A Rice cis-Natural Antisense RNA Acts as a Translational Enhancer for Its Cognate mRNA and Contributes to Phosphate Homeostasis and Plant Fitness  

Abstract

cis-natural antisense transcripts (cis-NATs) are widespread in plants and are often associated with downregulation of their associated sense genes. We found that a cis-NAT positively regulates the level of a protein critical for phosphate homeostasis in rice (Oryza sativa). PHOSPHATE1;2 (PHO1;2), a gene involved in phosphate loading into the xylem in rice, and its associated cis-NAT_PHO1;2 are both controlled by promoters active in the vascular cylinder of roots and leaves. While the PHO1;2 promoter is unresponsive to the plant phosphate status, the cis-NAT_PHO1;2 promoter is strongly upregulated under phosphate deficiency. Expression of both cis-NAT_PHO1;2 and the PHO1;2 protein increased in phosphate-deficient plants, while the PHO1;2 mRNA level remained stable. Downregulation of cis-NAT_PHO1;2 expression by RNA interference resulted in a decrease in PHO1;2 protein, impaired the transfer of phosphate from root to shoot, and decreased seed yield. Constitutive overexpression of NAT_PHO1;2 in trans led to a strong increase of PHO1;2, even under phosphate-sufficient conditions. Under all conditions, no changes occurred in the level of expression, sequence, or nuclear export of PHO1;2 mRNA. However, expression of cis-NAT_PHO1;2 was associated with a shift of both PHO1;2 and cis-NAT_PHO1;2 toward the polysomes. These findings reveal an unexpected role for cis-NAT_PHO1;2 in promoting PHO1;2 translation and affecting phosphate homeostasis and plant fitness.

INTRODUCTION

Recent studies of eukaryotic genomes using experimental approaches, such as deep RNA sequencing and tiling microarrays, have revealed that a large part of the genome of plants, fungi, and metazoans is transcribed and that noncoding regulatory RNAs (ncRNAs) play key roles in modulating gene expression. One group of such RNAs encompasses the small ncRNAs generated via processing from longer precursors and includes microRNAs (miRNAs) and short interfering RNAs (siRNAs). These small ncRNAs have well established roles in various gene silencing mechanisms involving the regulation of DNA methylation, mRNA processing, or translation (Ghildiyal and Zamore, 2009; Chen, 2010).

Long ncRNAs are typically >200 nucleotides in length and are generated without extensive processing except for splicing, capping, and polyadenylation. Natural antisense transcripts (NATs) represent one class of long RNAs containing sequences that are complementary to sense mRNAs (Lavorgna et al., 2004; Lapidot and Pilpel, 2006). While some NATs are known to encode proteins, the majority of NATs in eukaryote genomes are thought to be noncoding. NATs can be grouped into two categories, cis-NATs and trans-NATs. cis-NAT pairs are transcribed from opposing DNA strands at the same genomic locus and have a variety of orientations and differing lengths of overlap between the regions having perfect sequence complementarity, whereas trans-NAT pairs are transcribed from different loci and form partial complementarity to the sense transcript (Lapidot and Pilpel, 2006; Wang et al., 2006; Li et al., 2008).

Genome-wide analyses of numerous organisms, including human, mouse, Drosophila melanogaster, Arabidopsis thaliana, and rice (Oryza sativa) have revealed the widespread existence of NATs in eukaryotes, with ∼10 to 30% of mRNA-encoding genes having an associated NAT (Misra et al., 2002; Kiyosawa et al., 2003; Osato et al., 2003; Yelin et al., 2003; Katayama et al., 2005; Wang et al., 2005; Li et al., 2006; Zhang et al., 2006). Studies performed mainly in yeast and animals have revealed that NATs can participate in a broad range of regulatory events, including transcriptional interference, RNA masking-induced alternative splicing, X chromosome inactivation, genomic imprinting, RNA editing, DNA methylation, and siRNA-induced gene silencing (Faghihi and Wahlestedt, 2009; Au et al., 2011; Rinn and Chang, 2012). While the expression of some NATs has been associated with transcriptional activation, NAT expression primarily results in the suppression of gene expression and involves pathways leading to transcriptional downregulation, such as histone modification and the recruitment of chromatin modifiers, or posttranscriptional downregulation, such as the generation of siRNAs (Ponting et al., 2009).

In plants, despite the large number of NATs predicted or identified in Arabidopsis and rice, our current knowledge of their role in gene expression is limited to a few loci that are mainly involved in developmental control or adaptive responses to changing environmental conditions. All documented effects of NAT expression in plants have so far been associated with downregulation of gene expression. For example, the control of flowering in Arabidopsis by cold treatment was shown to involve expression of cis-NATs associated with the FLOWERING LOCUS C (FLC) locus, leading to FLC silencing via chromatin modification (Swiezewski et al., 2007, 2009; Liu et al., 2010). Expression of a noncoding cis-NAT associated with a cellulose synthase gene in barley (Hordeum vulgare) or a gene modulating plant immunity against a bacterial pathogen in Arabidopsis has been shown to trigger siRNA synthesis and lead to a downregulation of their respective target gene (Katiyar-Agarwal et al., 2006; Held et al., 2008). A similar mechanism involving the generation of siRNA and target downregulation was shown in two cases of convergent protein-coding genes overlapping in their 3′ end and involving male gametophyte function and plant response to salt stress (Borsani et al., 2005; Ron et al., 2010). The involvement of a cis-NAT in regulating the activity of the SHOOTING gene responsible for cytokinin synthesis has also been suggested to occur via degradation of Sho double-stranded RNA (Zubko and Meyer, 2007).

Here, we find a role for a cis-NAT in the homeostasis of Pi, one of the main nutrients limiting plant growth in most environments. Plants have evolved elaborate mechanisms to sense and adapt to low Pi. For example, microarray studies have revealed that several hundred genes are either induced or repressed following Pi starvation (Misson et al., 2005; Morcuende et al., 2007; Müller et al., 2007). Some of the important genes that are regulated by Pi deficiency are involved in the uptake of Pi from the environment and its controlled transport throughout the plant, such as members of the PHOSPHATE TRANSPORTER gene family, which encode H⁺−Pi cotransporters (Misson et al., 2005; Morcuende et al., 2007; Müller et al., 2007; Woo et al., 2012). Arabidopsis PHO1 is primarily expressed in root vascular tissues and is involved in the export of Pi to the apoplastic space of xylem vessels (Hamburger et al., 2002; Stefanovic et al., 2011; Arpat et al., 2012). The Arabidopsis pho1 null mutant shows a strong reduction of Pi loading into the xylem and displays a typical Pi-deficient phenotype in aerial parts of the plant, resulting in an overall reduction in shoot growth and seed yield (Hamburger et al., 2002). The expression of Arabidopsis PHO1 and the steady state level of PHO1 are increased under Pi deficiency (Stefanovic et al., 2007).

The rice genome contains three homologs of PHO1, and mutant analysis revealed that Os-PHO1;2 was the functional ortholog of At-PHO1, being the main gene responsible for the loading of Pi into the xylem vessel in rice (Secco et al., 2010). An interesting feature distinguishing the three rice PHO1 genes from their Arabidopsis homologs is the presence of cis-NATs associated with all of them (Secco et al., 2010). These cis-NATs overlap with the 5′ coding exons of the genes. Analysis of the expression profiles of the sense and cis-NAT pairs of the rice PHO1 genes revealed distinct patterns of expression in response to Pi deficiency. In particular, while the Os-PHO1;2 transcript level remained stable under Pi deficiency, its associated cis-NAT_PHO1;2 was strongly and specifically upregulated under the same nutrient deficiency (Secco et al., 2010). This expression pattern led us to investigate a potential role for cis-NAT_PHO1;2 in regulating PHO1;2 expression and to study its consequences on Pi homeostasis in rice.

RESULTS

Expression Pattern of PHO1;2 and Its Associated cis-NAT_PHO1;2

To examine the relationship between the expression pattern of PHO1;2 and its associated cis-NAT_PHO1;2, we generated transgenic rice lines expressing the β-glucuronidase (GUS) reporter under the control of the promoter sequence of the Os-PHO1;2 sense gene (Pro_{OsPHO1;2  -S}:GUS) or the cis-NAT_PHO1;2 (Pro_{cis-  NAT}:GUS) (Figure 1A). For each construct, 10 independent transgenic lines were examined by GUS staining. Four representative lines for each construct were selected for detailed analysis (Figure 1; see Supplemental Figure 1 online). Transgenic plants used for staining were cultured for 30 d in nutrient solution containing 1 mM Pi (+Pi treatment) or for 7 d on Pi-sufficient medium and then transferred to Pi-deficient medium for 23 d (−Pi treatment). Plants grown in medium without Pi were effectively starved for the nutrient, as determined by the low shoot Pi content and the induction of INDUCED BY PHOSPHATE STARVATION1 (IPS1), a gene known to be strongly upregulated by Pi deficiency (Hou et al., 2005) (see Supplemental Figure 2 online). Pro_{OsPHO1;2  -S}:GUS lines grown in Pi-deficient medium had GUS signal in the stele of the crown root (Figure 1B), as well as in the primary and lateral roots (see Supplemental Figure 3A online). Root tips containing meristematic tissues showed no GUS expression (see Supplemental Figure 3C online). Sections of roots revealed GUS staining primarily in the xylem parenchyma cells (Figure 1C). In shoots, GUS staining occurred in the large and small vascular bundles (Figure 1D). Cross sections of GUS-stained leaf blades showed high expression in vascular tissues, including xylem parenchyma, phloem sieve elements, and companion cells (Figure 1E). No GUS staining was detectable in floral structures. The same pattern of GUS expression in the different tissues was found in Pro_{OsPHO1;2  -S}:GUS lines grown in Pi-sufficient medium, with only a slight decrease in GUS activity relative to plants grown in Pi-deficient medium (Figure 1F).

Figure 1.

Expression Pattern and Quantification of GUS Activity in Pro_{OsPHO1;2  -S}:GUS and Pro_{cis-  NAT}:GUS Transgenic Lines.

(A) Schematic showing the genomic organization of the PHO1;2 locus. The 2-kb promoter regions of PHO1;2 and cis-NAT_PHO1;2 used to direct the expression of the GUS reporter gene are indicated by blue boxes. Exons are indicated by open red boxes, 3′ and 5′ UTRs by red hatched boxes, and introns as black lines. The DNA region giving rise to the cis-NAT_PHO1;2 transcript is indicated by the green box. Small arrows indicate the location of the primers used for strand-specific quantitative RT-PCR.

(B) to (E) and (G) to (J) Representative staining of GUS activity in 30-d-old Pi-deficient plants expressing Pro_{OsPHO1;2  -S}:GUS ([B] to [E]) or Pro_{cis-  NAT}:GUS ([G] to [J]). ab ep, abaxial epidermis; ad ep, adaxial epidermis; c, cortex; e, endodermis; ex, exodermis; lvb, large vascular bundle; mes, mesophyll; MxI, metaxylem I; pe, pericycle; ph, phloem; Px, protoxylem; svb, small vascular bundle; vc, vascular cylinder; xy, xylem. Bars = 500 µm in (B) and (G), 100 µm in (C), (E), (H), and (J), and 1 mm in (D) and (I).

(B) and (G) Intact crown root.

(C) and (H) Cross section of the primary root 2 cm from the tip and magnification (inset) of the central region of the vascular cylinder.

(D) and (I) Flag leaf blade.

(E) and (J) Cross section of the flag leaf blade and magnification of the small vascular bundle region.

(F) Fluorometric quantification of GUS activity in Pro_{OsPHO1;2  -S}:GUS and Pro_{cis-  NAT}:GUS transgenic lines under Pi-sufficient (+Pi) or Pi-deficient (−Pi) conditions. GUS activity was measured using crude extracts. Five transgenic plants from lines transformed with each construct were employed for GUS quantification. Data are mean ± se of GUS activity from three independent experiments.

Analysis of the Pro_{cis-  NAT}:GUS transgenic lines showed no GUS staining in shoots, roots, or floral structures for plants grown in medium with Pi. In sharp contrast, strong GUS staining was observed for plants grown in medium without Pi. For leaves and roots, the pattern of GUS expression was identical to the Pro_{OsPHO1;2  -S}:GUS lines, with strong staining in the large and small vascular bundles of shoots and the stele of the crown root (Figures 1G to 1J) and primary and lateral roots (see Supplemental Figure 3B online), but not in the root tip (see Supplemental Figure 3D online). In flowers, GUS staining was observed in pollen and the vascular cylinder of the anther (see Supplemental Figure 4 online). For plants grown under Pi-deficient conditions, GUS activity observed in roots and leaves of Pro_{cis-  NAT}:GUS lines was ∼5.5- and 7.8-fold higher, respectively, than for Pro_{OsPHO1;2  -S}:GUS lines (Figure 1F). These results revealed that the activities of the promoters of both PHO1;2 and its associated cis-NAT_PHO1;2 largely overlapped, driving gene expression in cells of the vascular cylinder in both shoots and roots.

No Detectable Expression of Proteins from Open Reading Frames Present in cis-NAT_PHO1;2

The cis-NAT_PHO1;2 transcript was analyzed for the presence of open reading frames (ORFs) that could encode polypeptides composed of at least 40 amino acids. Three such ORFs were found, with ORF1, ORF2, and ORF3 potentially encoding polypeptides of 160, 136, and 103 amino acids in length, respectively (Figure 2A). ORF2 is enclosed within the sequence of ORF1 but uses a different reading frame. Analysis of the ORF sequences by BLAST did not reveal any proteins with significant similarity to ORF1 and ORF3. For ORF2, the only similarity found was a putative polypeptide encoded by a cis-NAT associated with a PHO1 homolog in maize (Zea mays) (LOC100278095) (see Supplemental Figure 5 online). The region harboring the ORF in the maize cis-NAT overlaps with the third exon of maize PHO1 (see Supplemental Figure 5 online). Since the third base of each codon of the rice cis-NAT_PHO1;2 ORF2 and of the maize cis-NAT ORF aligns with the third base for codons of their respective PHO1 protein, the apparent conservation in the ORFs of the maize and rice cis-NATs simply could be due to the high similarity between the rice and maize PHO1 proteins in this region.

Figure 2.

Expression of an Epitope-Tagged cis-NAT_PHO1;2 Construct.

(A) Schematic of the modified cis-NAT_PHO1;2 cDNA (modcis-NAT_PHO1;2) used to generate overexpressing lines containing epitope-tagged ORFs (top panel) and the overexpression cassette in the construct pMDC32-modcisNAT_PHO1;2 (bottom panel). The synthetic fragment was generated by adding distinct epitope tags (FLAG, HA, and MYC) at the 3′ end of each of the three longest ORFs. Transcription is derived from the double cauliflower mosaic virus (2×35S) promoter. LB, left T-DNA border; NOS-t, nopaline synthase terminator; HPT, hygromycin phosphotransferase; RB, right T-DNA border. Small arrowheads indicate position of primers F3 and E3 used to detect the expression of the transgene.

(B) Quantitative RT-PCR analysis of cis-NAT_PHO1;2 (endogenous and transgenic modcis-NAT_PHO1;2) and PHO1;2 expression level. RNA was extracted from the shoots of OX transgenic lines showing high (OX31 and OX8) or no detectable (OX32 and OX26) expression of the transgenic modcis-NAT_PHO1;2 transcript. Transgenic lines and the corresponding null segregants are 4-week-old plants grown in Pi-sufficient conditions. Data are means ± se (n = 4)

(C) Immunoblot analysis on shoot protein extract of two overexpressing lines (OX8 and OX31) using antibodies against the epitope tags. As controls, the fusions ORF1:FLAG, ORF2:HA, and ORF3:Myc were independently expressed in the bacterial strain BL21 (DE3) using a vector inducible by β-d-1-thiogalactopyranoside. MW, molecular mass.

(D) Immunoblot analysis of PHO1;2 in the shoots of modcisNAT_PHO1;2-OX lines. Wild-type plants grown in the greenhouse (WT-G) and the pho1;2 null mutant were used as controls. The induction of PHO1;2 synthesis in wild-type plants after 0 d (t_0-d) and 30 d (t_30-d) of Pi starvation is also shown. The position of PHO1;2 is indicated by the black arrow.

To determine if proteins were produced from any of these ORFs in plants, transgenic lines were created that overexpressed a modified NAT_PHO1;2 by adding a distinct epitope tag (HA, MYC, or FLAG) at the 3′ end of each of the three ORFs (Figure 2A). Although the addition of an HA tag to ORF2 modifies the sequence of ORF1 by adding 10 codons before the terminal FLAG epitope, such a modification of the 3′ end of ORF1 would be unlikely to affect its translation potential into protein. The modified NAT_PHO1;2 was under the control of the constitutive 2x cauliflower mosaic virus 35S promoter and transformed in rice. Quantitative RT-PCR analysis revealed that the transgene was strongly expressed in several independent lines, reaching a 10-fold higher level compared with the expression of the endogenous cis-NAT_PHO1;2 in shoots (Figure 2B). Analysis by immunoblot of the shoot protein extracts from these transgenic lines using antibodies recognizing each of the three epitopes failed to detect any polypeptide of the expected size, while the recombinant proteins expressed in bacteria were readily detected (Figure 2C). The detection limit of the immunoblot assay was estimated at 0.3 ng of epitope tag polypeptide per microgram of total protein for ORF1 and ORF2 and 1.5 ng of epitope tag polypeptide per microgram of total protein for OFR3. Thus, there was no evidence for significant expression of polypeptides from the cis-NAT_PHO1;2, indicating that the transcript was likely to be noncoding.

Correlation between cis-NAT_PHO1;2 Expression and PHO1;2 Protein Level

The relationship between the expression of PHO1;2 and its associated cis-NAT_PHO1;2 with the synthesis of PHO1;2 protein was analyzed in shoots and roots of plants grown in Pi-deficient medium. Rice seedlings grown for 14 d in Pi-sufficient medium were transferred to Pi-deficient medium for 5 to 30 d, and one set of plants was switched back to Pi-sufficient medium for 24 h at 30 d (R+1, where R represents recovery). Immunoblot analysis was performed using microsomal extracts from roots and shoots and polyclonal antibodies against PHO1;2 (see Supplemental Figure 6 online). In plants grown under Pi-sufficient media for 14 d (day 0), PHO1;2 was weakly expressed in both roots and shoots, but the protein level started to increase 15 to 20 d after transfer to Pi-deficient medium and reached a maximum at 30 d (Figures 3A and 3B). Upon a 24-h resupply of Pi to 30-d-old Pi-deficient plants, the amount of PHO1;2 strongly decreased, reaching levels similar to Pi-sufficient plants (Figures 3A and 3B) and revealing that the steady state level of PHO1;2 was directly affected by the Pi supply.

Figure 3.

Analysis of the Relationship between cis-NAT_PHO1;2 Expression Level and PHO1;2 Protein Level.

(A) and (B) Immunoblot analysis of PHO1;2 protein synthesis in roots (A) or shoots (B) of wild-type plants grown in Pi-deficient medium from 0 to 30 d (−Pi, 0 to 30) or in Pi-deficient medium for 30 d followed by 1 d in medium with 1 mM Pi (+Pi, R+1). Fifty micrograms of microsomal proteins was loaded per lane. Immunoblots were probed with anti-OsPHO1;2 antibody. Ponceau S staining of the blot is shown in the bottom panel as a loading control. The position of PHO1;2 is indicated by the black arrow. MW, molecular mass.

(C) and (D) Expression analysis of PHO1;2 and cis-NAT_PHO1;2 in roots (C) or in shoots (D) of the same plants used for immunoblot analysis. Strand-specific quantitative RT-PCR was performed to detect PHO1;2 (white bar) and cis-NAT_PHO1;2 (black bar) transcripts level. The position of the primers used for quantitative RT-PCR is indicated in Figure 1A. Data are means ± se (n = 3 to 4).

Analysis of the expression of PHO1;2 and cis-NAT_PHO1;2 was performed by quantitative RT-PCR using a subset of the same plants used for protein analysis (Figures 3C and 3D). In Pi-sufficient conditions (0 d), PHO1;2 and cis-NAT_PHO1;2 were both expressed in roots at a level 3.5-fold higher than in shoots. After 15 and 30 d of growth in Pi-deficient medium, the sense transcript levels remained at a fairly constant level, while the cis-NAT_PHO1;2 transcript levels increased gradually, for both roots and shoots. In the last day of Pi starvation, cis-NAT_PHO1;2 transcripts were increased by 4 times in the roots and 8 times in the shoots compared with 0 d. Upon a 1-d Pi resupply, cis-NAT_PHO1;2 expression decreased strongly in both roots and shoots, reaching levels slightly below those of Pi-sufficient plants. The kinetics of changes in PHO1;2 and cis-NAT_PHO1;2 expression resulted in a strong shift in the ratios of antisense to sense transcripts, with a ratio of ∼1 for roots and shoots of Pi-sufficient plants, and a ratio of 9 in shoots and 4.5 in roots after 30 d of Pi starvation. Overall, these results show that the steady state level of PHO1;2 protein followed the same abundance profile as the antisense transcript, revealing a positive relationship between cis-NAT_PHO1;2 transcript expression and PHO1;2 accumulation.

Downregulation of cis-NAT_PHO1;2 Expression Reduced PHO1;2 Levels via a Posttranscriptional Mechanism

To better understand the relationship between cis-NAT_PHO1;2 expression and the synthesis of PHO1;2 protein, we generated transgenic plants in which cis-NAT_PHO1;2 expression was downregulated by RNA interference (RNAi) using a chimeric 447-bp target sequence that was complementary to the cis-NAT_PHO1;2 transcript (which is an intronless transcript) but not to the exons of the spliced sense PHO1;2 transcript (Figure 4A). The hairpin RNAi construct contained two inverted repeats of the target sequence linked by a 920-bp fragment of the Escherichia coli uidA gene, and the whole cassette was under the control of the maize ubiquitin1 promoter, which has been shown to drive high expression of foreign genes in transgenic rice (Figure 4A) (Uchimiya et al., 1993).

Figure 4.

Modulation of cis-NAT_PHO1;2 Expression Affects PHO1;2 Protein Synthesis without Affecting Sense PHO1;2 Transcript.

(A) Schematic diagram of the RNAi cassette in the pANDA-RNAicisNAT_PHO1;2 construct. A chimeric 447-pb cis-NAT_PHO1;2 trigger was synthesized corresponding to four stretches of sequences (shown in dark green) that were present in cis-NAT_PHO1;2 transcript (pale and dark green) but absent from PHO1;2 mRNA (plain red are exons and hatched red are untranslated sequences) (left diagram). The synthetic fragment was then cloned in two orientations in the pANDA vector to trigger RNAi (right diagram). The RNAi trigger was expressed constitutively under the control of the pUbq promoter of the maize ubiquitin gene. Downstream of pUbq, the two unlabeled boxes represent exons and the thick line separating the exons marks an intron of the Ubq gene.

(B) and (C) Quantitative RT-PCR analysis of PHO1;2 and cis-NAT_PHO1;2 transcripts in roots (B) and shoots (C) of wild-type (WT) plants and RNAi lines (Ri1 and Ri2) grown in Pi-deficient or Pi-sufficient conditions. Data are means ± se (n = 5).

(D) and (E) Immunoblot analysis of PHO1;2 synthesis in roots (D) or shoots (E) of wild-type plants and RNAi lines grown under Pi-deficient or Pi-sufficient conditions. Microsomal proteins were extracted at different time points during the Pi starvation (0, 15, and 30 d) and 1 d after Pi recovery (R+1). As a control, 30-d-old wild-type plants were used. Immunoblots were probed with anti-OsPHO1;2 antibody. Ponceau S staining of the blot is shown in the bottom panel as a loading control. The position of PHO1;2 is indicated by the black arrow. MW, molecular mass.

(F) and (G)  RPA analysis of PHO1;2  (F) or cis-NAT_PHO1;2  (G) transcripts in wild-type plants and RNAi lines. A schematic representation of ³²P-labeled full-length single-stranded RNA probes used to detect both the sense PHO1;2 RNA and cis-NAT_PHO1;2 is shown in the top panels. Autoradiographs revealing the hybridizations corresponding to the undigested probe (P) (1% of the amount used in the RPA) as well as the protected fragments in both roots and shoots are shown in the bottom panels. Yeast tRNA (Y) was used as a negative control. nt, nucleotides.

(H) and (I) Alternative splicing analysis of the 5′-coding region of PHO1;2 by RT-PCR.

(H) Map of the relative primer positions (arrowheads) used in alternative splicing analysis shown in (I).

(I) Analysis of RT-PCR products resulting from amplification across all four exons E1 to E4 in wild-type and RNAi lines. The rice ubiquitin gene (Ubq) was used as internal control, and DNA plasmid containing full-length PHO1;2 cDNA was used as a positive control (C). The figure shows a typical result from a duplicate technical replicate.

Sixteen independent transgenic lines were examined by PCR for the presence of the T-DNA vector (HPT gene) and the RNAi cassette (GUS linker) and by RT-PCR for expression of the RNAi cassette (GUS linker) and its effectiveness in downregulating cis-NAT_PHO1;2 expression (see Supplemental Figure 7 online). cis-NAT_PHO1;2 expression was reduced in 14 independent RNAi lines that had inherited the transgene, and two of these lines (named Ri1 and Ri2) were selected for further analysis. RNA was isolated from shoots and roots of plants grown for 7 d on Pi-sufficient medium and then transferred to either Pi-deficient (−Pi) or Pi-sufficient (+Pi) medium for 21 d. Quantitative RT-PCR confirmed that the cis-NAT_PHO1;2 transcript level was strongly decreased in lines Ri1 and Ri2 in comparison to that in wild-type plants in both roots (Figure 4B) and shoots (Figure 4C) of plants grown in Pi-sufficient or Pi-deficient media. By contrast, the level of the sense PHO1;2 transcript was similar between wild-type control and transgenic RNAi lines in both Pi treatments (Figures 4B and 4C). Expression of the RNAi cassette thus led to a reversal in the antisense to sense transcript ratio, which became ∼0.05 in roots and 0.2 in shoots.

Next, the effect of a reduction of cis-NAT_PHO1;2 expression on the synthesis of PHO1;2 protein was analyzed. For this purpose, we compared the protein level of wild-type plants to the RNAi transgenic lines in both roots and shoots. Two-week-old rice seedlings were subjected to Pi starvation for 15 and 30 d, and one set of plants was switched back to Pi-sufficient medium for 24 h at 30 d (R+1). Microsomal proteins were extracted at days 0, 15, and 30 and R+1 from roots and shoots and analyzed by immunoblot with antibodies against PHO1;2. While PHO1;2 protein was clearly detected in roots and shoots of wild-type plants grown for 30 d in Pi-sufficient medium, no signal was detectable in the roots or shoots of the two RNAi lines, irrespective of Pi treatment (Figures 4D and 4E).

To ensure that the sense PHO1;2 transcript detected by quantitative RT-PCR was not cleaved by the RNAi targeting the cis-NAT_PHO1;2, we conducted an RNase protection assay (RPA) to detect full-length transcripts. ³²P-labeled full-length single-stranded RNA probe was used to detect both the sense PHO1;2 RNA and cis-NAT_PHO1;2. RNA samples were prepared from wild-type plants grown either on −Pi or +Pi medium and also from RNAi lines grown on −Pi medium. Using the probe detecting the PHO1;2 sense mRNA, a single RNase protected fragment of 2386 nucleotides was detected in both roots and shoots in the RNAi lines and in the wild type (Figure 4F), in agreement with the expression of a full-length mRNA. The signal strength obtained between wild-type and RNAi samples was similar, supporting the conclusion that the level of expression of the PHO1;2 sense mRNA was not influenced either by Pi nutritional status or expression of the RNAi directed toward the cis-NAT_PHO1;2.

RPA analysis using a single-strand probe against cis-NAT_PHO1;2 revealed a single band of 1652 nucleotides in wild-type plants in both roots and shoots, but no signal in shoots and only weak smeary signals in the root of the two RNAi lines, confirming the effectiveness and specificity of the RNAi construct toward cis-NAT_PHO1;2 (Figure 4G). Furthermore, wild-type plants grown under Pi starvation showed higher expression levels of cis-NAT_PHO1;2 transcript, consistent with previous findings.

Expression of certain cis-NATs has previously been shown to influence splicing of mRNA in regions of overlap between sense and antisense RNA (Hastings et al., 2000; Yan et al., 2005; Beltran et al., 2008). Although the RPA analysis described above showed only a full-length protected fragment for the sense PHO1;2 transcript, indicating the absence of alternative splicing products, the presence of potential splicing variants was further analyzed by RT-PCR, using a combination of oligonucleotides bordering the exon-intron junctions for the first four exons of PHO1;2, the region overlapping with cis-NAT_PHO1;2 (Figure 4H). No alternative splicing was observed (Figure 4I). Furthermore, cDNA of PHO1;2 using oligonucleotides that amplified the complete ORF region along with 174 and 356 bp of the 5′ and 3′ untranslated region (UTR), respectively, was also amplified from roots and shoots of the wild type and the two RNAi lines. Only a single PCR fragment of similar size was obtained for all samples (see Supplemental Figure 8 online). Direct DNA sequencing of the PCR products revealed identical nucleotide sequences for all samples, corresponding to PHO1;2 full-length cDNA (based on the cDNA clone AK100323.1), with no evidence of a mixed population of cDNAs indicating the presence of splice variants. The presence of full-length PHO1;2 transcript and the absence of splicing variants in the RNAi lines was also confirmed by RNA gel blot analysis using probes specific to the PHO1;2 RNA and the cis-NAT_PHO1;2 (see Supplemental Figure 9 online). Taken together, these results reveal that a reduction of cis-NAT_PHO1;2 expression led to a reduction of PHO1;2 protein level without affecting the steady state level or splicing of the PHO1;2 mRNA.

Double-stranded RNAs created by sense-antisense transcripts are potential substrates for RNA editing enzymes that lead to the deamination of either cytidine to create uridine or adenosine to create inosine (Bass, 2002; Meng et al., 2010). As such mRNA modifications could affect protein production by, for example, deleting or creating ATG start codons, the presence of edited transcript was examined in PHO1;2 transcript from the wild type and the RNAi line Ri1. Five independent RT-PCR reactions were performed on RNAs from the wild type and the Ri1 line using oligonucleotides that amplify the region from the 5′ UTR to exon 5 of PHO1.2, which includes the region of overlap with cis-NAT_PHO1;2. Ten clones derived from each RT-PCR reaction were sequenced, for a total of 50 clones for the wild type and 50 clones for the Ri1 line. Sequences of all clones were identical, thus indicating that RNA editing is unlikely to contribute to the reduction in PHO1;2 protein triggered by the downregulation of cis-NAT_PHO1;2 expression.

Overexpression of NAT_PHO1;2 in Trans Led to Increased PHO1;2 Protein Level

The link between NAT_PHO1;2 expression and PHO1;2 protein accumulation was further tested by overexpressing modified NAT_PHO1;2 containing epitope-tagged ORFs in shoots. Four transgenic lines were analyzed by quantitative RT-PCR and immunoblot analysis; two lines expressed only very low levels of transgenic NAT_PHO1;2 (lines OX26 and OX32), while lines OX8 and OX31 expressed high levels of transgenic NAT_PHO1;2 (Figure 2B). No change in the level of the sense PHO1;2 transcript was observed among all lines tested (Figure 2B). By contrast, immunoblot analysis of microsomes from shoots indicated that while the wild type and lines OX26 and OX32 had comparable levels of PHO1;2 protein, lines OX8 and OX31 showed a strong increase in steady state PHO1;2 level (Figure 2D). These results indicated that overexpression of NAT_PHO1;2 in trans led to increased PHO1;2 accumulation.

Increased Expression of NAT_PHO1;2 Leads to Enhanced Association of Both Sense and Antisense Transcripts to Polysomes

To gain insight into the positive correlation between PHO1;2 production and expression of cis-NAT_PHO1;2, export of PHO1;2 transcripts to the cytosol and its association with polysomes was examined. Comparison of transcripts present in a nuclear-enriched fraction compared with a cytosolic fraction indicated that more than 80% of both the PHO1;2 and cis-NAT_PHO1;2 transcripts were found in the cytosolic fraction for all lines tested, which included the wild type, the RNAi lines Ri1 and Ri2, as well as the two NAT_PHO1;2-overexpressing lines OX31 and OX8 (see Supplemental Figure 10 online).

Polysome profiles were generated from roots of wild-type plants grown in Pi-sufficient or Pi-deficient media, as well as of roots of the RNAi lines Ri1 and Ri2 and the overexpressing lines OX8 and OX31 grown in Pi-sufficient medium (Figure 5). Wild-type plants grown in Pi-deficient medium showed a clear enrichment of the PHO1;2 mRNA in the heavy polysome fraction compared with wild-type plants grown in Pi-sufficient medium (Figure 5A). No similar shift toward the heavier polysomal fraction was observed for transcripts of the PHO1;3 homolog (Secco et al., 2010), indicating a specificity of the polysomal enrichment for PHO1;2 mRNA. Similarly, for plants grown in Pi-sufficient media, an enrichment of PHO1;2 transcript in the polysomal fractions was observed for the OX lines compared with the wild type, while a reduction was observed for the RNAi lines (Figure 5B). Interestingly, the distribution of cis-NAT_PHO1;2 in both the wild type and the OX lines followed the same trend as for the sense PHO1;2 transcript, being enriched in the polysome fraction in Pi-deficient wild-type plants and in Pi-sufficient OX plants (Figure 5C). Furthermore, in the OX lines, the transgenic epitope-tagged NAT transcript was also enriched in the polysome fractions (Figure 5C). These data reveal that the increased expression of a NAT_PHO1;2 transcript, triggered either by phosphate deficiency or via transgenesis, leads to a shift of both the sense PHO1;2 and the antisense NAT toward the translationally active polysomes.

Figure 5.

Increased Polysome Association of PHO1.2 mRNA upon Pi Deficiency Treatment or NAT_PHO1;2 Overexpression.

Quantitative RT-PCR was performed on root RNAs purified from six Suc gradient fractions to quantify transcripts from PHO1;2 and PHO1;3 in wild-type (WT) plants grown in Pi-sufficient or Pi-deficient media (A), PHO1;2 in the wild type, in the cis-NAT_PHO1;2  RNAi lines Ri1 and Ri2, and in the lines OX8 and OX32 overexpressing a modified NAT_PHO1;2, with all plants grown in Pi-sufficient medium (B), and the endogenous cis-NAT_PHO1;2 in wild-type plants grown on Pi-sufficient or Pi-deficient media, and both the endogenous and transgenic NAT_PHO1;2 in the OX8 and OX32 lines grown on Pi-sufficient conditions (C). Data obtained for the two RNAi lines or the two OX lines were similar and thus pooled to simplify the graphical display. The association with each fraction is shown as a linear plot of the percentage of RNA present in each fraction; the plot is superimposed on a representative absorbance profile of the gradient. Fractions representing polysomes are indicated by a bracket. Statistically significant differences relative to either PHO1;2 for the +Pi treatment in (A), PHO1;2 for the wild type in (B), or endogenous cis-NAT_PHO1;2 for the +Pi treatment in (C) are indicated by asterisks (t test, P < 0.05, n = 3). For (C), the asterisks apply to all three points in close proximity.

Effect of cis-NAT_PHO1;2 Suppression on Plant Growth and Pi Homeostasis

To understand the role of the endogenous cis-NAT_PHO1;2 on plant growth and Pi homeostasis, the effect of a reduction of cis-NAT_PHO1;2 expression was analyzed in the RNAi lines Ri1 and Ri2. Growth and Pi content were assessed for the RNAi lines, the null mutant pho1;2 (Secco et al., 2010), and wild-type plants grown hydroponically under Pi-sufficient or Pi-deficient conditions for 4 weeks. Growth of the RNAi lines was indistinguishable from that of the wild type over the early vegetative period, whereas the pho1;2 mutant showed a net decrease in shoot and root biomass compared with the wild type (Figures 6A and 6B). Yet, in contrast with the near-wild-type growth behavior of the RNAi lines, the assessment of the steady state Pi levels from plants grown in Pi-deficient media showed differences between RNAi lines and wild-type plants (Figures 6C and 6D). Under this condition, the RNAi plants had approximately twofold less Pi in the shoots and threefold more Pi in the roots compared with the wild-type plants (Figures 6C and 6D). By contrast, under Pi-sufficient conditions, the RNAi mutants showed no significant difference in shoot or root Pi content compared with the wild type. The assessment of Pi transfer from the root to shoot in plants grown in Pi-deficient medium revealed a 1.7-fold reduction in the RNAi lines compared with the wild type (Figure 6E). However, the decrease was smaller than for the pho1;2 null mutant (Figure 6E). These results indicate that a low level of PHO1;2 expression must be present in the RNAi lines that is sufficient to maintain a shoot Pi level similar to that in wild-type plants grown in Pi-sufficient conditions, but not for plants grown under Pi-deficient conditions.

Figure 6.

Phenotypic Analysis of the cisNAT_PHO1;2  RNAi Lines.

(A) Fresh weight of shoots and roots of RNAi lines, pho1;2, and the wild type (WT) after 4 weeks of growth in Pi-sufficient medium.

(B) Morphological appearance of 30-d-old wild-type, pho1;2, and RNAi lines grown hydroponically in Pi-sufficient medium.

(C) and (D) Pi content assessed in shoots (C) and roots (D) of RNAi lines, pho1;2, and wild-type plants. FW, fresh weight.

(E) Transfer rate of ³³P from roots to shoots of 3-week-old plants grown hydroponically in Pi-deficient medium.

Data are mean ± se (n = 5), except for (E) (n = 4). Asterisks indicate statistically significant differences relative to the wild-type control (t test, P < 0.05).

[See online article for color version of this figure.]

For phenotypic observation during the entire plant growth period, experiments were performed with soil-grown plants. Plants were grown for 5 to 6 months with regular Pi fertilization and the phenotypes were examined. At the heading stage, RNAi lines showed no phenotypic differences compared with nontransformed rice plants, and the biomass of roots and shoots was comparable (Figures 7A and 7B). However, as previously observed during the early vegetative phase, Pi distribution between shoots and roots of the RNAi lines was strongly affected compared with wild-type plants (Figure 7C). Pi level was strongly increased in roots and decreased in shoots. Moreover, seed-setting rate was decreased in RNAi lines (Figure 7D, top panel) and grains were smaller and thinner compared with wild-type plants (Figures 7D and 7E). Altogether, these data demonstrate that cis-NAT_PHO1;2 acts as a positive regulator of Pi homeostasis through increased OsPHO1;2 mRNA translation.

Figure 7.

Phenotypic Analysis of the RNAi Lines at Maturity.

Measurements were made on rice plants grown for 6 months in a greenhouse under a day/night temperature regime of 30°C/25°C. Plants were fertilized weekly with a nutrient solution containing 1 mM Pi.

(A) Shoot and root biomass in wild-type (WT) and transgenic plants.

(B) Morphology at the heading stage of wild-type plants, RNAi-line1 (Ri1), and RNAi-line2 (Ri2). Bar = 10 cm.

(C) Pi content assessed in roots and shoots of RNAi lines and wild-type plants. Data are mean ± se (n = 4). FW, fresh weight.

(D) Comparison of panicle-filling performance and seed phenotype at harvest of RNAi lines and wild-type plants. Inset (a), seed-setting performance in RNAi lines and the wild type. Inset (b), magnified view of spikelet (boxed in [a]) on the primary rachis branch of the wild type (left), Ri1 (center), and Ri2 (right). Inset (c), comparison of the brown rice seeds. Bars = 0.25 cm.

(E) Weight of 1000 grains of wild-type and RNAi lines. All data are mean ± se (n = 7 to 10). Asterisks indicate statistically significant differences relative to the wild-type control (t test, P < 0.05).

[See online article for color version of this figure.]

DISCUSSION

In contrast with small ncRNAs (siRNAs and miRNAs), which generally have conserved structures and are involved in gene silencing via specific base-pairing with their target, the long ncRNAs do not share conserved features and are implicated in diverse mechanisms of gene regulation. Although only a limited number of long ncRNAs have been characterized in some detail in plants, fungi, and metazoans, the majority have been shown to affect mRNA transcript levels (Au et al., 2011; Kim and Sung, 2012; Rinn and Chang, 2012). Long ncRNAs involved in the repression of transcription are predominant and implicate diverse mechanisms, such as the recruitment of proteins modifying histone structure for X INACTIVE SPECIFIC TRANSCRIPT and HOTAIR in mammals (Rinn et al., 2007; Zhao et al., 2008) or COOLAIR in plants (Swiezewski et al., 2009), titrating transcription factors for PANDA or DIHYDRFOLATE REDUCTASE MINOR in mammals (Martianov et al., 2007; Hung et al., 2011) or transcriptional interference for INTERFERING CRIC RNA1 and ZIPPING2 in yeast (Bumgarner et al., 2009; Gelfand et al., 2011). A reduction of steady state mRNA levels by long ncRNAs has also been described that implicates posttranscriptional mechanisms, such as mRNA degradation via half-STAU1 binding sites in mammals (Gong and Maquat, 2011) or siRNA via Dicer and double-stranded sense-antisense duplexes in plants, Drosophila, and mammals (Katiyar-Agarwal et al., 2006; Held et al., 2008; Okamura et al., 2008; Watanabe et al., 2008). Long ncRNAs have also been implicated in increasing steady state levels of target mRNA, either by increasing transcription by chromatin remodeling, as for HOTTIP in mammals (Wang et al., 2011) and long ncRNAs that act as enhancers (Ørom et al., 2010), or by increasing mRNA stability, such as in the case of BETA-SECRETASE1 (Faghihi et al., 2008).

Cases where long ncRNAs affect protein level without corresponding changes in steady state mRNA are relatively rare, and none have been reported in plants. One example in mammals is the translational inhibition of the mRNA encoding the transcription factor PU.1 by its associated cis-NAT (Ebralidze et al., 2008). There are also a few cases where long ncRNAs, and particularly cis-NATs, regulate protein expression via mRNA splicing, such as the regulation of ZINC FINGER E-BOX BINDING HOMEOBOX2 expression in mammals through inhibition of the splicing of an intron containing a ribosome entry site (Beltran et al., 2008) and the control of alternative splicing variants of the apoptotic factor APOPTOSIS STIMULATING FRAGMENT (Yan et al., 2005). Recently, a unique example of a cis-NAT that enhances protein expression independently of mRNA level has been described in mammals (Carrieri et al., 2012). Expression of the cis-NAT associated with the mouse UBIQUITIN CARBOXY-TERMINAL HYDROLASE L1 (Uchl1) gene was shown to increase UCHL1 synthesis without changes in its transcript level. This activity of the cis-NAT was dependent on the presence of a 5′ base pairing with its cognate mRNA and of an inverted short interspersed nuclear element (SINEB2) at the 3′ end and could occur by expression of these elements in trans. It was proposed that the association of Uchl1 mRNA with its cis-NAT in the cytosol enhances its translation.

The expression pattern of the promoter of Os-PHO1;2 in the vascular cylinder is comparable to the expression pattern of the homologous At-PHO1 promoter in the same tissues in Arabidopsis roots and in agreement with the characterized role of Os-PHO1 and At-PHO1 in the transfer of Pi into the xylem vessel (Hamburger et al., 2002; Secco et al., 2010). The promoter of Os-PHO1;2 is marginally responsive to the Pi status of the plant, in agreement with a lack of change in Os-PHO1;2 transcript expression upon Pi deficiency. By contrast, the cis-NAT_PHO1;2 promoter activity is strongly increased upon Pi deficiency and correlates with a marked increase in cis-NAT_PHO1;2 transcript level under the same conditions, indicating a major transcriptional regulation of cis-NAT_PHO1;2 expression by the Pi status. An analysis of the sequence of the cis-NAT_PHO1;2 promoter did not reveal the presence of P1BS-like elements (GNATATNC) recognized by PHR1, a major transcription factor responsible for the transcriptional upregulation of numerous genes under Pi deficiency (Rubio et al., 2001). Thus, further work is needed to identify the cis-regulatory elements responsible for the transcriptional regulation of cis-NAT_PHO1;2. Importantly, the expression pattern of the cis-NAT_PHO1;2 promoter overlapped with the expression of the PHO1;2 promoter in both the shoot and root vascular cylinder, an important element for a regulatory role of cis-NAT_PHO1;2 on PHO1;2 expression.

Three lines of evidence support the conclusion that cis-NAT_PHO1;2 positively regulates PHO1;2 protein accumulation without affecting steady state mRNA level. First, time-course analysis during Pi deficiency stress showed strong correlation between cis-NAT_PHO1;2 transcript level and PHO1;2 protein accumulation. Second, specific downregulation of cis-NAT_PHO1;2 via RNAi led to a strong decrease in PHO1;2 protein accumulation under Pi-sufficient and Pi-deficient conditions. Third, overexpression of a modified NAT_PHO1;2 in transgenic plants caused an increased PHO1;2 protein accumulation. Importantly, under all experimental conditions tested, no significant changes occurred in PHO1;2 steady state mRNA level.

The RNAi strategy used in this work relied on the expression of a chimeric construct composed of sequences corresponding to the exon of the cis-NAT_PHO1;2 transcript but only to introns of the sense transcript (Figure 4A). Expression of this RNAi construct resulted in the selective degradation of only the cis-NAT_PHO1;2 mRNA, revealing that RNAi-mediated transcript degradation only targeted mature transcripts and not the nuclear pre-mRNA PHO1;2 transcript containing introns. These results are in contrast with previous work showing siRNA-directed degradation of the FATTY ACID DESATURASE2-1A transcript in soybean (Glycine max) using an RNAi construct containing only intronic sequences to the target gene (Hoffer et al., 2011). While ARGONAUTE-mediated target cleavage has been shown to occur in the nucleus in Schizosaccharomyces pombe, the subcellular location of ARGONAUTE loading in plants remains unclear (Castel and Martienssen, 2013). Our results imply that there are probably differences either between plant species or between genes in their sensitivity to siRNA-mediated degradation in the cytosol or the nucleus.

Analysis by RPA and RT-PCR of the PHO1;2 transcripts in plants grown under conditions of high or low cis-NAT_PHO1;2 expression level did not reveal any evidence of alternative splicing or editing of the PHO1;2 transcript. Furthermore, analysis of databases of cDNAs, ESTs, or whole-genome RNA sequencing from rice also did not reveal the presence of alternative splicing products for PHO1;2. Thus, expression of cis-NAT_PHO1;2 is unlikely to affect PHO1;2 production via modification of the PHO1;2 transcript. Instead, cis-NAT_PHO1;2 expression has a clear role in increasing PHO1;2 production by enhancing the association of mRNA with polysomes. The fact that polysome association of PHO1;2 was stimulated not only by the increased expression of cis-NAT_PHO1;2 in Pi-deficient wild-type plants, but also by the overexpression, in trans, of NAT_PHO1;2 in Pi-sufficient plants, reveals that NAT_PHO1;2 can positively regulate PHO1;2 synthesis independently of the metabolic and development changes that accompany Pi deficiency and that this regulation does not require a cis-configuration of the sense and antisense transcripts. Future work will need to focus on the mechanism by which cis-NAT_PHO1;2 can enhance the association of the PHO1;2 transcript with polysomes. One attractive hypothesis is that cis-NAT_PHO1;2 associates with PHO1;2 via its overlapping region and that the free 3′ end of cis-NAT_PHO1;2 can recruit initiation factors or ribosomes that will then be translocated to the 5′ end of the PHO1;2 transcript. This hypothesis would fit the observation that NAT_PHO1;2 is also found in the polysome fraction. Such a model would be reminiscent of the translational enhancers found at the 3′ UTR region of several plant RNA viruses (Simon and Miller, 2013).

Studies of the role of cis-NATs in the regulation of gene expression in plants have so far identified two main mechanisms. Downregulation of the target mRNA level via siRNAs appears predominant, with examples of siRNA generated from sense−cis-NAT double-stranded RNA being implicated in the response of plants to salt stress or pathogen infection, cellulose biosynthesis, flowering control, sperm cell development, and cytokinin biosynthesis (Borsani et al., 2005; Katiyar-Agarwal et al., 2006; Swiezewski et al., 2007; Zubko and Meyer, 2007; Held et al., 2008; Ron et al., 2010). A second mechanism described for only FLC is the transcriptional downregulation of mRNA synthesis by the recruitment of chromatin modifying protein and histone methylation (Swiezewski et al., 2009; Liu et al., 2010; Heo and Sung, 2011). Despite the apparent prevalent role of siRNA in mediating the downregulation of gene expression by cis-NATs in the literature, a recent genome-wide analysis of siRNAs in biotic and abiotic stress–challenged Arabidopsis and rice found that only 6.1 and 15.7% of the Arabidopsis and rice sense−cis-NAT pairs, respectively, gave rise to significant levels of siRNAs (Zhang et al., 2012). Similar studies performed for rice and six other plants found that while siRNAs were particularly enriched in the overlapping regions of trans-NATs, only low levels of enrichment were found for cis-NATs (Zhou et al., 2009; Chen et al., 2010). Together, these studies reveal that siRNA-mediated gene silencing is unlikely to be the main mechanism by which cis-NATs regulate gene expression. This work on cis-NAT_PHO1;2 thus highlights a novel mode of regulation of cis-NATs on their sense targets in plants that may be of relevance to other sense−cis-NAT pairs, in particular those that show no correlation between cis-NAT induction and the negative effect on the sense mRNA transcript.

The physiological importance of cis-NAT_PHO1;2 action is shown in the RNAi lines that have a specific decrease in cis-NAT_PHO1;2 expression. Indeed, these RNAi lines show reduced transfer of Pi from root to shoot, leading to a reduction in shoot Pi and a concomitant increase in root Pi content. The fact that decreased PHO1 activity and shoot Pi content did not affect shoot biomass production for plants grown in well-fertilized soil is fully consistent with previous results obtained in Arabidopsis showing that it is possible to uncouple low shoot Pi content from reduced shoot growth by modulating the expression of PHO1 (Rouached et al., 2011). However, our results show that rice lines with reduced cis-NAT_PHO1;2 expression show a severe reduction in seed yield and seed weight, highlighting the important role of cis-NAT_PHO1;2 in plant fitness.

The plant's response to Pi deficiency is already known to implicate two ncRNAs. One is miR399, an miRNA induced by Pi deficiency that targets the PHO2 transcript, which encodes a ubiquitin-conjugating E2 enzyme that interacts with PHO1 and promotes its degradation (Lin et al., 2008; Pant et al., 2009; Liu et al., 2012). IPS1 is another ncRNA that is induced under Pi deficiency and acts as a target mimic for miR399, thus providing a level of feedback control over miR399 action (Franco-Zorrilla et al., 2007). This work on cis-NAT_PHO1;2 adds an additional layer of complexity and reveals an important role for a cis-NAT in the regulation of PHO1 activity and phosphate homeostasis.

METHODS

Plant Material and Growth Conditions

Rice (Oryza sativa ssp japonica ‘Nipponbare’) was used for all experiments. For hydroponic culture, seeds were first surface sterilized and germinated as described previously (Jabnoune et al., 2009). For plants grown under Pi deficiency, 7-d-old seedlings were grown in full-strength Hoagland nutrient solution (1 mM Pi) and then subjected to Pi deficiency (0 mM Pi, KH₂PO₄ was replaced by KCl) or the control condition (1 mM Pi) for the desired time duration. In experiments involving transgenic plants, seeds were geminated and screened in a solution containing 50 mg L⁻¹ hygromycin for 7 d and then confirmed by PCR for the presence of the transgene before being transferred to the hydroponic system. Growth conditions were 28°C, 13-h-light/22°C, 11-h-dark cycle in a phytotron or 30°C, 12-h-light/22°C, 12-h-dark cycle in a greenhouse.

Plasmid Construction and Plant Transformation

To develop transgenic plants expressing the GUS reporter gene, 2 kb of the promoter sequence of PHO1;2 and cis-NAT_PHO1;2 were cloned into the vector pHGWFS7.0 (Karimi et al., 2002). To develop transgenic plants expressing an RNAi construct targeting cis-NAT_PHO1;2, a synthetic DNA fragment containing the sequence of the first, second, and third introns of PHO1;2 was synthesized (Invitrogen) and cloned into the pANDA vector containing the maize (Zea mays) ubiquitin promoter region (Miki and Shimamoto, 2004). To generate transgenic plants overexpressing a modified NAT_PHO1;2 with epitope-tagged ORFs, a synthetic NAT_PHO1;2 fragment was synthesized (Invitrogen) containing the FLAG, HA, and MYC epitopes inserted at the 3′ end of ORF1, ORF2, and ORF3, respectively. The modified NAT_PHO1;2 construct was introduced into the expression vector pMDC32 harboring the cauliflower mosaic virus 35S promoter (Curtis and Grossniklaus, 2003). All the constructs described above were introduced into rice by Agrobacterium tumefaciens–mediated transformation at the Iowa State University Plant Transformation Facility. To express each epitope-tagged NAT_PHO1;2 ORF separately into Escherichia coli, DNA fragments corresponding to each tagged ORF were amplified from the synthetic NAT_PHO1;2 fragment and cloned into the expression vector pLATE11 (Thermo Scientific).

Histochemical GUS Staining and Fluorometric Assays

Plant tissues were collected from wild-type and transgenic plants containing the Pro_{OsPHO1;2  -S}:GUS or Pro_{cis-  NAT}:GUS transgenes. The root and leaf sections were vacuum infiltrated in X-gluc solution [0.5 mM X-gluc, 0.1 M NaHPO₄, pH 8.0, 0.5 mM K₃Fe(CN)₆, 0.5 mM K₄Fe(CN)₆, 0.01 M EDTA, pH 8.0, 20% methanol, and 0.1% Triton X-100] twice for 5 min each, followed by incubation at 37°C for 16 h and cleared two or three times with 70% ethanol at 65°C before samples were whole mounted in 70% ethanol for microscopy. For cross sections, the stained samples (without clearing) were embedded in 4% agarose, and 50-μm sections were made using a Vibratome (Leica VT1000 S).

Quantitative GUS activity was determined by measuring the production of 4-methylumbelliferone. For this, five samples from lines transformed with each construct were pooled and used for a quantitative 4-methylumbelliferyl-β-d-glucuronide assay. Plant tissues were homogenized in 0.4 mL of protein extraction buffer (0.1 M NaPO₄, pH 8.0, 0.1% SDS, 10 mM EDTA, 10 mM β-mercaptoethanol, and 0.1% Triton X-100) followed by centrifugation at 13,000g and 4°C for 15 min. The supernatant was used for protein quantification and fluorometric assay. Fifty microliters of supernatant was transferred to a microcentrifuge tube containing 500 μL of GUS reaction buffer (protein extraction buffer containing 10 mM 4-methylumbelliferyl-β-d-glucuronide) prewarmed to 37°C and mixed. One hundred microliters of this mixture was immediately transferred into 900 μL of GUS stop buffer (0.2 M Na₂CO₃) to serve as a control. The sample reaction mixture was incubated at 37°C. One hundred microliters of aliquots was removed at 15-, 30-, 60-, and 120-min intervals and mixed with 900 μL of GUS stop buffer. GUS activity was measured in a fluorometer (VersaFluor; Bio-Rad) with excitation at 365 nm and emission at 455 nm. The amount of 4-methylumbelliferone was determined from a standard curve. Protein concentrations of the samples were determined using Bradford reagent (Bio-Rad) and BSA as a standard.

RNA Extraction and Transcript Analysis

Total RNA was extracted using an RNeasy plant mini kit (Qiagen) following the manufacturer's instructions. cDNA was synthesized from 1 µg of DNaseI-treated total RNA (Fermentas) using oligo(dT) and Moloney murine leukemia virus reverse transcriptase (Promega) according to the manufacturer's instructions. Strand-specific real-time PCR was performed in a 25-μL volume containing 5 μL of cDNA, 12.5 μL of 2× SYBR green mix (Axon Lab), and 0.25 µM each primer. Analysis was performed in an MX3000P real-time PCR system (Stratagene) with cycle conditions of an initial denaturing at 95°C for 15 min, followed by 40 cycles of 95°C for 30 s, 60°C for 1 min, and 72°C for 1 min. The transcript-specific primers used are listed in Supplemental Table 1 online. All data were normalized against the expression level of the rice ubiquitin gene.

Protein Preparation and Immunoblot Analysis

For extraction of the microsomal protein fractions, tissues from roots or shoots of wild-type or mutants plants were frozen in liquid nitrogen and ground with a mortar and pestle. The frozen powder was homogenized with three volumes of ice-cold extraction buffer composed of 100 mM Tris-HCl, pH 7.5, 400 mM Suc, 1 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride, and 1× complete protease inhibitor (Roche). Debris was pelleted by centrifugation at 1000g for 5 min (at 4°C). The pellet was discarded and the supernatant was centrifuged at 8000g for 15 min (at 4°C). The pellet was discarded and the supernatant was recentrifuged at 150,000g for 1 h, at 4°C, to produce a 150,000g membrane pellet (P150) and a soluble fraction (S150). The P150 pellets were resuspended in standard extraction buffer supplemented with 0.5% (v/v) Triton X-100 and were used as crude microsomal membranes. The protein concentration was determined by DC protein assay (Bio-Rad) according to the manufacturer's instructions using an Epoch Micro-Volume Spectrophotometer system (Biotech Systems). Equal amounts of proteins (50 to 80 µg) were used for immunoblot analyses.

Proteins were separated by 4 to 20% Mini-Protean TGX gels (Bio-Rad) and transferred to polyvinylidene difluoride membrane (Hypobond-PVDF; Amersham Lifesciences) by electroblotting in transfer buffer (25 mM Tris, 192 mM Gly, and 10% methanol, pH 8.3). Nonspecific binding was reduced with blocking solution (5% [w/v] BSA in 1× PBS-Tween-20 [1× PBST solution], pH 7.2) at room temperature for 1 h with agitation. Then, blots were hybridized with a rabbit polyclonal antibody against PHO1;2 diluted 1:10,000 in 1× PBST overnight (at 4°C) with agitation. After three washes for 15 min in 1× PBST, blots were incubated with the secondary antibody (rabbit IgG; Sigma-Aldrich) for 1 h at room temperature. Three further washing steps (15 min each) in PBS were followed by detection using an Enhanced Chemiluminescence Plus detection kit (enhanced chemiluminescence protein gel blotting detection; GE Healthcare) and the ImageQuant 300 imager (GE Healthcare). The protein transferred onto membranes was visualized with Ponceau staining solution (0.1% [w/v] Ponceau S and 5% [v/v] acetic acid).

Rabbit primary polyclonal antibody was raised against the N-terminal fragment of PHO1;2 corresponding to the amino acid residues 78 to 177 (Strategic Diagnostics) and was affinity purified using immunogenic peptide coupled to a SulfoLink column (Pierce).

Recombinant Protein Production in E. coli

The plasmids were transformed in E. coli strain BL21(DE3) cells, and the induction of recombinant protein expression was performed by adding 0.5 mM isopropyl β-d-1-thiogalactopyranoside (37°C, 3 h) to the growth medium.

RNase Protection Assay

Total RNA was extracted from rice roots or shoots using an RNeasy plant mini kit (Qiagen) following the manufacturer's instructions. RPAs were performed as described previously with some modifications (Young et al., 2003). To prepare cRNA probes specific for PHO1;2 or cis-NAT_PHO1;2, the full-length cDNA of PHO1;2 and cis-NAT_PHO1;2 was first subcloned into the pFLCI vector, and then the plasmid was linearized by SmaI (OsPHO1;2-AS-pFLCI) or SalI (OsPHO1;2-S-pFLCI). The linearized fragments were used as templates to generate a ³²P-labeled antisense ripoprobes using T3 RNA polymerase, [α-³²P]rCTP (10 mCi/mL), and the Riboprobe transcription kit (Promega) according to the manufacturer's instructions.

Before conducting nuclease protection assays, ribonucleotide probes were extracted with phenol:chloroform (1:1) and then chloroform. Unincorporated nucleotides were removed with a G-50 Sephadex column (Roche Molecular Biochemicals). Total RNA was hybridized to 3 × 10⁵ cpm of labeled antisense probe and subsequently treated with RNase A and RNase T1 to digest unhybridized probe and single-stranded RNA. Digestion was stopped, and hybridized RNA was precipitated by the addition of RNase inactivation/PPT reagent III (Ambion) and incubation for 30 min at −20°C. Hybridized RNA was pelleted by centrifugation at 14,000g for 15 min. Protected mRNA fragments were identified by their electrophoretic mobility through a 5% acrylamide, 8 M urea denaturating gel (250 V for 1 h). Gels were dried and autoradiography was performed using x-ray film (Biomax-MR; Kodak) for 24 to 48 h at −80°C with intensifying screens.

RNA Gel Blot Analysis

Total RNA was isolated from 300 mg of rice roots using an RNeasy plant mini kit (Qiagen) following the manufacturer's instructions. RNA samples were separated by electrophoresis on an agarose gel containing 1.2% formaldehyde and transferred onto a nylon membrane (Amersham). Digoxigenin (DIG)-labeled PHO1;2 and cis-NAT_PHO1.2 antisense RNA probes were prepared using a DIG RNA labeling kit (Roche). Hybridization was performed under high stringency according to the DIG Nucleic Acid Detection protocol. After washes, filters were exposed and quantified by a phosphor imager (GE Healthcare).

Polysome Gradient Preparation

For polyribosome isolation, roots from 30-d-old wild-type and transgenic plants (Cis-NAT_PHO1.2  RNAi and overexpressing lines) were harvested, frozen, and ground to powder in liquid nitrogen. To prepare cytoplasmic extracts, 150 mg of powder was combined with 1.2 mL of chilled polysome buffer (100 mM Tris-HCl, pH 8.4, 50 mM KCl, 25 mM MgCl₂, 5 mM EGTA, 18 μM cycloheximide, 15.5 μM chloramphenicol, and 0.5% [v/v] Nonidet P-40). Debris was removed by centrifugation at 16,000g for 15 min at 4°C. Aliquots of the resulting supernatant were loaded on 20 to 50% (w/w) continuous Suc gradients and centrifuged at 32,000 rpm for 165 min in a Beckman SW41 rotor at 4°C. After centrifugation, fractions were collected from the bottom to the top of the gradient with continuous monitoring of the absorbance at 254 nm. Six equal fractions were collected after centrifugation. Six fractions (4 mL per fraction) were collected, 4 mL of Trizol reagent (Invitrogen) was added to each, and RNA was extracted following the manufacturer's instructions. A fixed volume of each RNA sample was then reversed transcribed, and the percentage of mRNA in each fraction was calculated as the percentage of the copy number in a specific fraction to the total copies in the combined six fractions.

Protoplast Preparation and Nuclear Fractionation

For protoplast isolation, green tissues from the stem and sheath of 15 to 20 rice seedlings grown hydroponically in full Pi medium were cut into ∼0.5-mm strips. The strips were immediately transferred into 0.5 M mannitol, 4 mM MES-potassium hydroxide, pH 5.5, and 20 mM KCl for 10 min in the dark. The strips were then incubated in an enzyme solution (1.5% Cellulase R10, 0.4% Macerozyme R-10, 0.4 M mannitol, 20 mM MES, pH 5.5, 20 mM KCl, 10 mM CaCl₂, and 0.1% BSA) for 4 to 5 h in the dark with gentle shaking (60 to 80 rpm). After the enzymatic digestion, an equal volume of washing solution (WS) containing 154 mM NaCl, 125 mM CaCl₂, 5 mM KCl, and 2 mM MES at pH 5.7 was added, followed by vigorous shaking by hand for 10 s. Protoplasts were released by filtering through 40-μm nylon meshes into round-bottom tubes with three to five washes of the strips using WS. Pellets were collected by centrifugation at 1500 rpm for 5 min at 4°C with a swinging bucket. After washing once with WS, pellets were resuspended in nuclei isolation buffer solution (0.1 mM spermidine, 10 mM MES-potassium hydroxide, pH 5.5, 2.5 mM EDTA, 10 mM NaCl, 10 mM KCl, 0.2 M Suc, 0.15% Triton X-10, and 2.5 mM DTT) at a concentration of 10⁶ cells mL⁻¹. Samples were incubated on ice for 7 min and then passed four times through syringes with a 25G (5/8) gauge needle. The lysate was filtered through a 20-µm filter into a new tube followed by centrifugation at 400g for 10 min. The supernatant was used as the cytoplasmic fraction, and the pellet represents the nuclear fraction. RNA was extracted from both the cytoplasmic and nuclear fractions using 1 volume of Trizol reagent (Invitrogen), following the manufacturer's instructions. A fixed volume of each RNA sample was then reverse transcribed, and the preparation was further used as template for mRNA quantification by quantitative RT-PCR, as described above.

RNA Editing Detection by Direct Sequencing

We generated cDNA clones via RT-PCR from RNAs of wild-type and cis-NAT_PHO1.2  RNAi lines. Reverse transcription was carried out from 1 µg of DNaseI-treated total RNA using oligo(dT) and Moloney murine leukemia virus reverse transcriptase (Promega) according to the manufacturer's instructions. The cDNA produced was diluted with four parts of sterile deionized water. The subsequent PCR amplification of the PHO1;2 region overlapping with cisNAT_PHO1.2 was performed using the oligonucleotide primers listed in Supplemental Table 1 online, designed to span the introns 1 to 4 to prevent the amplification of residual contaminant genomic DNA. The PCR reaction was performed using KOD high-fidelity polymerase (Novagen) and results in a 1205-bp product. DNA fragment was purified using a Zymoclean Gel DNA recovery kit (Zymo Research) and cloned using the pJET cloning system (Fermentas).

Accession Numbers

Sequence data from this article can be found in the Arabidopsis Genome Initiative or GenBank/EMBL databases under the following accession numbers: AK100323, PHO1;2; AK071338, cis-NAT_PHO1;2; and AK121590.1, rice ubiquitin gene.

Supplemental Data

The following materials are available in the online version of this article.

• Supplemental Figure 1. Determination of the Activity of the Promoter of PHO1;2 and cis-NAT_PHO1;2 in Plants Grown under Pi-Sufficient and Pi-Deficient Conditions.

• Supplemental Figure 2. Assessment of the Pi Deficiency Status in the Transgenic GUS Lines.

• Supplemental Figure 3.  GUS Expression Pattern in the Roots of ProOsPHO1;2-S:GUS and Procis-NAT:GUS Transgenic Lines.

• Supplemental Figure 4. Histochemical Localization of GUS Activity in the Flower Organs of Rice Plants Transformed with the cis-NATPHO1;2 Promoter:GUS Vector.

• Supplemental Figure 5.  cis-NAT Associated with the Maize PHO1 Transcript.

• Supplemental Figure 6. Immunoblot Analysis of PHO1;2 Expression in the Wild Type and pho1;2 Null Mutants.

• Supplemental Figure 7. Silencing of cis-NATPHO1;2.

• Supplemental Figure 8. RT-PCR Analysis of PHO1;2 Transcripts.

• Supplemental Figure 9. RNA Gel Blot Analysis of Transgenic Lines Expressing an RNAi Construct against the cis-NAT_PHO1.2.

• Supplemental Figure 10. Analysis of PHO1.2 and NAT_PHO1.2 Export from the Nucleus.

• Supplemental Table 1. Primers Used in This Work.

Acknowledgments

We thank Jurek Paszkowski (University of Geneva), Edward Farmer (University of Lausanne), and members of the Poirier laboratory for helpful discussions. This work was funded by grants from the Swiss National Foundation (31003A-138339) and the Sino-Swiss Science and Technology Cooperation Program (IZLCZ3 123946 to Y.P. and 2009DFA32040 to Q.S.).

Glossary

 
• ncRNA

  noncoding regulatory RNA

 
• miRNA

  microRNA

 
• siRNA

  short interfering RNA

 
• NAT

  natural antisense transcript

 
• GUS

  β-glucuronidase

 
• ORF

  open reading frame

 
• RNAi

  RNA interference

 
• RPA

  RNase protection assay

 
• UTR

  untranslated region

 
• WS

  washing solution

AUTHOR CONTRIBUTIONS

M.J. and Y.P. designed and performed the experiments, analyzed the results, and wrote the article. D.S. and C.L. performed the experiments. C.R. and Q.S. designed the experiments, analyzed the results, and wrote the article.

Author notes

1

Current address: Australian Research Council Centre of Excellence in Plant Energy Biology, University of Western Australia, Crawley WA 6009, Australia.

2

Address correspondence to .

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Yves Poirier ().

Some figures in this article are displayed in color online but in black and white in the print edition.

Online version contains Web-only data.

www.plantcell.org/cgi/doi/10.1105/tpc.113.116251