An auxin research odyssey: 1989-2023.

The phytohormone auxin is at times called the master regulator of plant processes and has been shown to be a central player in embryo development, the establishment of the polar axis, early aspects of seedling growth, as well as growth and organ formation during later stages of plant development. The Plant Cell has been key, since the inception of the journal, to developing an understanding of auxin biology. Auxin regulated plant growth control is accomplished by both changes in the levels of active hormones and the sensitivity of plant tissues to these concentration changes. In this historical review, we chart auxin research as it has progressed in key areas and highlighting the role The Plant Cell played in these scientific developments. We focus on understanding auxin-responsive genes, transcription factors, reporter constructs, perception and signal transduction processes. Auxin metabolism is discussed from the development of tryptophan auxotrophic mutants, the molecular biology of conjugate formation and hydrolysis, indole-3-butyric acid metabolism and transport, and key steps in indole-3-acetic acid biosynthesis, catabolism and transport. This progress leads to an expectation of a more comprehensive understanding of the systems biology of auxin and the spatial and temporal regulation of cellular growth and development.

Defying Gravity: WEEP promotes negative gravitropism in peach trees by establishing asymmetric auxin gradients.

Trees with weeping shoot architectures are valued for their beauty and are a resource for understanding how plants regulate posture control. The peach (Prunus persica) weeping phenotype, which has elliptical downward arching branches, is caused by a homozygous mutation in the WEEP gene. Little is known about the function of WEEP despite its high conservation throughout Plantae. Here, we present the results of anatomical, biochemical, biomechanical, physiological, and molecular experiments that provide insight into WEEP function. Our data suggest that weeping peach trees do not have defects in branch structure. Rather, transcriptomes from the adaxial (upper) and abaxial (lower) sides of standard and weeping branch shoot tips revealed flipped expression patterns for genes associated with early auxin response, tissue patterning, cell elongation, and tension wood development. This suggests that WEEP promotes polar auxin transport toward the lower side during shoot gravitropic response, leading to cell elongation and tension wood development. In addition, weeping peach trees exhibited steeper root systems and faster lateral root gravitropic response. This suggests that WEEP moderates root gravitropism and is essential to establishing the set-point angle of lateral roots from the gravity vector. Additionally, size-exclusion chromatography indicated that WEEP proteins self-oligomerize, like other proteins with sterile alpha motif (SAM) domains. Collectively, our results from weeping peach provide insight into polar auxin transport mechanisms associated with gravitropism and lateral shoot and root orientation.

Molecular switching in transcription through splicing and proline-isomerization regulates stress responses in plants.

The Arabidopsis thaliana DREB2A transcription factor interacts with the negative regulator RCD1 and the ACID domain of subunit 25 of the transcriptional co-regulator mediator (Med25) to integrate stress signals for gene expression, with elusive molecular interplay. Using biophysical and structural analyses together with high-throughput screening, we reveal a bivalent binding switch in DREB2A containing an ACID-binding motif (ABS) and the known RCD1-binding motif (RIM). The RIM is lacking in a stress-induced DREB2A splice variant with retained transcriptional activity. ABS and RIM bind to separate sites on Med25-ACID, and NMR analyses show a structurally heterogeneous complex deriving from a DREB2A-ABS proline residue populating cis- and trans-isomers with remote impact on the RIM. The cis-isomer stabilizes an α-helix, while the trans-isomer may introduce energetic frustration facilitating rapid exchange between activators and repressors. Thus, DREB2A uses a post-transcriptionally and post-translationally modulated switch for transcriptional regulation.

Abscisic acid biosynthesis is necessary for full auxin effects on hypocotyl elongation.

In concert with other phytohormones, auxin regulates plant growth and development. However, how auxin and other phytohormones coordinately regulate distinct processes is not fully understood. In this work, we uncover an auxin-abscisic acid (ABA) interaction module in Arabidopsis that is specific to coordinating activities of these hormones in the hypocotyl. From our forward genetics screen, we determine that ABA biosynthesis is required for the full effects of auxin on hypocotyl elongation. Our data also suggest that ABA biosynthesis is not required for the inhibitory effects of auxin treatment on root elongation. Our transcriptome analysis identified distinct auxin-responsive genes in root and shoot tissues, which is consistent with differential regulation of growth in these tissues. Further, our data suggest that many gene targets repressed upon auxin treatment require an intact ABA pathway for full repression. Our results support a model in which auxin stimulates ABA biosynthesis to fully regulate hypocotyl elongation.

AUXIN RESPONSE FACTOR protein accumulation and function.

Auxin is a key regulator of plant developmental processes. Its effects on transcription are mediated by the AUXIN RESPONSE FACTOR (ARF) family of transcription factors. ARFs tightly control specific auxin responses necessary for proper plant growth and development. Recent research has revealed that regulated ARF protein accumulation and ARF nucleo-cytoplasmic partitioning can determine auxin transcriptional outputs. In this review, we explore these recent findings and consider the potential for regulated ARF accumulation in driving auxin responses in plants.

Auxin and abiotic stress responses.

Plants are exposed to a variety of abiotic stresses; these stresses have profound effects on plant growth, survival, and productivity. Tolerance and adaptation to stress require sophisticated stress sensing, signaling, and various regulatory mechanisms. The plant hormone auxin is a key regulator of plant growth and development, playing pivotal roles in the integration of abiotic stress signals and control of downstream stress responses. In this review, we summarize and discuss recent advances in understanding the intersection of auxin and abiotic stress in plants, with a focus on temperature, salt, and drought stresses. We also explore the roles of auxin in stress tolerance and opportunities arising for agricultural applications.

Defying Gravity: WEEP promotes negative gravitropism in Prunus persica (peach) shoots and roots by establishing asymmetric auxin gradients.

Trees with weeping shoot architectures are valued for their beauty and serve as tremendous resources for understanding how plants regulate posture control. The Prunus persica (peach) weeping phenotype, which has elliptical downward arching branches, is caused by a homozygous mutation in the WEEP gene. Until now, little was known about the function of WEEP protein despite its high conservation throughout Plantae. Here, we present the results of anatomical, biochemical, biomechanical, physiological, and molecular experiments that provide insight into WEEP function. Our data suggest that weeping peach does not have defects in branch structure. Rather, transcriptomes from the adaxial (upper) and abaxial (lower) sides of standard and weeping branch shoot tips revealed flipped expression patterns for genes associated with early auxin response, tissue patterning, cell elongation, and tension wood development. This suggests that WEEP promotes polar auxin transport toward the lower side during shoot gravitropic response, leading to cell elongation and tension wood development. In addition, weeping peach trees exhibited steeper root systems and faster root gravitropic response, just as barley and wheat with mutations in their WEEP homolog EGT2. This suggests that the role of WEEP in regulating lateral organ angles and orientations during gravitropism may be conserved. Additionally, size-exclusion chromatography indicated that WEEP proteins self-oligomerize, like other SAM-domain proteins. This oligomerization may be required for WEEP to function in formation of protein complexes during auxin transport. Collectively, our results from weeping peach provide new insight into polar auxin transport mechanisms associated with gravitropism and lateral shoot and root orientation.

Nitric oxide-mediated S-nitrosylation of IAA17 protein in intrinsically disordered region represses auxin signaling.

The phytohormone auxin plays crucial roles in nearly every aspect of plant growth and development. Auxin signaling is activated through the phytohormone-induced proteasomal degradation of the Auxin/INDOLE-3-ACETIC ACID (Aux/IAA) family of transcriptional repressors. Notably, many auxin-modulated physiological processes are also regulated by nitric oxide (NO) that executes its biological effects predominantly through protein S-nitrosylation at specific cysteine residues. However, little is known about the molecular mechanisms in regulating the interactive NO and auxin networks. Here, we show that NO represses auxin signaling by inhibiting IAA17 protein degradation. NO induces the S-nitrosylation of Cys-70 located in the intrinsically disordered region of IAA17, which inhibits the TIR1-IAA17 interaction and consequently the proteasomal degradation of IAA17. The accumulation of a higher level of IAA17 attenuates auxin response. Moreover, an IAA17C70W nitrosomimetic mutation renders the accumulation of a higher level of the mutated protein, thereby causing partial resistance to auxin and defective lateral root development. Taken together, these results suggest that S-nitrosylation of IAA17 at Cys-70 inhibits its interaction with TIR1, thereby negatively regulating auxin signaling. This study provides unique molecular insights into the redox-based auxin signaling in regulating plant growth and development.

Plants use molecular mechanisms mediated by biomolecular condensates to integrate environmental cues with development.

This review highlights recent literature on biomolecular condensates in plant development and discusses challenges for fully dissecting their functional roles. Plant developmental biology has been inundated with descriptive examples of biomolecular condensate formation, but it is only recently that mechanistic understanding has been forthcoming. Here, we discuss recent examples of potential roles biomolecular condensates play at different stages of the plant life cycle. We group these examples based on putative molecular functions, including sequestering interacting components, enhancing dwell time, and interacting with cytoplasmic biophysical properties in response to environmental change. We explore how these mechanisms could modulate plant development in response to environmental inputs and discuss challenges and opportunities for further research into deciphering molecular mechanisms to better understand the diverse roles that biomolecular condensates exert on life.

The complexity of transferring genetic information.

The Central Dogma has been a useful conceptualization of the transfer of genetic information, and our understanding of the detailed mechanisms involved in that transfer continues to evolve. Here, we speak to several scientists about their research, how it influences our understanding of information transfer, and questions for the future.

ARF19 Condensation in the Arabidopsis Stomatal Lineage.

The phytohormone auxin regulates nearly every aspect of plant development. Transcriptional responses to auxin are driven by the activities of the AUXIN RESPONSE FACTOR family of transcription factors. ARF19 (AT1G19220) is critical in the auxin signaling pathway and has previously been shown to undergo protein condensation to tune auxin responses in the root. However, ARF19 condensation dynamics in other organs has not yet been described. In the Arabidopsis stomatal lineage, we found that ARF19 cytoplasmic condensates are enriched in guard cells and pavement cells, terminally differentiated cells in the leaf epidermis. This result is consistent with previous studies showing ARF19 condensation in mature root tissues. Our data reveal that the sequestration of ARF19 into cytoplasmic condensation in differentiated leaf epidermal cells is similar to root-specific condensation patterns.

Intrinsic and extrinsic regulators of Aux/IAA protein degradation dynamics.

The plant hormone auxin acts through regulated degradation of Auxin/INDOLE-3-ACETIC ACID (Aux/IAA) proteins to regulate transcriptional events. In this review, we examine the composition and function of each Aux/IAA structural motif. We then focus on recent characterization of Aux/IAA N-terminal disordered regions, formation of secondary structure within these disordered regions, and post-translational modifications (PTMs) that affect Aux/IAA function and stability. We propose how structural variations between Aux/IAA family members may be tuned for differential transcriptional repression and degradation dynamics.

Regulation of AUXIN RESPONSE FACTOR condensation and nucleo-cytoplasmic partitioning.

Auxin critically regulates plant growth and development. Auxin-driven transcriptional responses are mediated through the AUXIN RESPONSE FACTOR (ARF) family of transcription factors. ARF protein condensation attenuates ARF activity, resulting in dramatic shifts in the auxin transcriptional landscape. Here, we perform a forward genetics screen for ARF hypercondensation, identifying an F-box protein, which we named AUXIN RESPONSE FACTOR F-BOX1 (AFF1). Functional characterization of SCFAFF1 revealed that this E3 ubiquitin ligase directly interacts with ARF19 and ARF7 to regulate their accumulation, condensation, and nucleo-cytoplasmic partitioning. Mutants defective in AFF1 display attenuated auxin responsiveness, and developmental defects, suggesting that SCFAFF1 -mediated regulation of ARF protein drives aspects of auxin response and plant development.

Beating the heat: Phase separation in plant stress granules.

Plants form stress granules made of RNA binding proteins and RNA in response to various stresses. In this issue of Developmental Cell, Zhu et al. identify two RNA-binding proteins, RBGD2/4, that phase, separate, and localize stress granules to promote heat stress tolerance.

Structural Aspects of Auxin Signaling.

Auxin signaling regulates growth and developmental processes in plants. The core of nuclear auxin signaling relies on just three components: TIR1/AFBs, Aux/IAAs, and ARFs. Each component is itself made up of several domains, all of which contribute to the regulation of auxin signaling. Studies of the structural aspects of these three core signaling components have deepened our understanding of auxin signaling dynamics and regulation. In addition to the structured domains of these components, intrinsically disordered regions within the proteins also impact auxin signaling outcomes. New research is beginning to uncover the role intrinsic disorder plays in auxin-regulated degradation and subcellular localization. Structured and intrinsically disordered domains affect auxin perception, protein degradation dynamics, and DNA binding. Taken together, subtle differences within the domains and motifs of each class of auxin signaling component affect signaling outcomes and specificity.

Creativity comes from interactions: modules of protein interactions in plants.

Protein interactions are the foundation of cell biology. For robust signal transduction to occur, proteins interact selectively and modulate their behavior to direct specific biological outcomes. Frequently, modular protein interaction domains are central to these processes. Some of these domains bind proteins bearing post-translational modifications, such as phosphorylation, whereas other domains recognize and bind to specific amino acid motifs. Other modules act as diverse protein interaction scaffolds or can be multifunctional, forming head-to-head homodimers and binding specific peptide sequences or membrane phospholipids. Additionally, the so-called head-to-tail oligomerization domains (SAM, DIX, and PB1) can form extended polymers to regulate diverse aspects of biology. Although the mechanism and structures of these domains are diverse, they are united by their modularity. Together, these domains are versatile and facilitate the evolution of complex protein interaction networks. In this review, we will highlight the role of select modular protein interaction domains in various aspects of plant biology.

Direct photoresponsive inhibition of a p53-like transcription activation domain in PIF3 by Arabidopsis phytochrome B.

Photoactivated phytochrome B (PHYB) binds to antagonistically acting PHYTOCHROME-INTERACTING transcription FACTORs (PIFs) to regulate hundreds of light responsive genes in Arabidopsis by promoting PIF degradation. However, whether PHYB directly controls the transactivation activity of PIFs remains ambiguous. Here we show that the prototypic PIF, PIF3, possesses a p53-like transcription activation domain (AD) consisting of a hydrophobic activator motif flanked by acidic residues. A PIF3mAD mutant, in which the activator motif is replaced with alanines, fails to activate PIF3 target genes in Arabidopsis, validating the functions of the PIF3 AD in vivo. Intriguingly, the N-terminal photosensory module of PHYB binds immediately adjacent to the PIF3 AD to repress PIF3's transactivation activity, demonstrating a novel PHYB signaling mechanism through direct interference of the transactivation activity of PIF3. Our findings indicate that PHYB, likely also PHYA, controls the stability and activity of PIFs via structurally separable dual signaling mechanisms.