ARF5/MONOPTEROS directly regulates miR390 expression in the Arabidopsis thaliana primary root meristem

Abstract The root meristem is organized around a quiescent center (QC) surrounded by stem cells that generate all cell types of the root. In the transit‐amplifying compartment, progeny of stem cells further divides prior to differentiation. Auxin controls the size of this transit‐amplifying compartment via auxin response factors (ARFs) that interact with auxin response elements (AuxREs) in the promoter of their targets. The microRNA miR390 regulates abundance of ARF2, ARF3, and ARF4 by triggering the production of trans‐acting (ta)‐siRNA from the TAS3 precursor. This miR390/TAS3/ARF regulatory module confers sensitivity and robustness to auxin responses in diverse developmental contexts and organisms. Here, we show that miR390 is expressed in the transit‐amplifying compartment of the root meristem where it modulates response to exogenous auxin. We show that a single AuxRE located in miR390 promoter is necessary for miR390 expression in this compartment and identify that ARF5/MONOPTEROS (MP) binds miR390 promoter via the AuxRE. We show that interfering with ARF5/MP‐dependent auxin signaling attenuates miR390 expression in the transit‐amplifying compartment of the root meristem. Our results show that ARF5/MP regulates directly the expression of miR390 in the root meristem. We propose that ARF5, miR390, and the ta‐siRNAs‐regulated ARFs modulate the response of the transit‐amplifying region of the meristem to exogenous auxin.

The authors' conclusion (Line 144) that miR390 expression in the primary root meristem is modulated by exogenous auxin is not supported by the data. In fact they showed that expression persists in the root meristem as in control ( Fig 1C). However, the size of the meristem is reduced with auxin application. The data more likely reflects the effects of ARF2,3,4 on the auxin response. R/ We agree with the reviewer. Yet, our data show that miR390 marks the transient amplifying compartment and that auxin and miR390 expression are functionally connected. We have changed the sentence to "Altogether, these results indicate that miR390 is expressed in the primary root meristem and is involved in the modulation of root growth in response to exogenous auxin.". Furthermore, ARF4 is known to be upregulated in response to auxin, but there is no discussion of how this might affect the phenotypic readout. Therefore, additional experiments are required to elucidate the phenotypic contribution of miR390 regulation by ARF5, or potentially other ARFs.
R/ While we agree with the reviewer suggestion, such analysis falls out of the scope of the current manuscript that support that miR390 is a target of at least ARF5/MP. We have added a sentence in the discussion to make this point clear.
Y1H is known to produce false negatives, especially for transcription factors that have strong repression domains that can override the GAL4 activation domain. Therefore, this data alone cannot rule out the effects of other root expressed ARFs, such ARF2, 3 ARF6, 7, 19. Nulls of these alleles should be included in the analyses for miR390 expression in Fig 4A. The role of other ARFs should not be ruled out, especially as the expression pattern of ARF5 (Supp. Fig.2) does not mirror that of pmiR390:GUS (Fig1; Fig 4B). Furthermore, ARF18 was shown to bind the PRE in yeast, and to be expressed in the root, but was not examined further.
In the discussion the authors write 'It would be interesting to study whether in other developmental contexts where the miR390/TAS3/ARF regulatory network has been implicated such as lateral root and leaf patterning, similar network motif involving ARF5/MP or other ARF have also been co-opted to regulate miR390 expression' (Line 297). Given the genetic resources at hand these questions could be quite easily addressed.
R/ Although we agree with the reviewer suggestion, such analysis falls out of the scope of the current manuscript. Minor: In the discussion (line 320) it is also stated that miR396 non-cell autonomously represses GFFs in the root, but the referenced paper does not demonstrate nor imply non-cell autonomous behaviour of this miRNA.

R/
The sentence has been changed to "miR396 is transcribed in the QC and columella where it represses a set of GROWTH REGULATING FACTORs (GRFs) which are transcription factors that promote cell division. miR396 ensures the exclusion of these GRFs from the stem cell niche and contribute to the transition between the stem cell niche and transit amplifying compartment of the root meristem" Depending on where the 35S promoter is active, the MIR390 OE line may miss-express rather than over express. R/ We have changed the wording on l136 to "Plants with mis-expressed miR390 (OXMIR390, Figure 1E) had overall similar response to wild type (…)"

Reviewer #2
This manuscript presents experiments showing that MP/ARF5 regulates the MIR390a promoter in the root meristem. While this seems correct and the work is well done, the overall interest of the work seems low because the mir390a mutant only has a mild phenotype in the presence of exogenous auxin, and no phenotype under normal growth conditions.

Specific points:
It seems possible that other ARFs in addition to MP also regulate the promoter, as they have not tested some obvious candidates such as ARF7 and ARF19 (exclusion based on a negative Y1H result seems overly stringent). The argument that ectopic stabilized bdl affects expression does not mean that it acts only through MP/ARF5, as BDL should interact with multiple ARFs. R/ We agree with the reviewer and do not exclude that other ARFs may regulate the expression of miR390. The data presented here support that at least ARF5 is contributing to miR390 expression in the meristem.
In Figure 1E they present root length data relative to length in the absence of exogenous auxin. Perhaps meristem length would be more relevant. Also, are the baseline root lengths without auxin the same? R/ As the root lengths of the gain and loss of function miR390 mutants are slightly, albeit statistically different from wild type, normalization of root length was necessary. The size of the meristem were not recorded in these experiments.
Do ARF2, ARF3, and ARF4 have roles in root meristem function? Such evidence might indicate how relevant miR390 regulation might be. R/ arf2, arf3 and arf4 mutants have longer primary root than wild type (Marin et al. Plant Cell 10.1105/tpc.109.072553, supplemental material)

Reviewer #3
However, the primary and quite significant weakness of this paper is that the authors have no evidence that miR390 actually plays a role in root development. They show that miR390 affects the response of the root to exogenous auxin, however they were unable to find a root phenotype for a loss-of-function mutation in miR390. Consequently, the conclusion that "miR390...is necessary to maintain the size of the transit-amplifying region of the meristem" is not supported by the results presented in this paper.

R/
We have modified the abstract and the last paragraph of the introduction explicitly mentioning that miR390 affects the response of the root to exogenous auxin.