This is the accepted version of the article: Monte Collado, Elena. «Plant biology : AHL transcription factors inhibit growth- promoting PIFs». Current Biology, Vol. 30, issue 8 (April 2020), p. R354-R356. DOI 10.1016/j.cub.2020.03.035 This version is avaible at https://ddd.uab.cat/record/222101 under the terms of the license DISPATCH Plant Biology: AHL Transcription Factors Inhibit Growth-Promoting PIFs Elena Monte1 How do plants respond to abiotic stresses such as drought, salt or cold? A new study in Arabidopsis reveals that the stress-responsive AHLs antagonize the function of the PIF transcription factors to restrict rosette growth and allow resource reallocation for stress-adaptive responses. Plants have evolved many fascinating strategies overcome their lack of mobility. Thanks to their plasticity, plants can dynamically adjust their growth and development to variations and challenges in their immediate environment. In contrast to animals, plants continuously generate new tissues and organs such as leaves, which arise from undifferentiated stem cells called meristems. In Arabidopsis, new leaves emerge from the vegetative shoot apical meristem in a spiral pattern with very short internodes between them, to form a rosette optimized for light capture. Central to growth, the PHYTOCHROME-INTERACTING FACTORS (PIFs) are basic-helix-loop helix domain-containing transcription factors that promote growth throughout the life cycle of plants [1,2]. During leaf growth, PIFs promote petiole length, and consequently pif mutants display reduced petiole length and more compact rosettes [3–5]. PIFs directly regulate the expression of genes involved in growth regulation, including hormones and other signaling pathways [6,7], and PIF abundance and activity is highly regulated in accordance with the environment [8,9]. When exposed to stress, plants generally respond by reducing their growth to divert resources to stress- adaptive responses. Under biotic stress caused by pathogens and herbivores, this growth–defense tradeoff involves accumulation of the defense plant hormone jasmonate, which leads to an increase in DELLA protein levels [10]. DELLAs then interact with PIFs and block their action to inhibit growth [11–13]. Plants also restrict their growth when facing abiotic stresses such as cold or drought. However, in contrast to biotic stresses, how plants inhibit growth in response to abiotic challenges is not well understood. In this issue of Current Biology, Favero et al. [14] now describe that the abiotic stress-responsive SOB3/ AHL29, a member of the AT-HOOK MOTIF NUCLEAR LOCALIZED (AHL) family of transcription factors, restricts petiole elongation by antagonizing the growth-promoting PIFs. 1 In this paper, the authors observed that the SOB3 mutant SOB3-D, with increased SOB3 levels, had a short-petiole phenotype under long days (LD) at 22 ºC, while the dominant-negative sob3-6 mutant had enhanced petiole growth. Studies using bright-field microscopy indicated that both cell division and elongation were affected in SOB3-D, while only cell division was altered in sob3-6. They then sought to identify genes directly downstream of SOB3 which could be implementing these petiole phenotypes. Favero et al. [14] analyzed LD-grown juvenile (14 day-old) rosettes at ZT4, approximately the time of day with maximum petiole elongation in these conditions [15], using a combination of RNA-seq and ChIP-seq. For ChIP- seq, a ProSOB3::SOB3-GPP expressing line in a SOB3-null background was used. These studies identified 1,386 genes differentially expressed between SOB3-D and sob3-6 that were bound by SOB3, of which approximately half corresponded to SOB3-induced and half to SOB3-repressed genes, suggesting that SOB3 can act as both transcriptional activator and repressor. Enriched gene ontology terms in the repressed gene set included “regulation of cell size”, and response to “auxin” and “brassinosteroid stimulus”, while “response to abiotic stimulus” was enriched in the induced set. This result was in agreement with a role for SOB3 in growth repression and in the activation of stress responses. Motif analysis identified the TCP-binding-like motif GGHCCA as the most enriched cis-element, consistent with previous reports of TCP and AHL interaction and co-binding to DNA in the regulation of hypocotyl growth [16]. Interestingly, the second most enriched motif was CACRYG, resembling the PIF- binding motifs G- (CACGTG) and PBE- (CACATG) boxes [17–20]. Given the described role of PIFs as promoters of petiole elongation, this result was a hint that SOB3 might be regulating petiole growth by antagonizing PIFs. Moreover, a pif4 mutant grown under LD conditions phenocopied SOB3-D with short petioles and reduced cell length and number. Authors then re-analyzed previous PIF4 and PIF5 ChIP-seq experiments [6,19] to compare with their SOB3 ChIP-seq data. Interestingly, they found that PIF binding was enriched in the vicinity of SOB3 peak summits in co-bound loci. Subsequent analysis by RNA-seq of the PIF- regulated genes in juvenile rosettes of a pif4 pif5 pif7 mutant grown under LD conditions, and comparison with SOB3-regulated genes and ChIP-seq data, identified a significant number of SOB3 and PIF4/5 co-bound genes that corresponded with genes induced by PIFs and repressed by AHL. Among them are growth and hormone-associated genes like ATHB2, IAA19, PIN3, SAUR24, BRI1, ACS8 and YUC8, known to promote petiole growth. Some of them were validated for direct binding by PIF4 at ZT4 under the LD conditions. Based on these results, authors proposed that SOB3 might regulate petiole growth by directly antagonizing PIF activity in inducing hormone signaling pathways. A prediction from this was that the effect of SOB3 2 on petiole growth would therefore be dependent on PIF4. To test this possibility, authors next examined the genetic interactions between SOB3 and PIF4 by generating sob3-6 pif4 double mutants, where pif4 would be expected to be epistatic over sob3-6. Indeed, compared to pif4, no significant increase in petiole length was observed in sob3-6 pif4. Finally, Favero et al. [14] began to examine the mechanistic nature of SOB3 inhibition of PIF function. Yeast-two hybrid assays failed to detect interaction between PIF4 or PIF5 and SOB3, which led authors to propose that PIFs and SOB3 are likely not part of the same DNA-binding complex, although direct PIF–SOB3 binding in planta or indirect binding through bridging factors cannot be ruled out. Next, authors tested whether binding of PIF4 to its target genes might be affected by SOB3. This was performed by ChIP–qPCR using a ProPIF4::PIF4–myc XVE::SOB3 line, in which SOB3 expression was induced by β-estradiol. A decrease in PIF4 binding to the PIF4–SOB3 co-bound ATHB2 and ACS8 regulatory sequences was observed compared to the control ProPIF4::PIF4–myc. Importantly, the binding decrease to these SOB3–PIF4 co-targets was more pronounced compared with the binding reduction to PIF4- only targets. This was significant because induction of SOB3 in the ProPIF4::PIF4–myc XVE::SOB3 line led to a drastic 70% reduction of PIF4–myc protein levels. This decrease in PIF4 levels would be sufficient to explain the reduction in PIF4 DNA binding in the presence of SOB3, but the specific enhanced decrease in binding to SOB3–PIF4 co-targets compared with PIF4-only targets supports an additional specific effect of SOB3 in restricting PIF4 binding to DNA. Moreover, these lines also showed a significant reduction of PIF4 transcript levels of approximately 30%, and ChIP data showed that SOB3 binds both upstream and downstream of PIF4, suggesting that SOB3 might directly repress PIF4 transcription under certain conditions. However, this SOB3 regulation of PIF transcription was not detected in their RNA-seq data, and therefore it might only take place under specific conditions such as those used for the SOB3-induction experiment, which used light of very high intensity to compensate for the fact that the ProPIF4::PIF4–myc XVE::SOB3 line contain extra copies of PIF4. Together, these data suggest that SOB3 might inhibit PIF4 at the transcriptional and protein levels, including PIF accumulation and binding activity. Future studies will be necessary to refine these findings under more physiological conditions and establish how SOB3 affects PIF abundance and activity. Interestingly, this work found that the transcription of BIN2 and RGA were directly regulated by SOB3, and that SOB3 could directly interact with the transcription factor HY5. The possibility that SOB3 indirectly regulates PIFs through one or more of these factors, well known to be involved in the regulation of PIF degradation and activity [8], awaits to be investigated. 3 To summarize, the current study by Favero et al. [14] provides novel exciting insight into the inhibition of PIF-promoted rosette growth by the AHL family of transcription factors. This adds to a number of described factors that regulate PIF accumulation and activity in response to a variety of stimuli, like light, temperature, hormones or photoperiod, and include direct interaction of PIFs with phytochromes, circadian clock components, DELLAs, or transcription factors like HY5 and BZR1 [8]. Importantly, because AHLs are abiotic stress-responsive genes, the new work described by Favero et al. [14] establishes a novel link between abiotic stresses such as drought or cold and the modulation of growth. The balance between growth and abiotic stress responses could be seen as analogous to the growth–defense tradeoff in response to biotic stresses, and provides a new framework to understand how plants optimize resources to face abiotic environmental challenges like salt, cold or drought, which could be of increased relevance in the current scenario of climate change. Figure 1. Schematic representation of the proposed model by Favero et al. Top, under normal conditions, PIFs promote petiole elongation during vegetative development in Arabidopsis by directly inducing genes promoting petiole growth, such as ATHB2, IAA19 or YUC8. ATH factors like SOB3, expressed at low levels, inhibit PIF function moderately. This is exemplified by the long petiole phenotype of the dominant-negative mutant sob3-6. Bottom, under abiotic stress conditions, SOB3 is induced and represses PIF4 function. A loss in PIF4 function results in short petioles and more compact rosette. This is shown by the short petiole phenotype of the pif4 mutant. Because the effect of SOB3 on petiole growth is dependent on PIF4, no significant increase in petiole length is seen in the double mutant pif4 sob3-6 with respect to pif4. The mechanism by which SOB3 antagonizes PIF function is not well defined, but it does not seem to require direct ATH–PIF interaction and it likely involves promotion of PIF degradation and restriction of PIF binding to its target genes. 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Interaction between BZR1 and PIF4 integrates brassinosteroid and environmental responses. Nat. Cell Biol. 14, 802-809. 20. Zhang Y., Mayba O., Pfeiffer A., Shi H., Tepperman J.M., Speed T.P., and Quail P.H. (2013). A quartet of PIF bHLH factors provides a transcriptionally centered signaling hub that regulates seedling morphogenesis through differential expression-patterning of shared target genes in Arabidopsis. PLoS Genet. 9, e1003244. 1 Centre for Research in Agricultural Genomics (CRAG) CSIC-IRTA-UAB-UB, Campus UAB, Bellaterra, Barcelona, Spain. Consejo Superior de Investigaciones Científicas (CSIC), Barcelona, Spain. E-mail: elena.monte@cragenomica.es In Brief How do plants respond to abiotic stresses such as drought, salt or cold? A new study in Arabidopsis reveals that the stress-responsive AHLs antagonize the function of the PIF transcription factors to restrict rosette growth and allow resource reallocation for stress- adaptive responses. 6 No stress PIFs SOB3 PETIOLE GROWTH GENES Abiotic stress PIFs SOB3 PETIOLE GROWTH GENES Current Biology