Charting the evolutionary path of the SUMO modification system in plants reveals molecular hardwiring of development to stress adaptation.

SUMO modification is part of the spectrum of Ubiquitin-like (UBL) systems that give rise to proteoform complexity through post-translational modifications (PTMs). Proteoforms are essential modifiers of cell signaling for plant adaptation to changing environments. Exploration of the evolutionary emergence of Ubiquitin-like (UBL) systems unveils their origin from prokaryotes where it is linked to the mechanisms that enable sulfur uptake into biomolecules. We explore the emergence of the SUMO machinery across the plant lineage from single-cell to land plants. We reveal the evolutionary point at which plants acquired the ability to form SUMO chains through the emergence of SUMO E4 ligases hinting at its role in facilitating multicellularity. Additionally, we explore the possible mechanism for the neofunctionalization of SUMO proteases through the fusion of conserved catalytic domains with divergent sequences. We highlight the pivotal role of SUMO proteases in plant development and adaptation, offering new insights into target specificity mechanisms of SUMO modification during plant evolution. Correlating the emergence of adaptive traits in the plant lineage with established experimental evidence for SUMO in developmental processes we propose that SUMO modification has evolved to link developmental processes to adaptive functions in land plants.

DELLA proteins recruit the Mediator complex subunit MED15 to coactivate transcription in land plants.

DELLA proteins are negative regulators of the gibberellin response pathway in angiosperms, acting as central hubs that interact with hundreds of transcription factors (TFs) and regulators to modulate their activities. While the mechanism of TF sequestration by DELLAs to prevent DNA binding to downstream targets has been extensively documented, the mechanism that allows them to act as coactivators remains to be understood. Here, we demonstrate that DELLAs directly recruit the Mediator complex to specific loci in Arabidopsis, facilitating transcription. This recruitment involves DELLA amino-terminal domain and the conserved MED15 KIX domain. Accordingly, partial loss of MED15 function mainly disrupted processes known to rely on DELLA coactivation capacity, including cytokinin-dependent regulation of meristem function and skotomorphogenic response, gibberellin metabolism feedback, and flavonol production. We have also found that the single DELLA protein in the liverwort Marchantia polymorpha is capable of recruiting MpMED15 subunits, contributing to transcriptional coactivation. The conservation of Mediator-dependent transcriptional coactivation by DELLA between Arabidopsis and Marchantia implies that this mechanism is intrinsic to the emergence of DELLA in the last common ancestor of land plants.

PIF4 enhances the expression of SAUR genes to promote growth in response to nitrate.

Nitrate supply is fundamental to support shoot growth and crop performance, but the associated increase in stem height exacerbates the risks of lodging and yield losses. Despite their significance for agriculture, the mechanisms involved in the promotion of stem growth by nitrate remain poorly understood. Here, we show that the elongation of the hypocotyl of Arabidopsis thaliana, used as a model, responds rapidly and persistently to upshifts in nitrate concentration, rather than to the nitrate level itself. The response occurred even in shoots dissected from their roots and required NITRATE TRANSPORTER 1.1 (NRT1.1) in the phosphorylated state (but not NRT1.1 nitrate transport capacity) and NIN-LIKE PROTEIN 7 (NLP7). Nitrate increased PHYTOCHROME INTERACTING FACTOR 4 (PIF4) nuclear abundance by posttranscriptional mechanisms that depended on NRT1.1 and phytochrome B. In response to nitrate, PIF4 enhanced the expression of numerous SMALL AUXIN-UP RNA (SAUR) genes in the hypocotyl. The growth response to nitrate required PIF4, positive and negative regulators of its activity, including AUXIN RESPONSE FACTORs, and SAURs. PIF4 integrates cues from the soil (nitrate) and aerial (shade) environments adjusting plant stature to facilitate access to light.

Ratiometric gibberellin biosensors for the analysis of signaling dynamics and metabolism in plant protoplasts.

Gibberellins (GAs) are major regulators of developmental and growth processes in plants. Using the degradation-based signaling mechanism of GAs, we have built transcriptional regulator (DELLA)-based, genetically encoded ratiometric biosensors as proxies for hormone quantification at high temporal resolution and sensitivity that allow dynamic, rapid and simple analysis in a plant cell system, i.e. Arabidopsis protoplasts. These ratiometric biosensors incorporate a DELLA protein as a degradation target fused to a firefly luciferase connected via a 2A peptide to a renilla luciferase as a co-expressed normalization element. We have implemented these biosensors for all five Arabidopsis DELLA proteins, GA-INSENSITIVE, GAI; REPRESSOR-of-ga1-3, RGA; RGA-like1, RGL1; RGL2 and RGL3, by applying a modular design. The sensors are highly sensitive (in the low pm range), specific and dynamic. As a proof of concept, we have tested the applicability in three domains: the study of substrate specificity and activity of putative GA-oxidases, the characterization of GA transporters, and the use as a discrimination platform coupled to a GA agonists' chemical screening. This work demonstrates the development of a genetically encoded quantitative biosensor complementary to existing tools that allow the visualization of GA in planta.

The plant POLYMERASE-ASSOCIATED FACTOR1 complex links transcription and H2B monoubiquitination genome wide.

The evolutionarily conserved POLYMERASE-ASSOCIATED FACTOR1 complex (Paf1C) participates in transcription, and research in animals and fungi suggests that it facilitates RNA POLYMERASE II (RNAPII) progression through chromatin. We examined the genomic distribution of the EARLY FLOWERING7 (ELF7) and VERNALIZATION INDEPENDENCE3 subunits of Paf1C in Arabidopsis (Arabidopsis thaliana). The occupancy of both subunits was confined to thousands of gene bodies and positively associated with RNAPII occupancy and the level of gene expression, supporting a role as a transcription elongation factor. We found that monoubiquitinated histone H2B, which marks most transcribed genes, was strongly reduced genome wide in elf7 seedlings. Genome-wide profiling of RNAPII revealed that in elf7 mutants, RNAPII occupancy was reduced throughout the gene body and at the transcription end site of Paf1C-targeted genes, suggesting a direct role for the complex in transcription elongation. Overall, our observations suggest a direct functional link between Paf1C activity, monoubiquitination of histone H2B, and the transition of RNAPII to productive elongation. However, for several genes, Paf1C may also act independently of H2Bub deposition or occupy these genes more stable than H2Bub marking, possibly reflecting the dynamic nature of Paf1C association and H2Bub turnover during transcription.

Origin and evolution of gibberellin signaling and metabolism in plants.

Gibberellins modulate multiple aspects of plant behavior. The molecular mechanism by which these hormones are perceived and how this information is translated into transcriptional changes has been elucidated in vascular plants: gibberellins are perceived by the nuclear receptor GID1, which then interacts with the DELLA nuclear proteins and promote their degradation, resulting in the modification of the activity of transcription factors with which DELLAs interact physically. However, several important questions are still pending: how does a single molecule perform such a vast array of functions along plant development? What property do gibberellins add to plant behavior? A closer look at gibberellin action from an evolutionary perspective can help answer these questions. DELLA proteins are conserved in all land plants, and predate the emergence of a full gibberellin metabolic pathway and the GID1 receptor in the ancestor of vascular plants. The origin of gibberellin signaling is linked to the exaptation by GID1 of the N-terminal domain in DELLA, which already acted as a transcriptional coactivator domain in the ancestral DELLA proteins. At least the ability to control plant growth seems to be encoded already in the ancestral DELLA protein too, suggesting that gibberellins' functional diversity is the direct consequence of DELLA protein activity. Finally, comparative network analysis suggests that gibberellin signaling increases the coordination of transcriptional responses, providing a theoretical framework for the role of gibberellins in plant adaptation at the evolutionary scale, which further needs experimental testing.

BBX24 Interacts with JAZ3 to Promote Growth by Reducing DELLA Activity in Shade Avoidance.

Shade avoidance syndrome (SAS) is a strategy of major adaptive significance and typically includes elongation of the stem and petiole, leaf hyponasty, reduced branching and phototropic orientation of the plant shoot toward canopy gaps. Both cryptochrome 1 and phytochrome B (phyB) are the major photoreceptors that sense the reduction in the blue light fluence rate and the low red:far-red ratio, respectively, and both light signals are associated with plant density and the resource reallocation when SAS responses are triggered. The B-box (BBX)-containing zinc finger transcription factor BBX24 has been implicated in the SAS as a regulator of DELLA activity, but this interaction does not explain all the observed BBX24-dependent regulation in shade light. Here, through a combination of transcriptional meta-analysis and large-scale identification of BBX24-interacting transcription factors, we found that JAZ3, a jasmonic acid signaling component, is a direct target of BBX24. Furthermore, we demonstrated that joint loss of BBX24 and JAZ3 function causes insensitivity to DELLA accumulation, and the defective shade-induced elongation in this mutant is rescued by loss of DELLA or phyB function. Therefore, we propose that JAZ3 is part of the regulatory network that controls the plant growth in response to shade, through a mechanism in which BBX24 and JAZ3 jointly regulate DELLA activity. Our results provide new insights into the participation of BBX24 and JA signaling in the hypocotyl shade avoidance response in Arabidopsis.

BBX21 integrates brassinosteroid biosynthesis and signalling in the inhibition of hypocotyl growth under shade.

B-Box-containing zinc finger transcription factors (BBX) are involved in light-mediated growth, affecting processes such as hypocotyl elongation in Arabidopsis thaliana. However, the molecular and hormonal framework that regulates plant growth through BBX proteins is incomplete. Here, we demonstrate that BBX21 inhibits the hypocotyl elongation through the brassinosteroid (BR) pathway. BBX21 reduces the sensitivity to 24-epiBL, a synthetic active BR, principally at very-low concentrations in simulated shade. The biosynthesis profile of BRs showed that two active BR -brassinolide (BL) and 28-homobrassinolide (28-homoBL)- and 8 of 11 intermediates can be repressed by BBX21 under white light (WL) or simulated shade. Furthermore, BBX21 represses the expression of CYTOCHROME P450 90B1 (DWF4/CYP90B1), BRASSINOSTEROID-6-OXIDASE 1 (BR6OX1, CYP85A1) and BR6OX2 (CYP85A2) genes involved in the BR biosynthesis in WL while specifically promoting DWF4 and PHYB ACTIVATION TAGGED SUPPRESSOR 1 (CYP2B1/BAS1) expression in WL supplemented with far-red (WL+FR), a treatment that simulates shade. In addition, BBX21 represses BR signalling genes such as PACLOBUTRAZOL RESISTANCE1 (PRE1), PRE3 and ARABIDOPSIS MYB-LIKE 2 (MYBL2), and auxin-related and expansin genes, such as INDOLE-3-ACETIC ACID INDUCIBLE 1 (IAA1), IAA4 and EXPANSIN 11 (EXP11) in short-term shade. By a genetic approach we found that BBX21 acts genetically upstream of BRASSINAZOLE-RESISTANT 1 (BZR1) for the promotion of DWF4 and BAS1 gene expression in shade. We propose that BBX21 integrates the BR homeostasis and shade-light signalling allowing the fine-tuning of hypocotyl elongation in Arabidopsis.

DELLA proteins regulate spore germination and reproductive development in Physcomitrium patens.

Proteins of the DELLA family integrate environmental signals to regulate growth and development throughout the plant kingdom. Plants expressing non-degradable DELLA proteins underpinned the development of high-yielding 'Green Revolution' dwarf crop varieties in the 1960s. In vascular plants, DELLAs are regulated by gibberellins, diterpenoid plant hormones. How DELLA protein function has changed during land plant evolution is not fully understood. We have examined the function and interactions of DELLA proteins in the moss Physcomitrium (Physcomitrella) patens, in the sister group of vascular plants (Bryophytes). PpDELLAs do not undergo the same regulation as flowering plant DELLAs. PpDELLAs are not degraded by diterpenes, do not interact with GID1 gibberellin receptor proteins and do not participate in responses to abiotic stress. PpDELLAs do share a function with vascular plant DELLAs during reproductive development. PpDELLAs also regulate spore germination. PpDELLAs interact with moss-specific photoreceptors although a function for PpDELLAs in light responses was not detected. PpDELLAs likely act as 'hubs' for transcriptional regulation similarly to their homologues across the plant kingdom. Taken together, these data demonstrate that PpDELLA proteins share some biological functions with DELLAs in flowering plants, but other DELLA functions and regulation evolved independently in both plant lineages.

Plant size directly correlates with water use efficiency in Arabidopsis.

Plant transpiration is a fundamental process that determines plant water use efficiency (WUE), thermoregulation, nutrition, and growth. How transpiration impacts on such essential physiological aspects and how the environment modulates these effects are fundamental questions about which little is known. We investigated the genetic and environmental factors underlying natural variation in plant transpiration and water use efficiency in a population of natural Arabidopsis thaliana accessions grown under homogeneous conditions. As expected, we observed large variation of total transpiration capacity, transpiration per surface unit, and WUE among A. thaliana accessions. Despite the variation of stomatal density and ABA content in the population, WUE did not correlate with any of these parameters. On the contrary, a surprising direct correlation was found between WUE and projected leaf area, with bigger plants displaying a more efficient use of water. Importantly, genome-wide association studies further supported our observations through the identification of several loci involved in WUE variation, mutations in which caused a simultaneous reduction in plant size and a decrease in WUE. Altogether, our results strongly suggest that, although WUE depends on many parameters, plant size is an adaptive trait with respect to water use in A. thaliana.

Spatial regulation of plant hormone action.

Although many plant cell types are capable of producing hormones, and plant hormones can in most cases act in the same cells in which they are produced, they also act as signaling molecules that coordinate physiological responses between different parts of the plant, indicating that their action is subject to spatial regulation. Numerous publications have reported that all levels of plant hormonal pathways, namely metabolism, transport, and perception/signal transduction, can help determine the spatial ranges of hormone action. For example, polar auxin transport or localized auxin biosynthesis contribute to creating a differential hormone accumulation across tissues that is instrumental for specific growth and developmental responses. On the other hand, tissue specificity of cytokinin actions has been proposed to be regulated by mechanisms operating at the signaling stages. Here, we review and discuss current knowledge about the contribution of the three levels mentioned above in providing spatial specificity to plant hormone action. We also explore how new technological developments, such as plant hormone sensors based on FRET (fluorescence resonance energy transfer) or single-cell RNA-seq, can provide an unprecedented level of resolution in defining the spatial domains of plant hormone action and its dynamics.

COP1 destabilizes DELLA proteins in Arabidopsis.

DELLA transcriptional regulators are central components in the control of plant growth responses to the environment. This control is considered to be mediated by changes in the metabolism of the hormones gibberellins (GAs), which promote the degradation of DELLAs. However, here we show that warm temperature or shade reduced the stability of a GA-insensitive DELLA allele in Arabidopsis thaliana Furthermore, the degradation of DELLA induced by the warmth preceded changes in GA levels and depended on the E3 ubiquitin ligase CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1). COP1 enhanced the degradation of normal and GA-insensitive DELLA alleles when coexpressed in Nicotiana benthamiana. DELLA proteins physically interacted with COP1 in yeast, mammalian, and plant cells. This interaction was enhanced by the COP1 complex partner SUPRESSOR OF phyA-105 1 (SPA1). The level of ubiquitination of DELLA was enhanced by COP1 and COP1 ubiquitinated DELLA proteins in vitro. We propose that DELLAs are destabilized not only by the canonical GA-dependent pathway but also by COP1 and that this control is relevant for growth responses to shade and warm temperature.

Reversion of fruit-dependent inhibition of flowering in Citrus requires sprouting of buds with epigenetically silenced CcMADS19.

In Citrus, the response to environmental floral inductive signals is inhibited by the presence of developing fruits. The mechanism involves epigenetic activation of the CcMADS19 locus (FLC orthologue), encoding a floral repressor. To understand how this epigenetic regulation is reverted to allow flowering in the following season, we have forced precocious sprouting of axillary buds in fruit-bearing shoots, and examined the competence to floral inductive signals of old and new leaves derived from them. We have found that CcMADS19 is enriched in repressive H3K27me3 marks in young, but not old leaves, revealing that axillary buds retain a silenced version of the floral repressor that is mitotically transmitted to the newly emerging leaves, which are able to induce flowering. Therefore, we propose that flowering in Citrus is necessarily preceded by vegetative sprouting, so that the competence to respond to floral inductive signals is reset in the new leaves.

A genetic approach reveals different modes of action of prefoldins.

The prefoldin complex (PFDc) was identified in humans as a co-chaperone of the cytosolic chaperonin T-COMPLEX PROTEIN RING COMPLEX (TRiC)/CHAPERONIN CONTAINING TCP-1 (CCT). PFDc is conserved in eukaryotes and is composed of subunits PFD1-6, and PFDc-TRiC/CCT folds actin and tubulins. PFDs also participate in a wide range of cellular processes, both in the cytoplasm and in the nucleus, and their malfunction causes developmental alterations and disease in animals and altered growth and environmental responses in yeast and plants. Genetic analyses in yeast indicate that not all of their functions require the canonical complex. The lack of systematic genetic analyses in plants and animals, however, makes it difficult to discern whether PFDs participate in a process as the canonical complex or in alternative configurations, which is necessary to understand their mode of action. To tackle this question, and on the premise that the canonical complex cannot be formed if one subunit is missing, we generated an Arabidopsis (Arabidopsis thaliana) mutant deficient in the six PFDs and compared various growth and environmental responses with those of the individual mutants. In this way, we demonstrate that the PFDc is required for seed germination, to delay flowering, or to respond to high salt stress or low temperature, whereas at least two PFDs redundantly attenuate the response to osmotic stress. A coexpression analysis of differentially expressed genes in the sextuple mutant identified several transcription factors, including ABA INSENSITIVE 5 (ABI5) and PHYTOCHROME-INTERACTING FACTOR 4, acting downstream of PFDs. Furthermore, the transcriptomic analysis allowed assigning additional roles for PFDs, for instance, in response to higher temperature.

Prefoldins contribute to maintaining the levels of the spliceosome LSM2-8 complex through Hsp90 in Arabidopsis.

Although originally identified as the components of the complex aiding the cytosolic chaperonin CCT in the folding of actins and tubulins in the cytosol, prefoldins (PFDs) are emerging as novel regulators influencing gene expression in the nucleus. Work conducted mainly in yeast and animals showed that PFDs act as transcriptional regulators and participate in the nuclear proteostasis. To investigate new functions of PFDs, we performed a co-expression analysis in Arabidopsis thaliana. Results revealed co-expression between PFD and the Sm-like (LSM) genes, which encode the LSM2-8 spliceosome core complex, in this model organism. Here, we show that PFDs interact with and are required to maintain adequate levels of the LSM2-8 complex. Our data indicate that levels of the LSM8 protein, which defines and confers the functional specificity of the complex, are reduced in pfd mutants and in response to the Hsp90 inhibitor geldanamycin. We provide biochemical evidence showing that LSM8 is a client of Hsp90 and that PFD4 mediates the interaction between both proteins. Consistent with our results and with the role of the LSM2-8 complex in splicing through the stabilization of the U6 snRNA, pfd mutants showed reduced levels of this snRNA and altered pre-mRNA splicing patterns.

Transition from mild cognitive impairment to normal cognition: Determining the predictors of reversion with multi-state Markov models.

INTRODUCTION: The theoretical framework of the Alzheimer's disease continuum considers transition between stages in a unidirectional manner. Here we examine the rate of reversion from mild cognitive impairment (MCI) to normal cognition (NC) and explore a set of potential variables associated with this phenomenon. METHODS: A total of 985 Spanish community-dwelling individuals aged 70 years and over at baseline were monitored for 5 years. During this time, 173 MCI and 36 dementia cases were identified. Multi-state Markov models were performed to characterize transitions between states through the dementia continuum. RESULTS: The rate of reversion from MCI to NC was 11%. There were significant non-modifiable (age, socioeconomic status, or apolipoprotein E) and modifiable factors (cognitive training or absence of affective symptoms) associated with reversion. DISCUSSION: Overall, our results highlight that the likelihood of progression from MCI to dementia is very similar to that of reversion from MCI to NC.

Regulation of xylem fiber differentiation by gibberellins through DELLA-KNAT1 interaction.

The thickening of plant organs is supported by secondary growth, a process by which new vascular tissues (xylem and phloem) are produced. Xylem is composed of several cell types, including xylary fibers, parenchyma and vessel elements. In Arabidopsis, it has been shown that fibers are promoted by the class-I KNOX gene KNAT1 and the plant hormones gibberellins, and are repressed by a small set of receptor-like kinases; however, we lack a mechanistic framework to integrate their relative contributions. Here, we show that DELLAs, negative elements of the gibberellin signaling pathway, physically interact with KNAT1 and impair its binding to KNAT1-binding sites. Our analysis also indicates that at least 37% of the transcriptome mobilized by KNAT1 is potentially dependent on this interaction, and includes genes involved in secondary cell wall modifications and phenylpropanoid biosynthesis. Moreover, the promotion by constitutive overexpression of KNAT1 of fiber formation and the expression of genes required for fiber differentiation were still reverted by DELLA accumulation, in agreement with post-translational regulation of KNAT1 by DELLA proteins. These results suggest that gibberellins enhance fiber development by promoting KNAT1 activity.

Plant vascular development: mechanisms and environmental regulation.

Plant vascular development is a complex process culminating in the generation of xylem and phloem, the plant transporting conduits. Xylem and phloem arise from specialized stem cells collectively termed (pro)cambium. Once developed, xylem transports mainly water and mineral nutrients and phloem transports photoassimilates and signaling molecules. In the past few years, major advances have been made to characterize the molecular, genetic and physiological aspects that govern vascular development. However, less is known about how the environment re-shapes the process, which molecular mechanisms link environmental inputs with developmental outputs, which gene regulatory networks facilitate the genetic adaptation of vascular development to environmental niches, or how the first vascular cells appeared as an evolutionary innovation. In this review, we (1) summarize the current knowledge of the mechanisms involved in vascular development, focusing on the model species Arabidopsis thaliana, (2) describe the anatomical effect of specific environmental factors on the process, (3) speculate about the main entry points through which the molecular mechanisms controlling of the process might be altered by specific environmental factors, and (4) discuss future research which could identify the genetic factors underlying phenotypic plasticity of vascular development.

Residence, Clinical Features, and Genetic Risk Factors Associated with Symptoms of COVID-19 in a Cohort of Older People in Madrid.

BACKGROUND: The older population has been especially affected by the severe acute respiratory syndrome coronavirus 2 pandemic (COVID-19). OBJECTIVE: The aim of the study was to explore the incidence, severity, mortality rate, clinical features, and risk factors of symptoms of COVID-19 in home-dwelling older people, and its association with type of residence, cognitive deterioration, and neurodegenerative diseases. METHODS: Data about symptoms of COVID-19 were collected through a telephone survey in the cohort of 913 older volunteers of the Vallecas Project, aged 75-90 years, most of them (902) home-dwelling, in Madrid, Spain. The association of demographic and anthropometric measures, genetic polymorphisms, comorbidities, life habits, type of residence, and frailty surrogates were explored as potential risk factors for the incidence, severity, and mortality of COVID-19 in the older population. FINDINGS: Sixty-two cases reported symptoms compatible with COVID-19; 6 of them had died, 4 in their home and 2 in the nursing home. Moderate/severe cases were significantly older and more frequently males. The APOE ε4 allele was associated with the presence of symptoms of COVID-19. Higher systolic blood pressure, more intense smoking habit, more alcohol intake, lower consumption of coffee and tea, and cognitive impairment were associated with disease severity. CONCLUSIONS: The estimated incidence of symptomatic COVID-19 in this older cohort of Madrid was 6.8%, with an overall mortality rate of 0.7% (18.2% in those living in a nursing home) and a fatality rate of 9.9%. Our exploratory study indicates that life habits, other clinical conditions and, the ε4 variant of the APOE gene are associated with the presence and clinical severity of coronavirus infection.

Auxin methylation is required for differential growth in Arabidopsis.

Asymmetric auxin distribution is instrumental for the differential growth that causes organ bending on tropic stimuli and curvatures during plant development. Local differences in auxin concentrations are achieved mainly by polarized cellular distribution of PIN auxin transporters, but whether other mechanisms involving auxin homeostasis are also relevant for the formation of auxin gradients is not clear. Here we show that auxin methylation is required for asymmetric auxin distribution across the hypocotyl, particularly during its response to gravity. We found that loss-of-function mutants in Arabidopsis IAA CARBOXYL METHYLTRANSFERASE1 (IAMT1) prematurely unfold the apical hook, and that their hypocotyls are impaired in gravitropic reorientation. This defect is linked to an auxin-dependent increase in PIN gene expression, leading to an increased polar auxin transport and lack of asymmetric distribution of PIN3 in the iamt1 mutant. Gravitropic reorientation in the iamt1 mutant could be restored with either endodermis-specific expression of IAMT1 or partial inhibition of polar auxin transport, which also results in normal PIN gene expression levels. We propose that IAA methylation is necessary in gravity-sensing cells to restrict polar auxin transport within the range of auxin levels that allow for differential responses.