APUM5 is a pathogen-responsive gene but does not affect susceptibility or resistance to Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) and Alternaria brassicicolainfections
APUM5 is involved in susceptibility to CMV [22, 23]. An APUM5 gene transcript analysis was performed via the Arabidopsis eFP Browser to further elucidate the functional role of APUM5 [24]. Interestingly, APUM5 was induced by biotic and abiotic stresses in the bioinformatics analysis. Furthermore, APUM5 gene expression was enhanced in the mpk4-1 mutant background compared with accession Landsberg erecta (Ler) control plants (Additional file 1). Arabidopsis MAP kinase 4 (AtMPK4) is a negative regulator involved in salicylic acid-dependent disease resistance. mpk4-1 exhibits enhanced resistance to biotrophs and increased susceptibility to necrotrophs [25]. Based on these results, we postulated that APUM5 might be associated with the biotic/abiotic defense response pathway. First, we confirmed whether APUM5 expression is responsive to infection by bacterial pathogens such as Pst DC3000. As a result, APUM5 mRNA expression increased upon Pst DC3000 infection at 24 h post infection (hpi) and started to diminish at 48 and 72 hpi (Figure 1A). AtPR1 was used as a positive control and was successfully induced upon Pst DC3000 infection (Figure 1A). Thus, we checked the possibility that APUM5 might be involved in the pathogen defense response and could affect bacterial growth. However, APUM5 transgenic plants (both 35S-APUM5 and APUM5-RNAi plants) did not show changes in susceptibility or resistance in the Pst DC3000 growth assay (Figure 1C). These data indicate that APUM5 is not required for bacterial pathogen resistance although APUM5 was induced upon bacterial infection.
Next, we checked whether APUM5 affected fungal pathogen infection. When Col-0 plants were inoculated with A. brassicicola, APUM5 mRNA expression levels increased at the 24 and 48 h time points (Figure 1B). PDF1.2, which is a positive control for fungal pathogen infection, was induced by the A. brassicicola infection (Figure 1B). We evaluated the necrotic lesion size in Col-0 and APUM5 transgenic plants upon A. brassicicola infection. The APUM5 transgenic plants did not exhibit any significant increase or decrease in necrotic lesion sizes compared with those in Col-0 plants (Figure 1D). Thus, APUM5 overexpression or knockdown did not change the fungal pathogen growth phenotype, although APUM5 is a fungal pathogen-responsive gene. These results suggest that APUM5 is not associated with defense against Pst DC3000 and A. brassicicola infections even though APUM5 expression response to bacterial and fungal pathogens.
Expression of APUM5increases strongly following mannitol, salt, and ABA treatments
We found that APUM5 is a pathogen-responsive gene following bacterial and fungal pathogen infections. However, the function of APUM5 was not associated with defense against these pathogens, although APUM5 inhibits CMV infection by binding to the CMV 3′ UTR [22, 23]. Furthermore, APUM5 transgenic plants did not show enhanced or repressed PR gene expression [22]. These results might explain that APUM5 did not directly regulate defense-related genes.
APUM5 is significantly induced by abiotic stressors such as mannitol, salt, ABA treatments [24]. We verified that APUM5 expression increased rapidly in response to mannitol treatment in 10-day-old seedlings (Figure 2A). The RD29A promoter contains both a dehydration-responsive element (DRE) and an ABA-responsive element (ABRE), which are two major cis-acting elements involved in ABA-independent and -dependent gene expression, respectively [26]. RD29A was used as a positive control and was successfully induced by mannitol stress (Figure 2A). To further investigate the effects of other osmotic stressors and ABA treatment on APUM5 expression, the expression levels of APUM5 in 10-day-old seedlings treated with NaCl and ABA were measured by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). APUM5 expression increased gradually and strongly following NaCl treatment (Figure 2B). We also evaluated the effect of ABA. As expected, APUM5 gene expression was enhanced by ABA treatment (Figure 2C). RD29A expression was also increased by the NaCl and ABA treatments (Figure 2). These observations suggest that APUM5 may play a role during the osmotic and ABA stress response.
Tissue-specific expression of GUS in APUM5pro-GUStransgenic plants
A 1.3-kb fragment of the APUM5 promoter region was fused to the GUS reporter gene, and this construct was introduced into Arabidopsis to analyze the spatial expression of APUM5. APUM5pro-GUS expression was analyzed in the 10-day-old seedling stage of T3 transgenic plants, with strong GUS activity in the root tip, primary root, lateral root, and shoot apical meristem region (Figure 3A, Sections 1, 3, 4, and 5). Although low GUS activity was detected in leaf tissue, much higher activity was observed in the hydathodal cells (Figure 3A, Section 2). High levels of GUS activity were also detected in cauline leaves, flowers, and silique ends (Additional file 2). These observations confirmed results described previously [27].
Previous results showed that APUM5 expression was highly enhanced under the osmotic stress condition (Figure 2), indicating that APUM5 expression could be observed in the leaf tissue in the GUS activity assay following osmotic stress treatment. Thus, the APUM5 promoter cis-elements were examined using Athena, the Arabidopsis promoter analysis tool [28]. The promoter analysis revealed that the APUM5 promoter contains ABA response elements (ABFs binding motif, ABREATRD22) and MYB recognition elements (MYB4 binding motif, MYB1AT), all of which are cis-acting elements often found in ABA- or environmental stress-related genes (Additional file 3) [26]. APUM5pro-GUS transgenic plants were treated with mannitol, NaCl, and exogenous ABA in soil to further investigate APUM5 expression. After applying of NaCl, mannitol, and ABA, most of the leaf tissues showed strong GUS activity and were activated in the entire plant vasculature (Figure 3B, C). This was consistent with qRT-PCR analyses of the APUM5 gene expression pattern during osmotic stress and exogenous ABA applications (Figure 2). Interestingly, GUS activity was not detected in guard cells (Figure 3C, Section 4), whereas strong GUS activity was detected in the hydathodes, trichomes, mesophyll cells, main veins, and vascular tissues (Figure 3C, Sections 1–3). These results indicate that APUM5 may not directly affect stomatal regulation through an ABA-dependent pathway of stomatal closure and opening. In contrast, APUM5 may affect the physical endurance of the plants under osmotic or drought stress via hydathodes and trichomes.
Overexpression of APUM5 leads to hypersensitivity and down-regulation to reduced susceptibility to salt and mannitol in Arabidopsis
The above results demonstrated that osmotic stress and exogenous ABA treatment up-regulated APUM5 expression (Figure 2) and this result was consistent with APUM5pro-GUS expression under similar stress conditions (Figure 3B and C). Additionally, these results are supported by the APUM5 promoter cis-acting elements analysis (Additional file 3). These data suggest that APUM5 may be involved in osmotic, drought, and ABA sensitivity. Germination and post-germination growth efficiency of APUM5 transgenic and Col-0 wild-type plants were examined when the plants were treated with various concentrations of salt or mannitol to determine whether the physiological role of APUM5 in Arabidopsis is associated with salt or dehydration stress. The germination rate of Col-0, 35S-APUM5, and APUM5-RNAi plants was similar on the control 1/2 MS medium plate (Additional file 4). However, the germination rate of APUM5-RNAi line #1 and #2 plants was about 20% higher than that of Col-0 on the 1/2 MS medium supplemented with 150 mM NaCl, whereas no obvious difference in germination rate was observed between the Col-0 and 35S-APUM5 transgenic plants (Figure 4A). However, primary root elongation of 35S-APUM5 transgenic plants was hypersensitive in the 1/2 MS plate containing 100 mM NaCl (Additional file 5A). On the other hand, the germination rate of APUM5-RNAi line #1 and #2 plants increased about 18% compared with that of the Col-0 plants on 1/2 MS medium supplemented with 400 mM mannitol, whereas the germination rate of 35S-APUM5 transgenic line #1 and #2 plants decreased significantly (about 35%) by 7 days (Figure 4B). Primary root elongation of 35S-APUM5 was also hypersensitive on the 1/2 MS plate containing 300 mM mannitol (Additional file 5B). These results suggest that APUM5 may negatively contribute to salt and dehydration stress tolerance.
Fully germinated 3-day-old seedlings in normal 1/2 MS medium were transferred to plates supplemented with 150 mM NaCl or 400 mM mannitol to further assess the effect of salt or dehydration on growth of APUM5 transgenic seedlings, and primary root elongation was monitored after 7 days. Primary root elongation of the APUM5 transgenic plants was similar to that of the Col-0 plants under normal conditions (Figure 4C). However, primary root length of APUM5-RNAi plants was longer than that of Col-0 plants under the 150 mM NaCl supplement condition, whereas that of 35S-APUM5 transgenic plants was shorter (Figure 4D). This phenotype is thus concentration-dependent effect in root elongation of 35S-APUM5 plants. In contrast, APUM5-RNAi transgenic plants showed enhanced primary root growth under the salinity stress condition (Figure 4D). The primary root length of APUM5-RNAi transgenic plants was 14–15% longer compared with that of Col-0 plants when the plants were grown on plates supplemented with 400 mM mannitol, whereas 35S-APUM5 plants showed reduced primary root growth of 16–17% compared to that of Col-0 plants (Figure 4E).
We assessed whether germination and primary root growth were affected by an exogenous ABA application. The germination rate of 35S-APUM5 transgenic plants decreased about 39% and 49% compared with that of wild-type and APUM5-RNAi plants following 0.5 μM and 0.7 μM ABA treatments, respectively (Additional file 6A). Primary root length of 35S-APUM5 plants was shorter than that of the wild-type and APUM5-RNAi plants following ABA treatment (Additional file 6B). These results show that APUM5-overexpressing plants are more hypersensitive to dehydration or salt stress, suggesting that APUM5 might regulate the abiotic stress response.
APUM5 seems to be directly involved in the osmotic stress response. Phenotypes of wild type and APUM5 transgenic plants treated with salt were evaluated in soil to investigate the possible role of APUM5 in the salt stress response. A high-salinity treatment resulted in symptoms on Col-0 leaves such as chlorosis, leaf burn, and senescence as well as a decrease in leaf area compared with those in non-stressed plants [29]. Wild-type and APUM5 transgenic plants showed similar normal growth at the vegetative stage. However, 35S-APUM5 transgenic plants exhibited a slightly more shrinking phenotype compared with Col-0 and APUM5-RNAi plants when irrigated with 150 mM NaCl for 5 days (Figure 5A). 35S-APUM5 transgenic plants showed significantly enhanced chlorosis, leaf burn, and reduced leaf area at 10 days, compared with that in Col-0 and APUM5-RNAi plants (Figure 5A). To further analyze the effect of salt stress, chlorophyll contents and chlorophyll a/b ratio were measured because chlorosis was enhanced in APUM5-overexressing plants. Chlorophyll content decreased but the chlorophyll a/b ratio remained unchanged in 35S-APUM5 transgenic plants compared to those in Col-0 and APUM5-RNAi plants (Figure 5B and C). Taken together, these results indicate that APUM5-overexpressing plants exhibit hypersensitivity to salt stress at the vegetative and primary root elongation stage, suggesting that APUM5 may act as a negative regulator when plants are subjected to salt stress.
APUM5-overexpressing plants are more hypersensitive whereas APUM5-knockdown plants are more tolerant to drought stress compared to wild-type plants in soil
We further investigated whether APUM5 transgenic plants showed an altered phenotype to drought tolerance. Both Col-0 and 35S-APUM5 plants became severely wilted when water was withheld from soil for 14 days (Figure 6A). However, down-regulation of APUM5 by APUM5-RNAi resulted in the enhanced tolerant phenotype at the same stage compared with that of Col-0 (Figure 6A). Survival rate was examined after re-watering the 14 day-water-withheld plants. Approximately 77% of the APUM5-RNAi plants survived, whereas 11–13% of the 35S-APUM5 and 58% of the Col-0 plants survived (Figure 6B). Additionally, water loss rate of the wild-type and APUM5 transgenic plants was measured in detached leaves. Wild-type plants exhibited similar weight loss of detached leaves as APUM5-RNAi plants, whereas 35S-APUM5 transgenic plants had highly enhanced water loss rates (Figure 6C). Thus, similar to the salt stress results, APUM5-overexpressing plants exhibited more drought sensitivity, whereas repressing of APUM5 expression led to more drought tolerance compared with that of wild-type plants in soil.
Altered transcription levels of abiotic stress-responsive genes in APUM5-overexpressing plants under drought stress
A gene expression analysis of various genes was performed by qRT-PCR to examine whether the enhanced sensitivity of 35S-APUM5 transgenic plants to salt and drought stresses was accompanied by changed transcription levels of abiotic stress-responsive genes. The transcription levels of some abiotic stress-responsive genes decreased in 35S-APUM5 transgenic plants under the drought stress condition compared to those in wild type plants (Figure 7A). The putative APUM5 binding site was searched in the 3′ UTR region of abiotic stress-responsive genes to investigate the possible role of APUM5 as a post-transcriptional repressor. We conducted this search because mammalian Pumilio proteins directly interact with target transcripts containing the conserved ‘UGUA’ tetranucleotide motif [9, 15, 17]. Some of 3′ UTRs of abiotic stress-responsive genes contained the conserved ‘UGUA’ tetranucleotide motif (Figure 7B and Additional file 7B). The 3′ UTRs of significantly downregulated genes in 35S-APUM5 transgenic plants by drought stress were re-analyzed and compared with the Drosophila hbNRE2 sequence. The ‘UGUA’ tetranucleotide motif of the 3′ UTRs was highly conserved among the most abiotic stress-responsive genes and hbNRE2 (Figure 7B). ERD10 and ABI4 transcript levels also decreased about 52% and 42% in APUM5-overexpressing plants compared with wild-type plants upon drought stress, respectively (Figure 7A). However, ERD10 and ABI4 did not have the ‘UGUA’ motif in the 3′ UTR region (Figure 7B). In contrast, KIN1, AtMYB6, AAO3, and RD29B did not contain the ‘UGUA’ tetranucleotide motif and their transcript levels did not change significantly in 35S-APUM5 plants upon drought stress (Additional file 7). APUM5-RNAi plants did not exhibit a dramatic alteration in ABA-response genes expression partly because APUM5-RNAi transgenic plants showed only 50% silencing levels of the APUM5 gene and the effect may be similar to that of the heterozygote mutant (Huh et al., 2013). These results suggest that transcripts of some abiotic stress-responsive genes could be negatively regulated by binding of the APUM5 protein in their 3′ UTR regions.
APUM5-PHD binds to the 3′ UTR motifs of abiotic stress-responsive putative target genes
An electrophoretic mobility shift assay (EMSA) was performed to determine if the APUM5-PHD binds to the putative target RNAs. Genes that were highly down-regulated by APUM5 and had a ‘UGUA’ binding motif were selected. 32P-labeled synthetic 30 nucleotide RNAs along with ‘UGUA’ core sequence mutants were incubated with recombinant GST-APUM5-PHD protein. GST was used as a negative control. The EMSA results revealed that GST-APUM5-PHD bound effectively to DREB2A, RD22, COR15, and RAB18 but not to mutant RNAs, whereas the GST protein did not interact with these RNAs (Figure 8A–D). Furthermore, APUM5-PHD also showed strong binding affinity for hbNRE2 RNA (Figure 8E). APUM5 recognized the 8–10 nucleotide ‘UGUA’ core motifs. These results indicate that APUM5 binding affinity might be flexible for target binding motif recognition and this flexibility could contribute to multi-regulation of abiotic stress-responsive genes by destabilizing target mRNAs. This result confirmed that the Arabidopsis APUM5 protein has RNA binding activity and that the binding is important for regulating putative target 3′ UTRs.
APUM5 negatively regulates the RD22 and RAB183′ UTR reporters
We found that the APUM5-PHD protein directly bound to 3′ UTRs of ABA-responsive genes in vitro. This phenomenon might explain that APUM5 negatively regulates ABA-responsive genes via binding to target 3′ UTRs. We made reporter constructs with 3′ UTRs of ABA-responsive genes and expressed reporter constructs in Col-0 and 35S-APUM5 transgenic protoplasts to identify the function of this binding. The RD22-3′ UTR reporter normally expressed GFP signals in Col-0 protoplasts, whereas the RD22-3′ UTR reporter showed reduced GFP signals in 35S-APUM5 transgenic protoplasts (Figure 9A). Next, these GFP signals were quantified by confocal LSM700 microscopy and ImageJ software. The signal intensity of the RD22-3′ UTR reporter in 35S-APUM5 transgenic protoplasts decreased approximately 20% compared with that in Col-0 protoplasts (Figure 9C). Western blot with a GFP antibody and RT-PCR analyses were performed to further confirm these data. Both the GFP protein and RNA levels of the RD22-3′ UTR reporter in 35S-APUM5 transgenic protoplasts decreased compared with the levels in Col-0 protoplasts (Figure 9E). We also performed the reporter assay with the RAB18-3′ UTR reporter using a similar procedure. The RAB18-3′ UTR reporter in 35S-APUM5 transgenic protoplasts showed about a 45% reduction in GFP signal intensity compared with that in Col-0 protoplasts (Figure 9B and D). In the Western blot and RT-PCR analyses, GFP protein and RNA levels decreased slightly in 35S-APUM5 transgenic protoplasts compared with those in Col-0 protoplasts (Figure 9F).
Previous results showed that APUM5-PHD also bound to the DREB2A and COR15 3′ UTRs (Figure 8A and C), and these putative target RNAs also contained the PHD binding RNA motif. Thus, we expected that these reporters would also exhibit a decrease of the GFP signal in 35S-APUM5 transgenic protoplasts. However, the GFP protein and RNA levels of the DREB2A and COR15 3′ UTR reporters were not affected in 35S-APUM5 transgenic protoplasts compared with Col-0 protoplasts (Additional file 8). These data indicate that APUM5 negatively regulates some ABA-responsive genes by binding to 3′ UTR post-transcriptionally. Furthermore, RNA binding motifs may be very flexible and other RNA residues in addition to the ‘UGUA’ core motif or RNA structure could be important for the in vivo APUM5 binding system.