Alternative splicing of PIF4 regulates plant development under heat stress

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1. 

   eLife

   Mar 3, 2026

   eLife Assessment

   This manuscript identifies temperature-dependent alternative splicing of PIF4 in Arabidopsis thaliana and shows that heat stress promotes the accumulation of a short exon 5-skipping isoform that is predicted to encode a non-functional protein. This finding is important, and it provides an intriguing new layer of regulation for PIF4; however, the strength of the mechanistic conclusions is limited, and several key conclusions rely on indirect evidence. As a result, while the data robustly demonstrate heat-regulated alternative splicing of PIF4, the causal role of PIF4 isoforms' balance in shaping heat-induced developmental responses remains only partially supported. This work will be of interest to biologists working on alternative splicing.

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2. 

   eLife

   Mar 3, 2026

   Reviewer #1 (Public review):

   This manuscript by Niño-González and collaborators shows that PIF4 undergoes alternative splicing in response to elevated temperature, generating distinct isoforms that may contribute to early seedling responses of Arabidopsis thaliana to heat stress (37 {degree sign}C). This work provides an intriguing perspective on how PIF activity may be modulated under stress conditions.

   The authors report rapid heat-induced changes in seedling morphology, with cotyledon angle and hypocotyl length altered as early as 3 hours after transfer to 37 {degree sign}C. These responses correlate with a transient increase in PIF4 transcript levels, followed by a return to control values at later time points. Notably, heat induces preferential production of an exon 5-skipping isoform of PIF4. The resulting short protein variant …

   Reviewer #1 (Public review):

   This manuscript by Niño-González and collaborators shows that PIF4 undergoes alternative splicing in response to elevated temperature, generating distinct isoforms that may contribute to early seedling responses of Arabidopsis thaliana to heat stress (37 {degree sign}C). This work provides an intriguing perspective on how PIF activity may be modulated under stress conditions.

   The authors report rapid heat-induced changes in seedling morphology, with cotyledon angle and hypocotyl length altered as early as 3 hours after transfer to 37 {degree sign}C. These responses correlate with a transient increase in PIF4 transcript levels, followed by a return to control values at later time points. Notably, heat induces preferential production of an exon 5-skipping isoform of PIF4. The resulting short protein variant (PIF4-S) lacks part of the bHLH domain and is therefore unlikely to be transcriptionally active.

   To explore functional consequences, the authors expressed the exon 5 inclusion (functional) isoform, PIF4-L, in the pif4-101 mutant background. Some heat-induced phenotypes, such as protochlorophyllide accumulation and subsequent photobleaching, were reduced or absent in these lines. Interestingly, pif4-101 mutants themselves largely resemble WT plants for most heat-responsive traits, with the exception of hypocotyl length. PIF4-L expression specifically attenuates the cotyledon angle response to heat, without strongly affecting hypocotyl elongation.

   An important point is that PIF4 itself is not essential for the observed heat responses, as pif4 mutants respond largely like wild-type plants. This implies that the phenotypes described are likely controlled by multiple PIFs acting redundantly. In this context, the generation of the PIF4-S isoform may represent one of several mechanisms by which heat stress reduces overall functional PIF levels, rather than a PIF4-specific regulatory switch.

   Other caveats should be considered when interpreting the work. The functional relevance of the PIF4-S isoform under heat stress is not tested, as heat responses of these transgenic lines were not examined. Transcriptome analysis of heat-stressed WT, pif4-101 mutant, and PIF4-L-expressing plants revealed an enrichment of PIF-regulated genes, supporting a possible role for this family of transcription factors in the heat stress response. Notably, the heat responsiveness of the mutant and of the transgenic lines differs only marginally from that of WT plants. In addition, the study relies primarily on total transcript-level analyses, without quantitative assessment of individual PIF isoforms or direct measurement of PIF protein abundance. Given that other PIFs are also expressed and may be subject to alternative RNA processing, it needs to be determined whether PIF4-S alone could exert a dominant effect, counteracting all the other functional PIFs by itself, under heat stress. Hence, the proposed model is a plausible but still incomplete framework that requires further experimental validation and analysis.

   Altogether, the results presented in this manuscript could also be interpreted as follows: multiple PIFs contribute to the observed phenotypes in response to heat, with overlapping (redundant) functions. Heat stress may reduce functional PIF levels through different mechanisms, one of which is the regulation of alternative splicing, as shown here for PIF4, leading to the production of non-functional proteins or protein variants that could act as negative competitors (such as PIF4-S). Restoring PIF levels to values of control conditions could therefore reverse heat-induced phenotypes, as observed in the PIF4-L expression lines.

   Main concerns:

   (1) The existence of a shorter isoform of PIF4 and PIF6 is relevant, and PIF4 could indeed play a role in the context of heat stress, as it does in thermomorphogenesis. In this sense, the interplay between PIF4-S and PIF4-L might be linked to plant morphological responses to heat; however, the present work requires further investigation to determine whether this is indeed the case. It is important to note that pif4 mutants behave similarly to WT plants, indicating that PIF4 is not necessary for the observed responses. These phenotypes are therefore most likely related to several PIFs rather than to one specific family member. The results obtained with the transgenic lines expressing PIF4-L or PIF4-S support this interpretation, as increasing a functional PIF (PIF4-L) reduces some phenotypes, while expressing a dominant-negative version mimics heat-induced phenotypes under control conditions. Thus, it is reasonable to interpret that under heat stress, functional PIF levels are reduced through multiple mechanisms, alternative splicing and PIF4-S generation being one of them in the case of PIF4, but likely with additional effects on other family members. This clearly requires further study.

   (2) RT-qPCR quantification of total PIF4 transcripts, as well as the long and short isoforms under the tested conditions, is necessary. While we agree with the authors that PIF4-S could act as a dominant-negative factor, demonstrating this requires comparison of phenotypes under heat versus control conditions using the PIF4-S transgenic lines. Importantly, for the authors' hypothesis to be valid, PIF4-S must be able to outcompete other PIFs; therefore, accurate quantification of its expression levels across conditions is crucial. Combining the results shown in Figures 2A and Figure 2G suggests that the levels of the functional PIF4-L isoform are unchanged or even reduced after 3 h of heat treatment, as the increase in total PIF4 does not fully compensate for the diversion toward PIF4-S. Additionally, it would be equally relevant to quantify the expression of other PIFs (or at least those shown in Suppl. Fig. 6) to determine whether PIF4-S could exert such a strong effect even when expressed at relatively low levels. By "proper quantification", we refer specifically to functional protein-coding variants, as in the PIF4-L case. Supplemental Figure 6 shows that PIF3 and PIF5 appear unaffected by heat, while PIF1 expression is increased. However, JBrowse data for dark-grown seedlings indicate that PIF1 is subject to alternative transcription initiation, alternative splicing, and alternative polyadenylation at its 3′ end. A similar situation occurs for PIF3, at least at the 5′ end of the transcriptional unit. Therefore, alternative RNA processing mechanisms may play a key role in modulating functional PIF protein levels in response to heat. Without considering diverted isoforms of other PIFs, the interpretation becomes problematic, as PIF1 is upregulated by heat, and PIF4-S would therefore need to overcome its activity as well. This is particularly relevant given that the cotyledon angle phenotype at 37 {degree sign}C appears even stronger than in the pif1pif3pif5 triple mutant, if such a comparison is feasible.

   (3) In addition, PP2A is a well-established housekeeping gene for normalization across different light regimes, as its expression is not affected by light. However, we are not convinced this holds true under heat stress conditions (see Li et al., Plant Cell 2019 Jul 29;31(10):2353-2369. doi:10.1105/tpc.19.00519).

   (4) Furthermore, the mechanistic conclusions would be strengthened by directly assessing PIF protein levels, for example, by western blot analysis, to determine whether changes in transcript isoform abundance translate into corresponding changes in protein accumulation under heat stress.

   (5) Importantly, the authors' interpretation that "PIF4-L.1 expresses the long isoform at levels similar to those of WT plants (Supplemental Figure 9A), ruling out the possibility that the suppression of heat-induced phenotypes (cotyledon opening and Pchlide accumulation) is due to elevated PIF4 expression levels" is not correct. The RT-qPCR assay quantifies all isoforms containing exon 6, which include both long and short variants with respect to exon 5 inclusion. Since WT plants at 37 {degree sign}C express both isoforms (L/S ≈ 60/40), the PIF4-L lines actually express 2-4-fold higher levels of the functional PIF4 isoform, based on the values shown in the figures.

   (6) Figure 3B should include a statistical analysis, as it appears that PIF4-L expression does not significantly reduce photobleaching. Cotyledon angle is not affected by either the pif4 mutation or PIF4-L expression under 22 {degree sign}C conditions (Figure 3C). However, after 24 h at 37 {degree sign}C, there is a clear effect, with cotyledon angles closer to those observed in WT plants at 22 {degree sign}C. Regarding hypocotyl length, although statistical testing was not performed, it is evident that pif4-101 affects this parameter, while PIF4-L expression in this background does not substantially alter the mutant response.

   Other comments:

   (1) We do not believe that Figure 3E is an optimal way to demonstrate attenuation of transcriptional changes by PIF4-L expression in pif4 mutants. A heat map representation would likely be more direct and informative.
   The authors should consider expressing another functional PIF in the pif4 mutant background to determine whether the observed effects are specific to PIF4, as proposed, or whether they reflect a general PIF function.

   (2) It would also be informative to examine the response under Light + 37 {degree sign}C conditions. Since PIF4 mRNA accumulation is induced by light, the authors should test whether plants incubated in light show a similar response to heat or whether it is attenuated. Potential cross-regulation between light and heat responses would be worth exploring.

   (3) As the authors acknowledge in the introduction, most of our knowledge regarding PIFs in temperature signalling has focused on thermomorphogenesis. Therefore, we believe it is important to place these new findings (exon 5 skipping) within that framework, as they could help explain observations made under better-characterized conditions. In addition, would be interesting to see the phenotypes of the pifq mutant under heat stress. Even though this mutant line displays a heat-stress-like phenotype under control conditions, it may still respond to heat treatment. If so, this would indicate that PIFs are not fully determinative of this response.

   (4) The authors should clearly state the genetic background of the PIF4-S expression lines, which appear to be in the pif4-101 background but are not explicitly described as such in the manuscript.

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3. 

   eLife

   Mar 3, 2026

   Reviewer #2 (Public review):

   The manuscript "Alternative splicing of PIF4 regulates plant development under heat stress" by Niño-González et al. describes a heat-responsive alternative splicing (AS) event in PIF4 in Arabidopsis and its potential impact on seedling development. The authors observe that etiolated ings exposed to heat respond with a more photomorphogenic developmental behaviour, as reflected, for example, by increased cotyledon opening and reduced hypocotyl elongation. They propose that the AS event in PIF4 may contribute to this response, due to reduced formation of the full-length PIF4 protein and an increase in the shorter PIF4 protein with potentially dominant negative functions.

   Expressing the individual variants in a pif4 mutant background was used to further examine their function. In the case of the full-length …

   Reviewer #2 (Public review):

   The manuscript "Alternative splicing of PIF4 regulates plant development under heat stress" by Niño-González et al. describes a heat-responsive alternative splicing (AS) event in PIF4 in Arabidopsis and its potential impact on seedling development. The authors observe that etiolated ings exposed to heat respond with a more photomorphogenic developmental behaviour, as reflected, for example, by increased cotyledon opening and reduced hypocotyl elongation. They propose that the AS event in PIF4 may contribute to this response, due to reduced formation of the full-length PIF4 protein and an increase in the shorter PIF4 protein with potentially dominant negative functions.

   Expressing the individual variants in a pif4 mutant background was used to further examine their function. In the case of the full-length PIF4 variant, some of the heat-induced phenotypes were suppressed. For the lines overexpressing the shorter PIF4 variant, heat responses were not examined.

   The authors describe an interesting phenotype and present an appealing model of how AS of PIF4, a well-known key regulator of developmental processes including light- and temperature responses, might be involved. However, I don't think that the authors provide strong evidence for their model, and the unaltered heat response of pif4 mutants argues against a major role of this gene and its AS event under these conditions. Regarding the heat responses, it remains open how distinct those are from thermomorphogenesis.

   Weaknesses:

   (1) In the manuscript, it is emphasized that previous studies on PIFs' role in temperature responses have mainly focused on thermomorphogenesis under high ambient temperature and not under hot temperatures causing heat stress. How do the authors know that the effects they are looking at are specific to hot temperatures and do not also occur at more moderate temperature increases? So, what would PIF4 splicing look like upon a shift from 22{degree sign}C to 28{degree sign}C (instead of 37{degree sign}C as used in the manuscript)?

   (2) The potential role of PIF4 and its AS event in the heat response is the key point of this manuscript, as also reflected by the title. As summarized above, I don't see direct evidence for this and a functional characterization of the AS event is lacking. First, the pif4 mutant doesn't show an altered response, which argues against its requirement under these conditions, and in particular against the proposed model that a shortened version of PIF4 acts in a dominant negative manner. Second, the impact of AS on PIF4 protein levels remains open. Antibodies against PIF4 exist and have been used before, e.g. in Lee et al. (2021), Nat Comm, and Fan et al. (2025), Nat Comm - both studies address the role of PIF4 in thermomorphogenesis and should also be discussed in this manuscript. Detecting PIF4 proteins would allow testing if indeed both PIF4 protein variants are detectable and whether, upon heat stress, the longer variant decreases while the shorter variant increases. This could be expected based on transcript data; however, due to regulation at multiple steps, a correlation between transcript and protein levels might not exist. Third, the transgenic lines expressing either the short or long PIF4 variant do not really reflect the situation in the wild type and might be/are overexpression lines. Specifically, constructs for both variants lack the UTRs according to the description in the method section. Furthermore, is the short version expressed as GFP fusion, as I understood from the method description? The PIF4-L mutants have similar PIF levels as the WT (SFig. 9); however, this refers to total transcripts, which makes a difference in the wild type, in particular under heat stress. Comparing here only the PIF4-L levels would be more informative. Accordingly, the transgenic lines may overexpress PIF4-L compared to the wild type. All the PIF4-S lines show 4 to 5-fold overexpression (again for total transcripts) compared to WT. Including lines with lower overexpression levels would be needed for a direct comparison to the wild type. Moreover, immunoblot analysis of the PIF4 protein would be needed for a direct comparison between the wild type and the two types of mutants.

   (3) Apart from the question of what level of (over)expression the transgenic lines have, several aspects of the phenotyping experiments are not in line with a simple model of PIF4 regulation or have not been addressed. Expressing the long PIF4 variant in the pif4 mutant background suppresses some of the heat-induced changes, but not the hypocotyl shortening, suggesting that the hypocotyl effect is not caused by a heat-induced lack of PIF4.

   When expressing the short variant, the authors observe increased cotyledon opening in darkness, consistent with a suppression of skotomorphogenesis due to a negative function of PIF4-S, at least when it is overexpressed. For hypocotyl length, no consistent difference between wild type and PIF4-S lines was observed: seedlings grown for 3 d in darkness had identical lengths, for 4-d-old seedlings, the PIF4-S lines did not give consistent results: PIF4S.1 (which has highest transgene expression) had same length as wild type; a pronounced difference was only seen for PIF4-S.3, which is the line with lowest expression. Have the experiments been reproduced with independent seed badges? I'm also wondering why the authors haven't performed the heat stress experiments with these PIF4-S lines, as they did for the PIF4-L mutants. According to the authors' model, the PIF4-S lines might show an opposite response compared to the PIF4-L lines, i.e. an even more pronounced heat effect compared to the wild type.

   (4) Why was the heat effect on AS of PIF6 not further analysed? Previous work showed the role of PIF6 in seed development and germination; in line with this, PIF6 expression is particularly high in embryos and seeds, but it is also expressed and alternatively spliced in other tissues and conditions, as shown in Figure 1 and SFigure 2. From the data in Figure 1, it looks like the AS pattern in heat might also be different from other conditions. So, it would be interesting to see how AS of PIF6 changes in the control and heat samples that the authors analysed for PIF4 AS, in particular, if this response is distinct for PIF4 versus PIF6.

   (5) The presentation of the RNA-seq data is incomplete. According to the method section, WT, pif4-101, PIF4-L.1 and PIF4-L.2 seedlings upon 3 h heat/control treatment were analysed. Why are DE and DAS genes and comparisons of different genotypes not shown? The FC data displayed in Figure 2E and the overlap between heat-regulated genes (Fig. 3D; only in WT) and PIF regulation show only some aspects of the data.

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4. 

   eLife

   Mar 3, 2026

   Reviewer #3 (Public review):

   Summary:

   PIFs play a pivotal role not only in light and temperature signaling pathways, but in many other signaling pathways regulating plant development by modulating transcription of a large number of genes both directly and indirectly. Similarly, alternative splicing (AS) plays a critical role in shaping the splice isoforms of thousands of genes under different environmental conditions to regulate plant development. In fact, AS of PIF6 has been shown to be involved in seed development. PIF4 is a central transcription factor integrating light and temperature signaling pathways. However, AS of PIF4 has not been involved in any pathways. This story first describes how AS of PIF4 is regulated by heat stress, and this regulation is involved in heat stress signaling to regulate plant development. This is an …

   Reviewer #3 (Public review):

   Summary:

   PIFs play a pivotal role not only in light and temperature signaling pathways, but in many other signaling pathways regulating plant development by modulating transcription of a large number of genes both directly and indirectly. Similarly, alternative splicing (AS) plays a critical role in shaping the splice isoforms of thousands of genes under different environmental conditions to regulate plant development. In fact, AS of PIF6 has been shown to be involved in seed development. PIF4 is a central transcription factor integrating light and temperature signaling pathways. However, AS of PIF4 has not been involved in any pathways. This story first describes how AS of PIF4 is regulated by heat stress, and this regulation is involved in heat stress signaling to regulate plant development. This is an important finding of general interest.

   Strengths:

   The authors first describe AS of PIF4 is regulated by heat stress, and this regulation is involved in heat stress signaling to regulate plant development.

   Weaknesses:

   There are many loose ends in this story that need to be tied up.

   Major points:

   (1) The authors are showing only the AS transcripts by PCR, but no protein data. Given that the hypothesis is that the short form of PIF4 is functioning in a dominant negative fashion, the authors need to show that this short isoform expresses a protein. In addition, they need to show that this form is functioning in a dominant negative fashion with other PIFs, either by showing that this form reduces the DNA binding and/or transcriptional responses of other PIFs.

   (2) The two mutant alleles used for this study (pif4-100 and pif4-2) have T-DNA insertion after the AS exon. Do these alleles express any short version of the protein? The previous studies showed no protein production, and thus, they may not function as a dominant negative form. Usually, the T-DNA insertion alleles may express truncated transcripts, but many do not express any protein due to a lack of stop codon and/or degradation of the transcripts. But in this case, the mutants are behaving like WT. The authors need to show that these alleles are expressing a truncated version of the PIF4 protein.

   (3) Figure 4 shows phenotypes of independent lines expressing the PIF4 short version. The authors analyzed only the cotyledon and hypocotyl phenotypes, but not Pchlide or bleaching assays. The authors need to do a thorough phenotype analysis, including heat-stress phenotypes of these lines, to test if the data make sense with their hypothesis.

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5. 

   eLife

   Mar 3, 2026

   Author response:

   We would like to thank the Editor and the three Reviewers for their detailed assessment of our manuscript and their constructive feedback. We found the suggestions valuable for refining our work. Before presenting the fully updated manuscript, we would like to clarify a few points in this initial response. This manuscript identifies a heat-induced, alternativelyspliced short isoform of PIF4 (PIF4-S) that contributes to the physiological responses observed in heat-stressed etiolated seedlings. First, we agree with all Reviewers that including PIF4 protein data will strengthen our findings an more definitely demonstrate the generation of a protein-coding alternative isoform under heat stress. Therefore, this will be one of our main priorities in the revision. Evidence for the functionality of this alternative isoform is …

   Author response:

   We would like to thank the Editor and the three Reviewers for their detailed assessment of our manuscript and their constructive feedback. We found the suggestions valuable for refining our work. Before presenting the fully updated manuscript, we would like to clarify a few points in this initial response. This manuscript identifies a heat-induced, alternativelyspliced short isoform of PIF4 (PIF4-S) that contributes to the physiological responses observed in heat-stressed etiolated seedlings. First, we agree with all Reviewers that including PIF4 protein data will strengthen our findings an more definitely demonstrate the generation of a protein-coding alternative isoform under heat stress. Therefore, this will be one of our main priorities in the revision. Evidence for the functionality of this alternative isoform is clearly demonstrated by the distinct phenotypes exhibited by transgenic lines expressing either the long or the short versions of PIF4. Nevertheless, we agree that a more comprehensive characterization of these lines, as well as of the pif4 mutant lines, will further strengthen the demonstration of the functional relevance of this alternative splicing event. In addition, we will extend the phenotypic analysis of the PIF4-S lines to heat stress conditions. Importantly, the phenotypes observed in these lines suggest that additional molecular mechanisms may act in parallel with this alternative splicing event to regulate development in heat-stressed etiolated seedlings. As proposed by Reviewer #1, other PIFs may be involved in this response, and we will address this possibility. We will also provide new experimental data to show that alternative splicing in this gene is specific to heat stress and does not occur in other PIFs. Finally, we would like to clarify that the main scope of this manuscript is to demonstrate the functional relevance of the alternative isoform generated by splicing in PIF4 under heat stress. A detailed investigation of its molecular mode of action is beyond the scope of the present study. We sincerely appreciate the thoughtful feedback provided by all Reviewers. We will carefully consider their suggestions and use them to guide the inclusion of additional experiments and analyses in our revised manuscript to reinforce and clarify our conclusions.

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6. 

   Version published to 10.64898/2025.12.17.694898 on bioRxiv

   Dec 18, 2025