A shade-responsive microProtein in the Arabidopsis ATHB2 gene regulates elongation growth and root development

Reviewer #1 (Public Review):

Summary:

The authors report evidence for a microprotein of AtHB2-miP. The authors came across HB2 in a screen for alternative transcription start sites in Arabidopsis in response to white light or a white light followed by a far red light representative of shade. Out of 337 potential microproteins, authors selected AtHB2. At the beginning of the manuscript, it is investigated that an alternative transcription start site of HB2 gene can be used in response to far red light. The resulting shorter protein form seems to interact with HB2 protein forms, altering the localization of HB2 in transient expression assays. The functionality of HB2-miP overexpression has been addressed in transgenic Arabidopsis lines using a 35S promoter. The responses and phenotypes were compared with either WT or various types of athb2 mutant lines with disrupted HB2 gene. Such mutants and the 35S promoter-driven AtHB2-miP line showed various types of phenotypes versus each other that can be classified as mild or none, e.g. small effects on root growth, iron homeostasis gene expression, and iron contents.

Strengths:

The authors performed an interesting screen for alternative transcription start sites which resulted in 337 candidates (Figure 1A). Principally, it can be interesting to find that plants may use alternative start sites for HB2 in response to shading light. The authors provide evidence that alternative transcription start sites of HB2 can be present and used in response to FR. The possibility that potentially resulting small protein may have effects under FR light, causing alteration of root growth and physiology, is an interesting idea.

Weaknesses:

In the present manuscript, there are several signs of incomplete analysis.

(1) The transient expression experiments are not conducted with much detail to demonstrate that indeed HB2 miP is produced and can interact with regular protein. The localization of HB2 was found to be linked with condensates, but perhaps not in the presence of HB2 miP. Clearly, the lack of quantitative and qualitative analysis hampers a clear assessment of this point.

(2) The authors, unfortunately, did not provide the data of the screen to demonstrate which concrete candidates may have miPs and whether there is enrichment of certain functions. There is no supplemental table accompanying Figure 1A.

(3) One of the major unclear points that is also not addressed in the discussion is that the function of miR is studied in overexpression plants (35S promoter::miP). The effects are only compared to wild type and various lines of HB2 knockouts or knockdowns, partly with fairly uncharacterized phenotypes. It can now not be clearly determined whether the miP effects are due to a regular function of miP or due to overexpression of it. A needed control would be a 35S::AtHB2 line, or better at least two different lines (only a single miP overexpression line investigated). Since it has not been assessed by deletion mutant analysis to determine which protein parts of miP are involved in the protein regulation, it cannot be ruled out that the observed miP effects are not naturally occurring but the result of ectopic expression of a protein. Clearly, the effect of miP would be ideally studied in an environment where the levels can be controlled and the resulting phenotypes and protein levels quantified.

(4) It is not shown that the microprotein is generated in Arabidopsis in response to shade, e.g. through Western or fluorescence protein detection. The main idea that authors want to claim, namely that miP binds with regular protein and thereby controls its localization or activity has not been addressed in Arabidopsis. There are no localization experiments of HB2 protein data in the presence of miP in Arabidopsis.

(5) The plants with altered HB2 forms seem to grow well and the recorded phenotypes are rather minor. Photos are not shown. At some point, the authors discuss that there could be redundancy or that HB miP might interact with other HB proteins. However, such protein interactions have not been experimentally investigated.

Reviewer #2 (Public Review):

The first portion of the manuscript centered on identifying and confirming the ATHB2 microprotein (ATHB2miP), which constitutes the core message of this study. Overall, I find no issue with the selection criteria employed for identifying alternative microprotein mRNA transcripts. However, I do have some queries that I hope the authors can address for clarity.

(1) Upon reviewing the supplemental dataset where the authors listed the 377 unique novel miPs, along with those specifically in WL or shade treatments, I sought to comprehend the rationale behind focusing on ATHB2. Have the authors examined the shade response of all 377 potential microprotein candidates? Readers may be intrigued to learn how many of these candidates exhibit induction or repression under shade conditions, and whether such changes correlate positively or negatively with alterations in the full-length TSSs in response to shade. Essentially, I aim to discern the prevalence of microprotein production during shade responses and any shared characteristics among these microprotein transcripts. This inquiry also aims to uncover the existence of a common mechanism regulating microprotein transcription.

(2) To confirm that ATHB2miP stems from an independent transcription event, the authors sequenced full-length cDNAs using PacBio isoseq. However, I find the information regarding isoseq missing from the manuscript. My assumption is that the full-length cDNAs were reverse transcribed from mRNAs isolated from whole seedlings, where mature mRNAs in the cytoplasm predominate, making it challenging to evaluate whether a specific mRNA undergoes post-transcriptional processing. One approach to confirming ATHB2miP as a product of independent transcription involves examining nascent mRNA produced in the nucleus. The authors may need to isolate nascent mRNAs associated with RNA Polymerase II in the nucleus from seedlings treated with shade for 45 min, and then perform reverse transcription and PacBio isoseq.

(3) The authors noted the identification of two potential start codons, TTG and CTG, in the alternative TSS of ATHB2 using TISpredictor. Yet, it's imperative to identify the actual translation initiation site and the full-length sequence of ATHB2miP. I suggest the authors fuse an epitope tag (e.g., 3xFLAG) to the C-terminus of ATHB2 (utilizing the genomic sequence of ATHB2) and generate transgenic lines to be treated with shade to induce ATHB2miP-3xFLAG production. Affinity purification (anti-FLAG beads) and mass spectrometry can then identify the actual start site of ATHB2miP. This step is crucial, as the current ATHB2miP used may not be the exact sequence, and any observed phenotype could be artifacts arising from these lines.

(4) My confusion arose when analyzing the results in Figures 1E - G. The authors didn't specify whether these plants were subjected to shade treatment. What are the sequences within the second intron and third exon excluded from pATHB2control::GUS that promote transcription and translation? Have the authors examined the sequence features? This information is pivotal and related to the above question #1 because it may tell us whether the sequence feature is shared by other miP candidates.

The latter part of the manuscript focused on the functional characterization of ATHB2miP. The approaches adopted by the authors resemble those used in studying antimorphic (dominant negative) alleles. However, I have several concerns regarding the approaches and conclusions.

(5) Firstly, as mentioned in question #3, the authors did not map the actual translation initiation site of ATHB2miP. Therefore, all constructs involving ATHB2miP, such as eGFP-ATHB2miP, BD-ATHB2miP, and mCherry-ATHB2miP in Figure 2, and 35S::miP in Figures 3-5, may contain extra amino acids in the N-terminus, given that epitope tags were all added to the N terminus. These additional amino acids could potentially impact the behavior of ATHB2miP and lead to artifacts. Identifying the translation initiation site in ATHB2miP would facilitate the development of tools to disrupt ATHB2miP expression without affecting full-length ATHB2 expression. For instance, if the "CTG" before the leucine zipper domain is confirmed as the translation initiation site, mutating it to another Leu codon (e.g., TTA) could generate transgenic lines using the genomic sequence of ATHB2, including this mutation, to evaluate the impact of losing ATHB2miP on shade responses.

(6) Another concern pertains to the 35S::miP line utilized in Figures 3-5. The authors only presented results from one 35S::miP line, raising the possibility of T-DNA insertion disrupting an endogenous gene in the transgenic plant genome. It is essential to clarify how many individual T1 plants were generated and how many of them showed the same phenotype as the line used in the manuscript. Additionally, the use of the constitutive CaMV35S promoter could generate artifacts akin to neomorphic mutations. For example, the authors identified Cluster 1 genes that were only induced in 35S::miP, but not in t-athb2 or WT plants (Figure 3B); moreover, they found an overrepresentation of genes involved in root development in this cluster. This observation correlated well with the root phenotype of 35S::miP under the proximity shade (Figure 4D), in which the short-root phenotype was only observed in lines expressing 35S::miP. These data could be artifacts due to the constitutive expression of ATHB2miP in roots but didn't necessarily reflect the natural function of ATHB2miP.

(7) Furthermore, I seek clarification regarding the rationale behind employing different shade conditions, including deep shade, canopy shade, and proximity shade, and the significance of treating plants with these conditions. The results were challenging to interpret, and I have reservations about some statements made. The authors claimed that ATHB2 acts as a growth repressor in deep shade but a growth promoter in the canopy and proximity shade (Lines 366-368). However, it appears that regardless of the shade conditions, most mutant and transgenic lines were not significantly different from WT (Figure 4C). Additionally, the definition of proximity shade in this manuscript (R:FR = 0.06) differs from that in Roig-Villanova & Martinez-Garcia (Front. Plant Sci., 2016; R:FR, 0.5-0.3). Clarity on this disparity would be appreciated.

(8) In Figure 5, no statistical analyses were presented in Figure 5C. It remains unclear whether the differences observed are statistically significant. Moreover, the values appear quite similar among all three genotypes. Even if statistically significant, do these minor differences in Fe concentrations significantly impact plant physiology? Additionally, some statements related to Figure 5 do not align with the data presented. For instance, claims about longer hypocotyls in t-athb2, athb2∆, and atbh2∆LZ mutants compared to wild type under shade conditions on high iron media (lines 453-455) were not supported by the data in Figure 5D. Similarly, statements about the differences between mutants (lines 458-460) were not substantiated by the data.

Reviewer #3 (Public Review):

Summary and Strengths:

In this interesting manuscript, the authors identify a large number of alternative transcription start sites (TSS) and focus their functional analysis on an alternative TSS that is expected to produce a micro-protein (miP) encoding the C-terminus of ATHB2 (ATHB2miP). ATHB2miP is expected to comprise the leucine zipper part of ATHB2 and hence interact with the full-length protein through this dimerization motif. Such interactions are shown using yeast two-hybrid and FRET-FLIM assays. ATHB2 is a well-known shade-induced gene that has been implicated in shade-regulated growth responses. The authors then test the potential role for ATHB2miP genetically by comparing several athb2 loss-of-function (LOF) alleles: one does not express either full-length ATHB2 or the short ATHB2miP (t-ATHB2), two CRISPR alleles give rise to frameshift mutations in the full-length transcript but still express a potentially functional short ATHB2miP (athb2deltaLZ and athb2delta). The authors also use plants that over and ectopically express ATHB2miP (35S:miP). Overall, the results are consistent with the hypothesis that ATHB2miP inhibits the function of ATHB2, which constitutes a novel negative feedback loop. Potentially ATHB2miP may also inhibit the activity of other related HD ZIP proteins (based on 35S:miP). The effects of these genetic alterations on shade-regulated hypocotyl growth are relatively modest. Effects on root growth are also investigated and in one intriguing case, the negative feedback model does not appear to explain the data (Figure 4D, effect on lateral roots, because for this phenotype 35S:miP is very different from the lof alleles). The authors also identify a potentially interesting link between shade-regulated hypocotyl growth and iron uptake. A number of text changes and corrections to the figures would be important for clarity. They primarily concern three issues: names of the alleles, names of the studied shade conditions, and statements about significant differences between genotypes. Also, it would be interesting to know whether the effects of ATHB2 on iron uptake are due to local effects of ATHB2. Is ATHB2 expressed in roots?

Weaknesses:

(1) The naming of the different shade conditions is difficult to follow and not consistent with the way most authors in the field call such conditions. Deep shade is ok (low PAR and low R/FR, WL, PAR 13microE, R/FR 0.13). This condition is clearly defined for experiments in Figure 4. However, data in Figure 1 also use Deep shade (line 174) but PAR is not defined there. I suggest that all light conditions are clearly defined in the figure legends and in the M&M (not the case in this ms). Regarding Canopy shade (WL, PAR 45microE, R/FR 0.15) and proximity shade (WL, PAR 45microE, R/FR 0.06), see lines 355-357, this nomenclature is unclear. First proximity shade has a higher R/FR ratio than canopy shade. Second for canopy shade (compared to the WL control) PAR should decrease which is not what is done here. What is called proximity shade and canopy shade are 2 WL conditions with different R/FR ratios, which are compared to WL controls with the same PAR. It would make more sense to call them proximity shade and indicate the different R/FR ratios. Finally, extensive literature from many plant species and numerous labs has shown that hypocotyl elongation increases with R/FR decreasing. In the data shown in Figure 4, it is the opposite. Hypocotyls in Canopy shade (WL, PAR 45microE, R/FR 0.15) are longer than those in proximity shade (WL, PAR 45microE, R/FR 0.06), while with these R/FR ratios the opposite is expected. Could this be a mistake in the text? Please check.

(2) In several instances (in particular regarding data from Figures 4 and 5), the authors write that 2 genotypes are significantly different while the statistical analysis of the data does not support such statements. For example lines 392-395, the authors write that in WL the t-DNA mutant, both CRISPR mutants and 35S:miP lines all had significantly lower number of lateral roots than the WT. This is true for the t-DNA mutant (group bc, while the WT is in group a), however, all other genotypes are in group ab, hence not significantly different from the WT. Please carefully check all such statements about significant differences.

(3) The naming of the CRISPR mutants is problematic. In particular athb2delta, such a name suggests that the gene is deleted (also suggested by Figure 4A), which is not the case in this CRISPR allele leading to a frameshift early in the coding sequence. This is particularly problematic because in this allele ATHB2miP is still expressed, while based on such a name one would expect that in this mutant both the full length and the miP are lost. Both CRISPR alleles lead to a frameshift and this should be clarified in Figure 4A and in the text.

(4) Overall hypocotyl growth phenotypes of athb2 lof mutants and 35S:miP are similar and consistent with a model according to which ATHB2miP inhibits the full-length protein. However, this is not the case for the root phenotype described in 4D. It would be interesting to discuss this.

(5) The authors propose a role for ATHB2 in the root, in particular linked to iron uptake. Is this due to a local effect of ATHB2 in the roots? Is ATHB2 expressed in roots? It would be very informative if the authors would show such data, e.g. using the reporter lines used in Figure 1. Are both the FL and the miP expressed in roots?

(6) From the description regarding 5'PEAT.seq data presented in Figure 1 (see lines 174-177) it is not clear in which light conditions the seedlings were grown. It appears that samples were collected in 3 conditions. WL and after 45 and 90 minutes of low R/FR treatment. However, then the data is discussed collectively. Does the 12398 TSS correspond to what was found in all three conditions together? Are the authors showing shade-regulation of TSS? This is clearly the case for ATHB2miP. This needs to be clarified.

(7) The way gene expression of low F/FR effects is done might conflate circadian effects and low R/FR effects because the samples from different light conditions are not collected at the same ZT. This is how I understood the text. If I'm wrong please clarify the text. If I am right, this potential problem should be mentioned in the text.

(8) Could the authors envisage a way to genetically test the role of ATHB2miP by using an allele that makes the full length but not the miP? Currently, the authors use lof alleles that either make none of the transcripts (t-DNA) or potentially only the miP (CRISPR alleles). Overall, these alleles do not appear to differ in their phenotypes, suggesting that most of the effect of ATHB2miP is through ATHB2 FL. Having an allele only producing the FL would be nice (but technically I'm not sure how one could do that).