Salt Stress Induces an Endogenous siRNA from a Pair of Antisense Genes
Previously, we cloned miRNAs and putative siRNAs from Arabidopsis
plants treated with various abiotic stresses (Sunkar and Zhu, 2004
). One putative siRNA (clone # P96-F02) matched the overlapping region between the 3′ end of the P5CDH
ORF and the 3′UTR of an unknown gene on the opposite strand (At5g62520), which has recently been designated SRO5
(). These two genes generate convergent transcripts that overlap by 760-nt, and the cloned siRNA sequence matched a 21-nt region (1873–1893) of the SRO5
3′UTR and was complementary to the corresponding region of the P5CDH
Salt Stress Induces a 24-nt nat-siRNA from the SRO5-P5CDH cis-Antisense Overlapping Genes
Using an oligonucleotide probe complementary to the 21-nt siRNA, we could not detect any siRNAs in Arabidopsis plants grown under normal conditions (); however, a 24-nt siRNA (referred to hereafter as 24-nt SRO5-P5CDH nat-siRNA) was detected in NaCl-treated adult plants and seedlings. Occasionally, a very weak signal at 21-nt could also be seen in the salt-stressed plants (not shown); this signal likely corresponds to the cloned 21-nt siRNA. No 24-nt SRO5-P5CDH nat-siRNA signal was found in plants exposed to several other stress or homone treatments (), demonstrating that the induction of 24-nt SRO5-P5CDH nat-siRNA is highly specific to NaCl stress.
To define the sequence of the 24-nt SRO5-P5CDH
nat-siRNA, we hybridized the small RNA blots with oligonucleotide probe A (5′-GGGGACCCGAGAGGGGCCGGGATA
-3′) and oligonucleotide probe B (5′-GACCCGAGAGGGGCCG GGATA
GGG-3′) (the sequences in bold letters are complementary to the 21-nt cloned siRNA). Probe A but not B detected the 24-nt SRO5-P5CDH
nat-siRNA from salt-stressed plants (data not shown). This suggests that the 24-nt SRO5-P5CDH
nat-siRNA sequence is likely 5′-UAUCCCGGCCC CUCUCGGGUCCCC-3′ (). This sequence was confirmed by a modified small RNA cloning approach (Table S1
Although siRNAs are generated in the double-stranded form, often only one of the strands is incorporated into RISC and can be detected (Schwarz et al., 2003
; Khvorova et al., 2003
). Twenty-four nucleotide SRO5-P5CDH
nat-siRNA was detected with an antisense (relative to SRO5
) probe but not with a sense probe (), suggesting that the sense strand as shown in is stable and the other strand is rapidly degraded. Furthermore, examination of the 24-nt SRO5-P5CDH
nat-siRNA sequence indicates that the sense strand sequence conforms to the asymmetry rule for siRNA stability (Schwarz et al., 2003
24-nt SRO5-P5CDH nat-siRNA Is Produced by a Unique Biogenesis Pathway
To define the components required for the formation of 24-nt SRO5-P5CDH
nat-siRNA, it was examined in salt stressed plants of small RNA biogenesis mutants. The 24-nt SRO5-P5CDH
nat-siRNA was detected in salt-treated wild-type plants of all ecotypes tested such as Landsberg (Ler), Columbia, C24, and Nossen (). However, it was not detected in the hen1
mutant. Instead, a larger-sized signal was found in hen1
plants (). The 24-nt SRO5-P5CDH
nat-siRNA was present in dcl1
, and dcl4
but not in dcl2
. The sgs3
, and rdr6
mutations also blocked the accumulation of 24-nt SRO5-P5CDH
nat-siRNA (). In contrast, the rdr2
mutation did not have any effect on 24-nt SRO5-P5CDH
nat-siRNA, although it abolished the accumulation of siRNA 1003. The HYL1
gene has been shown to be required for the accumulation of several miRNAs (Han et al., 2004
; Vazquez et al., 2004a
), including miR172, and the level of 24-nt SRO5-P5CDH
nat-siRNA was reduced in the hyl1
mutant plants (). These results suggest that accumulation of the 24-nt SRO5-P5CDH
nat-siRNA is dependent on DCL2, RDR6, SGS3, and NRPD1A. This pathway is different from those producing heterochromatin-related siRNAs or tasiRNAs, suggesting that the SRO5-P5CDH
nat-siRNA defines a new class of siRNAs.
Accumulation of SRO5-P5CDH nat-siRNA Requires DCL2, RDR6, SGS3, NRPD1A and Is Dependent on Induction of SRO5 mRNA by Salt Stress
Salt Stress Regulates SRO5 and P5CDH Transcript Levels and P5CDH mRNA Cleavage
To understand the mechanism of salt stress induction of the SRO5-P5CDH nat-siRNA, we investigated whether the expression of SRO5 or P5CDH is regulated by salt stress. SRO5 was not expressed in plants grown under normal conditions, but its expression was induced by NaCl treatment (). In contrast, P5CDH expression was reduced by NaCl treatment (). These results suggest that, upon salt treatment, the SRO5 and P5CDH mRNAs can form a dsRNA that is then processed by DCL2 to generate the 24-nt SRO5-P5CDH nat-siRNA.
A possible role of the 24-nt SRO5-P5CDH nat-siRNA is to cause cleavage of P5CDH mRNA. In addition to detecting a full-length P5CDH transcript, a P5CDH 3′UTR probe also hybridized with a smaller band corresponding to the size of the putative 3′ cleavage product. This putative 3′ cleavage product accumulated only in NaCl-treated plants (). Importantly, in dcl2, sgs3, nrpd1a, and rdr6 mutants where the 24-nt SRO5-P5CDH nat-siRNA was not produced, there was less or no decrease in P5CDH full-length transcript level, and the 3′ cleavage product was not detected (). Interestingly, in hen1 plants, salt stress still reduced the level of full-length P5CDH transcript and caused accumulation of the 3′ cleavage product. This suggests that the larger-sized siRNA in hen1 mutant plants was still functional in causing P5CDH mRNA cleavage. Taken together, these results suggest that salt stress triggers the expression of SRO5, leading to dsRNA formation and consequently generation of 24-ntSRO5-P5CDHnat-siRNA, which then downregulates P5CDH transcript levels through mRNA cleavage.
Cleavage Site Mapping of P5CDH mRNA Reveals Phased Processing of 21-nt SRO5-P5CDH nat-siRNAs
To verify that the 24-nt SRO5-P5CDH
nat-siRNA indeed targets P5CDH
mRNA for endonucleolytic cleavage, we carried out 5′ RACE assays using mRNA from salt-treated plants to map potential cleavage sites. Among several cleavage sites found in the P5CDH
mRNA in wild-type plants, one site is between nucleotides 12 and 13 of the 24-nt SRO5-P5CDH
nat-siRNA (). This differs from other ARGONAUTE-catalyzed RNA cleavage sites which occur between nucleotides 10 and 11 (Allen et al., 2004
). No cleavage products were found when the assay was performed with mRNA isolated from salt stress treated tissue of rdr6
plants or with mRNA from unstressed wild-type plants (data not shown). The results support our hypothesis that the 24-nt SRO5-P5CDH
nat-siRNA can direct P5CDH
Cleavage Site Mapping of P5CDH mRNA and the Detection of 21-nt nat-siRNAs
Surprisingly, the 5′ RACE assay also revealed four additional cleavage sites in the P5CDH mRNA (). One of these was the most frequent cleavage site (28 out of 40). These additional cleavage sites are 21-nt apart and in-phase with each other. We hypothesized that cleavage guided by the 24-nt SRO5-P5CDH nat-siRNA may set the phase for processing of P5CDH transcript by RDR into dsRNA and then dicing by a different DCL, which generates new 21-nt P5CDH nat-siRNAs. We designed three oligonucleotide probes in an attempt to detect these predicted 21-nt P5CDH nat-siRNAs (1, 2, and 3; ). Each of these probes detected a 21-nt P5CDH nat-siRNA in salt-stressed plants. Analysis of one of these siRNAs (probe 2) in the same mutants analyzed above showed that generation of 21-nt P5CDH nat-siRNAs was dependent on DCL1, DCL2, HEN1, RDR6, SGS3, and NRPD1A ().
We also looked for, but failed to find, siRNAs deriving from sequences upstream of and in-phase with the 24-nt SRO5-P5CDH
nat-siRNA-directed cleavage site by using a mix of 21-nt oligonucleotide probes complementary to the upstream sequences. Additionally, we hybridized the small RNA blots with 24-nt oligonucleotide probes designed to detect other potential 24-nt siRNAs produced in-phase with (either downstream or upstream of) the 24-nt SRO5-P5CDH
nat-siRNA(probe sequences used are described in TableS2
). These probes failed to detect any siRNA signal (not shown), suggesting that only one 24-nt SRO5-P5CDH
nat-siRNA accumulates. Since these 24-nt probes are completely out of phase with the 21-nt P5CDH
nat-siRNAs, the result also suggests a lack of siRNAs not in-phase with the 24-nt SRO5-P5CDH
nat-siRNA-directed cleavage site, although the experiment cannot rule out the existence of other out-of-phase siRNAs.
The combined data suggest that the 21-nt P5CDH nat-siRNAs were generated by DCL1, and their production was dependent on production of the 24-nt SRO5-P5CDH nat-siRNA by DCL2. To directly test whether formation of the 24-nt SRO5-P5CDH nat-siRNA is required for the formation of 21-nt nat-siRNAs, we transiently expressed combinations of wild-type and mutated P5CDH and SRO5 transcripts (mP5CDH and mSRO5) in Nicotiana benthamiana leaves. mP5CDH and mSRO5 were mutated to introduce four mismatches with the complementary wild-type sequence in the region corresponding to the 24-nt SRO5-P5CDH nat-siRNA (). However, mP5CDH and mSRO5 were perfectly complementary to each other at the 24-nt SRO5-P5CDH nat-siRNA site. Small RNA blots from tissue coexpressing these transcripts were probed with oligonucleotides complimentary to the wild-type and m24-nt SRO5-P5CDH nat-siRNA and with a mixture of 21-nt P5CDH nat-siRNAs (probes 1, 2, and 3; ).
The results of this experiment support the hypothesis that 24-nt SRO5-P5CDH nat-siRNA is produced first and required for the formation of 21-nt P5CDH nat-siRNAs. When wild-type P5CDH and SRO5 transcripts were coexpressed in N. benthamiana leaves, both 24-nt and 21-nt SRO5-P5CDH nat-siRNAs were produced (, lane 3). Expression of either gene in combination with the unrelated GFP transcript did not produce any nat-siRNAs (, lanes 1 and 2), demonstrating that both the P5CDH and SRO5 transcripts must be present to generate nat-siRNAs. When wild-type SRO5 was coexpressed with mP5CDH, wild-type 24-nt SRO5-P5CDH nat-siRNA was still produced from the SRO5 transcript but no 21-nt P5CDH nat-siRNAs could be detected (, lane 4). This demonstrated that the initial 24-nt SRO5-P5CDH nat-siRNA could be produced from the long, double-stranded SRO5-P5CDH transcripts despite the mismatches. However, this wild-type 24-nt SRO5-P5CDH nat-siRNA was presumably unable to guide cleavage of mP5CDH due to the mismatches and thus no 21-nt nat-siRNAs could be formed. A similar result was observed from coexpression of P5CDH and mSRO5 (, lane 6). Coexpression of mP5CDH and mSRO5 led to the production of both m24-nt and 21-nt nat-siRNAs (, lane 5). Thus, while initial processing of the SRO5-P5CDH double-stranded RNA is tolerant of mismatches, subsequent production of 21-nt nat-siRNAs is dependent on perfect complementarity between the 24-nt SRO5-P5CDH nat-siRNA and the P5CDH transcript.
Role of SRO5-P5CDH nat-siRNAs in Proline Metabolism and Salt-Stress Tolerance
To determine the physiological role of the salt stress-induced SRO5-P5CDH nat-siRNAs, we compared the levels of salt stress-induced proline accumulation in various mutant plants. In dcl2, sgs3, rdr6, and nrpd1a, which lacked SRO5-P5CDH nat-siRNAs and cleavage of the P5CDH transcript, proline accumulation was not significantly induced by salt stress or was induced to a lesser extent than in the corresponding wild-type (). This result is consistent with their inability to downregulate P5CDH under stress, thereby causing continued proline catabolism and reduced proline accumulation. In contrast, the dcl1 and rdr2 mutants, which were able to degrade P5CDH mRNA, had wild-type levels of proline accumulation under salt stress (). The wild-type level of proline accumulation in dcl1 indicates that although the 21-nt P5CDH nat-siRNAs were not produced, the 24-nt SRO5-P5CDH nat-siRNA alone was sufficient to cause downregulation of P5CDH (). Whether or not the 21-nt P5CDH nat-siRNAs are required to downregulate P5CDH under other stress conditions will be of interest for future experiments.
Proline Accumulation and Salt and Proline Sensitivity in Mutants Affected in SRO5-P5CDH nat-siRNA Accumulation
We also examined the proline and salt-stress sensitivity of nrpd1a
, two mutants that do not have strong pleiotropic phenotypes and grow well under nonstressed conditions. Consistent with previous observations (Hellmann et al., 2000
), high levels of exogenous proline inhibited seedling growth of the C24 and Col wild-types (). The nrpd1a
mutants were clearly more tolerant of exogenous proline (). This proline tolerance may be caused by a reduced cleavage of P5CDH
mRNA leading to greater P5CDH activity and reduced P5C levels in the proline-treated seedlings. Conversely, these mutants were less tolerant of salt stress, which is consistent with their inability to control proline catabolism and accumulate protective amounts of proline.
To further examine the biogenesis of SRO5-P5CDH nat-siRNAs and their role in proline and salt-stress tolerance, we identified two homozygous Arabidopsis mutant plants with a T-DNA insertion in the ORF of SRO5 or P5CDH (). Northern blot analysis showed that expression of P5CDH and SRO5 was abolished in the respective mutants (). Neither mutant could produce the 24-nt SRO5-P5CDH nat-siRNA () or 21-nt P5CDH nat-siRNAs (data not shown) under salt stress. In the sro5 knockout mutant, P5CDH full-length transcript accumulated to a higher level and was not reduced by salt stress (). These results further demonstrate that production of SRO5-P5CDH nat-siRNAs is dependent on the presence of the P5CDH and SRO5 antisense transcripts, and that SRO5-P5CDH nat-siRNAs are responsible for the down-regulation of the P5CDH mRNA. Interestingly, SRO5 mRNA was detected in the p5cdh knockout mutant even without salt stress (). This indicates that either the p5cdh knockout causes certain physiological stress (e.g., oxidative stress) that can induce SRO5 expression, or that SRO5 transcription or transcript stability is normally suppressed by the P5CDH gene through an unknown mechanism.
SRO5-P5CDH nat-siRNA Accumulation and Salt and Proline Responses in sro5 and p5cdh Knockout Mutants
mutant accumulated more proline than wild-type under control conditions and slightly more under salt stress (). The mutant was slightly more tolerant to NaCl stress under our conditions, which is possibly related to its higher proline accumulation (). As reported previously (Deuschle et al., 2004
), the p5cdh
mutant was more sensitive than wild-type to exogenous proline (). In contrast, the sro5
mutant accumulated less proline under salt stress () and was more sensitive to NaCl stress () and more tolerant of exogenous proline (). These phenotypes are all consistent with the increased expression of P5CDH
Altered ROS Sensitivity and Accumulation in sro5 Indicates a Functional Link between the SRO5 and P5CDH Proteins
Our results suggest that nat-siRNA regulation of P5CDH
is dependent on the SRO5
mRNA. However, SRO5
is a coding gene, and we hypothesized that the SRO5 protein may also be functionally related to P5CDH. It has been shown that P5CDH
downregulation leads to the accumulation of its substrate, P5C (Deuschle et al., 2004
). P5C itself or glutamate semialdheyde, which is in spontaneous equilibrium with P5C, causes the accumulation of high levels of ROS (Deuschle et al., 2001
; Nomura and Takagi, 2004
). Therefore, the SRO5 protein may function in counteracting this ROS accumulation in a manner that balances the effects of reduced P5CDH activity.
We found that sro5
plants were more sensitive to H2
-mediated oxidative stress (). Also, there was substantially more accumulation of ROS, particularly H2
, in salt-stressed sro5
seedlings (). The increased ROS level in p5cdh
under salt stress was expected based on previous observations that P5C can cause ROS accumulation (Deuschle et al., 2001
; Nomura and Takagi, 2004
). The observation that sro5
had even greater ROS accumulation and sensitivity than p5cdh
suggests a role for SRO5 protein in counteracting the increased ROS production caused by decreased P5CDH activity, either by blocking ROS production or increasing ROS detoxification.
SRO5 Is Required for Oxidative Stress Response
A role for the SRO5 protein in ROS regulation is supported by its intracellular localization. SRO5 is predicted to be a mitochondrial protein, and we confirmed this by observing that transiently expressed SRO5-YFP fusion protein is targeted to the mitochondria (Figure S1
). Proline catabolism occurs in the mitochondria, and P5CDH is localized on the matrix side of the inner mitochondrial membrane (Deuschle et al., 2001
; Nomura and Takagi, 2004
). Also, SRO5 shares partial sequence homology to RADICLE INDUCED CELL DEATH 1 (RCD1). RCD1 is involved in controlling ROS-induced cell death, and rcd1
plants are hypersensitive to ozone, which causes oxidative stress (Ahlfors et al., 2004
). Further support for a role of SRO5 protein will require demonstration that an untranslatable SRO5
mRNA can cause P5CDH
cleavage but cannot suppress ROS accumulation.
Salt treatment causes oxidative stress, and we also found that application of H2O2 induced the expression of SRO5, induced the 24-nt SRO5-P5CDH nat-siRNA, and decreased P5CDH transcript levels (). The result suggests that the salt-stress induction of SRO5 and SRO5-P5CDH nat-siRNA formation might be mediated by increased ROS under salt stress.