The miR159-MYB33/65 module is ubiquitously present in Arabidopsis rosettes
To begin the characterisation of miR159-MYB pathway in Arabidopsis rosettes, two time-course experiments were performed to determine in what developmental stages and rosette tissues miR159 and MYB33/65 are expressed. Firstly, a qRT-PCR based transcript profiling was performed on a time-course of Arabidopsis rosettes grown over 60 days to determine the abundance of mature miR159a/miR159b and the mRNA levels of MYB33/MYB65 (Fig. 1a). However, MYB33/65 mRNA levels are not accurate indicators of their protein expression level due to the presence of a strong miR159-mediated translational repression mechanism [27]. Therefore, we investigated whether the mRNA levels of a downstream gene, CYSTEINE PROTEINASE1 (CP1; At4g36880) [15] would be an accurate indicator of MYB33/65 activity. We found CP1 mRNA levels tightly correlate with MYB33 and MYB65 mRNA levels in the absence of miR159 (the mir159ab mutant background; Additional file 1: Figure S1). Therefore, CP1 levels are used throughout the study as an indicator of MYB33/MYB65 activity.
It was found that both miR159a and miR159b were expressed throughout rosette development. Both miRNAs had similar developmental profiles, increasing approximately two-fold during the first half of rosette development and then decreasing slightly (Fig. 1a). Although for miR159a, there were no significant differences in miRNA levels at the different time points, independent time-courses confirmed this pattern and the approximate two-fold increase in miR159a abundance (Additional file 2: Figure S2). For MYB33 and MYB65, their transcript levels fluctuated throughout rosette development. However, their levels did not inversely correlate with the miR159 profile, and so these observed differences are likely to be independent of regulation by miR159 (Fig. 1b). Additionally, these wild-type MYB33/MYB65 transcript levels are approximately 10-fold lower compared to levels in 35-day-old mir159ab rosettes (Additional file 1: Figure S1), suggesting MYB33/MYB65 are being repressed throughout vegetative development. Supporting this notion, mRNA of the CP1 marker gene remains low throughout wild-type rosette development (Fig. 1c), as CP1 mRNA in 35-day-old mir159ab rosettes is at least an order of magnitude higher (Additional file 1: Figure S1). In mir159ab rosettes, the CP1 mRNA abundance is similar to that of three-day-old wild-type seedlings, tissue that is known to have high MYB protein activity due to weak miR159 activity in seeds [15, 16]. Based on all the above data, MYB33 and MYB65 mRNA is likely being continually repressed throughout wild-type rosette development, and that the fluctuations observed in their transcript levels may not have any functional significance.
To determine in what rosette tissues MIR159 and its target MYB genes are transcribed, a β-glucuronidase (GUS)-staining assay was carried out over a 60-day time course on two transgenic Arabidopsis lines; MIR159b:GUS and mMYB33:GUS. The MIR159b:GUS line was constructed by fusing the GUS gene downstream of the MIR159b promoter, to visualize the transcriptional domain of MIR159b [10], while the mMYB33:GUS line carries a miR159-resistant version of MYB33, which enables visualization of the MYB33 transcriptional domain [9]. The rosettes of each line were harvested and stained every ten days. It was found that the rosettes of both lines stained at all the tested time points, from young seedling (10-day-old) to the late reproductive (60-day-old) growth phases (Fig. 1d). Moreover, the staining appeared ubiquitous throughout MIR159b:GUS and mMYB33:GUS rosettes. Patches of unstained cells in the older plants did not reflect a developmental pattern, but rather appeared to correspond to dead cells or a leaf staining penetration problem. Hence, both MIR159b and MYB33 appear transcribed in all cells and at all rosette developmental stages. Together, the data suggests the strong constitutive expression of miR159 that suppresses the expression of the constitutively transcribed MYB33 and MYB65 genes throughout Arabidopsis rosette development.
MiR159 is functionally active throughout rosette development
Since 35S-miR159 Arabidopsis plants have no obvious vegetative defects [12] and miR159 appears to constantly silence MYB33 and MYB65 under normal growth conditions (Fig. 1), a major impact of the miR159-MYB pathway in the rosette only appears possible if miR159 levels can be decreased enabling MYB33/MYB65 expression. To test this idea, a XVE-MIM159 transgene was transformed into Arabidopsis (Fig. 2a). XVE is a transactivator that can be induced by estrogen (e.g. 17-β-estradiol), resulting in transcriptional activation of the downstream transgene [28], while the MIM159 gene carries a non-cleavable miR159 binding site that inhibits miR159 function [29]. Primary XVE-MIM159 transformants were selected and grown for 21 days so that rosettes were well established, and were then treated with either 10 μM 17-β-estradiol (inducer) or dimethyl sulfoxide (DMSO; dissolving solution, negative control). After two weeks of 17-β-estradiol treatment, rosettes contained upwardly curled leaves (Fig. 2a). This occurred in the older leaves of the plant, consistent with the older leaves of mir159ab displaying the strongest curl. Such phenotypes were not observed in XVE-MIM159 transformants treated with just DMSO or in any wild-type plants grown under our conditions. Additionally, MYB33, MYB65 and CP1 mRNA levels were elevated in 17-β-estradiol treated XVE-MIM159 plants (Fig. 2b). Together, this data suggests that miR159 function is constantly active in developing rosettes and perturbation of this function results in de-repression of MYB33/65 expression accompanied with morphological alterations to the rosette. This raises the possibility that the miR159-MYB module may be involved in response to environmental stress(es), where repression of miR159 by a stress may potentially activate MYB33/65 expression, resulting in morphological alterations in response to the stress.
The miR159-MYB33/MYB65 pathway has no major impact in the response to common abiotic stresses
MiR159 function has been implicated in plant response to abiotic stresses, as miR159 levels are repressed during heat stress in wheat [13] or in response to drought stress in potato [4]. For Arabidopsis, we searched the GENEVESTIGATOR platform (https://www.genevestigator.com/gv/) and eFP Browser (http://bar.utoronto.ca/efp/cgi-bin/efpWeb.cgi) for growth conditions that may activate CP1 transcription based on the assumption that this gene will be activated if miR159 function is compromised. However, CP1 mRNA levels were found to remain low under all examined growth conditions and stresses (data not shown). Hence, it is unclear whether the miR159-MYB33/MYB65 pathway is playing a major role in response to abiotic/biotic stresses in Arabidopsis.
To investigate whether miR159 is responsive to abiotic/biotic stresses in Arabidopsis, wild-type plants were grown under several common environmental stresses including ABA application, heat, high light intensity, drought, and cold and miR159 levels were measured. Additionally, mir159ab.myb33.myb65 quadruple mutant plants were grown alongside to determine whether there are alterations to growth when the miR159-MYB33/65 pathway is mutated. Also included in the analysis was the loss-of-function mir159a mutant in which miR159 abundance is reduced to approximately 10 % of wild type levels, but is morphologically indistinguishable from wild-type [10], possibly making such a genotype sensitized to subtle perturbations of miR159 function which may not manifest in wild-type plants.
None of the tested stress conditions induced a major or obvious observable phenotypic difference between wild-type (Col), mir159a and mir159ab.myb33.myb65 plants, where the two mutant genotypes appear indistinguishable from wild-type when grown under stress conditions (Fig. 3a). Consistent with this, none of the stress conditions, or the stress-related ABA hormone, resulted in suppression of miR159 to levels in which MYB33/65 expression would be predicted to be de-repressed (Fig. 3b). MiR159 levels in plants subjected to high temperature had the lowest relative miR159 abundance, but this appears due to a higher level of the normalizing reference gene (sno101) rather than a decrease in abundance of miR159 (Additional file 3: Figure S3). Therefore, of the different conditions, low-temperature treatment appeared to result in the lowest levels of miR159, although this reduction was not statistically different from untreated (Fig. 3b). Nevertheless, we investigated miR159 response in cold stress further (Fig. 4). However, the mRNA levels of CP1 remained unchanged between Col and the sensitized background, mir159a (Student’s Test: P > 0.05) (Fig. 4b), indicating that miR159-mediated silencing of MYB33/65 had not been strongly perturbed. This supports the observation that the Col, mir159a and mir159ab.myb33.myb65 plants displayed no strong morphological differences under low temperature stress (Fig. 4a). From these experiments, it appears that the miR159-MYB33/65 pathway plays no major role resulting in an obvious phenotypic impact in response to these common abiotic stress conditions.
Expression of viral silencing suppressors failed to strongly inhibit miR159
One likely biotic factor that could inhibit miR159 is the expression of viral silencing suppressor (VSS) proteins, which can interfere with one or more steps/factors of plant miRNA biogenesis/action. To test this idea, 35S-P19 and 35S-P0 transgenes encoding the VSSs P19 and P0 respectively were transformed into Arabidopsis and multiple transformants were obtained for both constructs. Most 35S-P19 transformants displayed reduced rosette sizes, indicating that P19 expression perturbs Arabidopsis development (Fig. 5a), a finding previously observed [30]. However, these rosettes displayed no obvious leaf-curling, suggesting that 35S-P19 expression was not strongly perturbing miR159 function. By contrast, many 35S-P0 transgenic plants developed severe morphological abnormalities, which were characterized by a reduced rosette size and curled leaves (Fig. 5a). These abnormalities were consistent with what had been previously reported for this 35S-P0 transgene [31], having characteristics similar to that of mir159ab rosettes and thus were further investigated.
First, the 35S-P0 primary transformants were grouped into four classes, based on the severity of rosette defects (Fig. 5b). Next, the P0 transcript level was measured in each class and was found to strongly correlate with the severity of morphological abnormalities (Fig. 5c), suggesting the P0-induced phenotypes are dose-dependent. To determine whether these phenotypes were potentially due to inhibition of miR159 function, MYB33 and MYB65 transcript levels were measured by qRT-PCR. With the exception of Class I (wild-type looking phenotype), mild increases (1–3 fold) of MYB33 and MYB65 transcript levels were observed in all other 35S-P0 classes, positively correlating with the severity of abnormalities and the level of P0 transcript (Fig. 5c). However, although increases in CP1 mRNA levels also positively correlated with both the P0 and MYB33/65 transcript levels (Fig. 4c), the fold change of CP1 mRNA level was much lower than that observed in mir159ab, both in this study (Fig. 4b) and in previous reports [15]. This suggests that perturbation of miR159 function by P0 expression is mild and de-regulation of MYB33/65 may not be strongly impacting the phenotype of the 35S-P0 plants.
To investigate this possibility, the 35S-P0 transgene was transformed into a loss-of-function myb33.myb65 mutant [35S-P0(myb33.myb65)] and grown alongside 35S-P0 Col transformants [35S-P0(Col)]. The 35S-P0(myb33.myb65) transformants developed similar phenotypes to those of 35S-P0(Col) and could be grouped into the same phenotypic classes (class I, II, III and IV as shown in Fig. 5b). Moreover, qRT-PCR data demonstrated that the P0 transcript levels were similar in comparable 35S-P0(Col) and 35S-P0(myb33,myb65) phenotypic classes (Fig. 5d). This finding indicated that similar P0 expression levels triggered similar phenotypic defects in both Col and myb33.myb65 plants. Hence, these P0-induced phenotypes are largely MYB33 and MYB65 independent, and not related to the mild increase of MYB33 and MYB65 mRNA levels in 35S-P0(Col). This agreed with the weak induction of CP1 (Fig. 5c). Together, these data suggest that P0 expression is unable to perturb miR159 sufficiently to result in strong de-repression of MYB33/65 expression.
The response of a myb33.myb65 mutant to Turnip Mosaic Virus is indistinguishable from wild-type
The failure of the transgenically expressed VSSs to strongly inhibit miR159 function may relate to their expression levels, which can be very high during viral infection [32]. Thus, to further investigate the possibility of perturbing miR159 function with a biotic stress, Arabidopsis was infected with Turnip Mosaic Virus (TuMV) that contains the VSS HELPER COMPONENT-PROTEINASE (HC-Pro), which sequesters sRNA duplexes [33, 34]. TuMV inoculations were made by infecting two leaves of three week-old wild-type (Col) plants, followed by two weeks of post-inoculation growth at 21 °C, followed by one week at 15 °C. This lower growth temperature was used as there is evidence that it promotes viral infections [35–37].
Three weeks post-inoculation, the infected rosettes developed symptoms including inhibited growth, upwardly-folded and twisted leaves, and exaggerated serrations of leaf edges and accelerated senescence (Fig. 6a). These symptoms vary in severity, which could be approximated as mild or severe with respect to the rosette size (Additional file 4: Figure S4). To explore the impact of TuMV infection on the miR159-MYB pathway, transcript levels of TuMV, MYB33 and CP1 were analysed in the TuMV-infected wild-type rosettes by qRT-PCR in two plants displaying mild symptoms and two plants displaying severe symptoms. First, analysis found that TuMV RNA accumulated to higher levels in the rosettes classified with severe symptoms, suggesting different levels of viral infection (Fig. 6b). Correlating with these TuMV transcript levels were MYB33 mRNA levels that were higher (~2.5 fold) in the TuMV-infected plants compared with uninfected controls (Fig. 6b). Consistent with possible MYB33 de-regulation, CP1 mRNA levels had increased (3–4 fold) in most of these infected rosettes. Generally, the abundance of mature miR159a/b were found to accumulate to higher levels in TuMV-infected rosettes (Fig. 6c), consistent with the role of HC-Pro in sequestrating sRNA duplexes, so an increased miR159 abundance likely reflects an accumulation of sequestered miR159 [38]. Although all these data suggest that viral infection can inhibit miR159, given the weak induction of CP1 in most infected plants, this would predict that MYB33/65 has only been weakly de-repressed.
To gauge the impact of TuMV infection on other miRNAs families, the mRNA levels of the canonical miRNA targets PHABULOSA (PHB; miR165/166 target), CUP-SHAPED COTYLEDON 1 (CUC1; miR164 target), AUXIN RESPONSE FACTOR 4 (ARF4; miR390 target) and TCP4 (miR319 target) were measured by qRT-PCR. The mRNA levels of PHB, CUC1 and ARF4 were found to increase (8–15 fold) in the rosettes showing severe TuMV symptoms (Fig. 6b). These were generally higher fold-increases than that of MYB33 (~3 fold, Fig. 6b). Only TCP4 (~2 fold) had mRNA levels increases similar to MYB33, possibly due to the low expression of miR319 in the rosette [39, 40]. Therefore, these data suggest that in comparison with miR159, other miRNA pathways might be more susceptible to de-regulation by TuMV infection, making a stronger contribution to the observed symptoms.
Finally, to determine the contribution of MYB33/65 de-regulation to the manifestation of TuMV symptoms, a comparison of TuMV-infected Col and myb33.myb65 plants was performed. Both TuMV-infected Col and myb33.myb65 plants developed similar abnormal leaves and rosettes that appeared indistinguishable from one another (Fig. 6a). Together, all this data suggest that, although TuMV can inhibit miR159, it may do only weakly, being of no major physiological consequence for the plant in response to viral infection.