression of floral development-related genes in plants under drought and assists in maintaining normal floral morphogenesis.



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*Source: 303451-2014-08-26.xml*