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SMAX1-LIKE (SMXL) proteins in plants are cellular signaling hubs, many of which are posttranslationally regulated by karrikins from smoke, the plant hormones strigolactones (SLs), and/or cues such as light and nutrients. SMXL proteins control diverse aspects of growth, development, and environmental adaptation in plants through transcriptional corepression and interactions with transcriptional regulator proteins. In flowering plants, the SMXL family comprises four phylogenetic clades with different roles. Functions of the aSMAX1 clade include control of germination and seedling development, while the SMXL78 clade controls shoot architecture. We investigated how SMXL roles are specified inArabidopsis thaliana.Through promoter-swapping experiments, we found thatSMXL7can partially replicateSMAX1function, butSMAX1cannot replaceSMXL7. This implies that the distinct roles of these genes are primarily due to differences in protein sequences rather than expression patterns. To determine which part of SMXL proteins specifies downstream control, we tested a series of protein chimeras and domain deletions of SMAX1 and SMXL7. We found an N-terminal region that is necessary and sufficient to specify control of germination, seedling growth, or axillary branching. We screened 158 transcription factors (TFs) for interactions with SMAX1 and SMXL7 in yeast two-hybrid assays. The N-terminal domain was necessary and/or sufficient for most of the 33 potential protein–protein interactions that were identified for SMAX1. This finding unlocks different ways to engineer plant growth control through cross-wiring SMXL regulatory “input” and developmental “output” domains from different clades and lays a foundation for understanding how functional differences evolved in the SMXL family. 

Strigolactones (SLs) are methylbutenolide molecules derived from β-carotene through an intermediate carlactonoic acid (CLA). Canonical SLs act as signals to microbes and plants, whereas noncanonical SLs are primarily plant hormones. The cytochrome P450 CYP722C catalyzes a critical step, converting CLA to canonical SLs in most angiosperms. Using synthetic biology, we investigated the function ofCYP722A, an evolutionary predecessor ofCYP722C. CYP722A converts CLA into 16-hydroxy-CLA (16-OH-CLA), a noncanonical SL detected exclusively in the shoots of various flowering plants. 16-OH-CLA application restores control of shoot branching to SL-deficient mutants inArabidopsis thalianaand is perceived by the SL signaling pathway. We hypothesize that biosynthesis of 16-OH-CLA by CYP722A was a metabolic stepping stone in the evolution of canonical SLs that mediate rhizospheric signaling in many flowering plants. 

ABSTRACT SMAX1-LIKE (SMXL) proteins are transcriptional co-repressors that regulate many aspects of plant growth and development. Proteins from the SMAX1- and SMXL78-clades of this family are targeted for degradation after karrikin or strigolactone perception, triggering downstream responses. We investigated how SMXL proteins control development.SMXL7can partially replicateSMAX1function in seeds and seedlings, butSMAX1cannot replaceSMXL7in shoot branching control. Therefore, the distinct roles of these genes arise from differences in protein activity more than expression. Analysis of chimeras and domain deletions of SMAX1 and SMXL7 proteins revealed that an N-terminal domain is necessary and sufficient to specify developmental functions. We screened 158 transcription factors for interactions with SMAX1. The N-terminal domain is necessary and/or sufficient for the majority of candidate interactions. These discoveries enable cross-wiring of karrikin and strigolactone control of plant development and lay a foundation for understanding how SMXL proteins evolved functional differences. 

Hormone-activated proteolysis is a recurring theme of plant hormone signaling mechanisms. In strigolactone signaling, the enzyme-receptor DWARF14 (D14) and an F-box protein, MORE AXILLARY GROWTH2 (MAX2), mark SUPPRESSOR OF MAX2 1- LIKE (SMXL) family proteins SMXL6, SMXL7, and SMXL8 for rapid degradation. Removal of these transcriptional corepressors initiates downstream growth responses. The homologous proteins SMXL3, SMXL4, and SMXL5, however, are resistant to MAX2- mediated degradation. We discovered that the smxl4 smxl5 mutant has enhanced responses to strigolactone. SMXL5 attenuates strigolactone signaling by interfering with AtD14-SMXL7 interactions. SMXL5 interacts with AtD14 and SMXL7, providing two possible ways to inhibit SMXL7 degradation. SMXL5 function is partially dependent on an EAR motif that typically mediates interactions with the TOPLESS family of transcriptional corepressors. However, we find that loss of the EAR motif reduces SMXL5-SMXL7 interactions and the attenuation of strigolactone signaling by SMXL5. We hypothesize that integration of SMXL5 into heteromeric SMXL complexes reduces the susceptibility of SMXL6/7/8 proteins to strigolactone-activated degradation, and that the EAR motif promotes the formation or stability of these complexes. This mechanism may provide a way to spatially or temporally fine-tune strigolactone signaling through the regulation of SMXL5 expression or translation. 

Abstract Karrikins (KARs) are chemicals in smoke that can enhance germination of many plants. Lettuce (Lactuca sativa) cv. Grand Rapids germinates in response to nanomolar karrikinolide (KAR1). Lettuce is much less responsive to KAR2 or a mixture of synthetic strigolactone analogs, rac-GR24. We investigated the molecular basis of selective and sensitive KAR1 perception in lettuce. The lettuce genome contains two copies of KARRIKIN INSENSITIVE2 (KAI2), which in Arabidopsis (Arabidopsis thaliana) encodes a receptor that is required for KAR responses. LsKAI2b is more highly expressed than LsKAI2a in dry achenes and during early stages of imbibition. Through cross-species complementation assays in Arabidopsis, we found that an LsKAI2b transgene confers robust responses to KAR1, but LsKAI2a does not. Therefore, LsKAI2b likely mediates KAR1 responses in lettuce. We compared homology models of KAI2 proteins from lettuce and a fire-follower, whispering bells (Emmenanthe penduliflora). This identified pocket residues 96, 124, 139, and 161 as candidates that influence the ligand specificity of KAI2. Further support for the importance of these residues was found through a broader comparison of pocket residues among 281 KAI2 proteins from 184 asterid species. Almost all KAI2 proteins had either Tyr or Phe identity at position 124. Genes encoding Y124-type KAI2 are more broadly distributed in asterids than in F124-type KAI2. Substitutions at residues 96, 124, 139, and 161 in Arabidopsis KAI2 produced a broad array of responses to KAR1, KAR2, and rac-GR24. This suggests that the diverse ligand preferences observed among KAI2 proteins in plants could have evolved through relatively few mutations. 

Summary Karrikins (KARs) are a class of butenolide compounds found in smoke that were first identified as seed germination stimulants for fire‐following species. Early studies of KARs classified the germination and postgermination responses of many plant species and investigated crosstalk with plant hormones that regulate germination. The discovery thatArabidopsis thalianaresponds to KARs laid the foundation for identifying mutants with altered KAR responses. Genetic analysis of KAR signalling revealed an unexpected link to strigolactones (SLs), a class of carotenoid‐derived plant hormones. Substantial progress has since been made towards understanding how KARs are perceived and regulate plant growth, in no small part due to advances in understanding SL perception. KAR and SL signalling systems are evolutionarily related and retain a high degree of similarity. There is strong evidence that KARs are natural analogues of an endogenous signal(s), KAI2 ligand (KL), which remains unknown. KAR/KL signalling regulates many developmental processes in plants including germination, seedling photomorphogenesis, and root and root hair growth. KAR/KL signalling also affects abiotic stress responses and arbuscular mycorrhizal symbiosis. Here, we summarise the current knowledge of KAR/KL signalling and discuss current controversies and unanswered questions in this field. 

Abstract Chemical signals known as strigolactones were discovered more than 50 years ago as host-derived germination stimulants of parasitic plants in the Orobanchaceae. Strigolactone-responsive germination is an essential adaptation of obligate parasites in this family, which depend upon a host for survival. Several species of obligate parasites, including witchweeds (Striga, Alectra spp.) and broomrapes (Orobanche, Phelipanche spp.), are highly destructive agricultural weeds that pose a significant threat to global food security. Understanding how parasites sense strigolactones and other host-derived stimulants will catalyze the development of innovative chemical and biological control methods. This review synthesizes the recent discoveries of strigolactone receptors in parasitic Orobanchaceae, their signaling mechanism, and key steps in their evolution. 

Karrikins (KARs) are chemicals in smoke that can enhance germination of many plants. Lactuca sativa cv. Grand Rapids (lettuce), germinates in the presence of nanomolar karrikinolide (KAR1). We found that lettuce is much less responsive to KAR2 or a mixture of synthetic strigolactone analogs, rac-GR24. We investigated the molecular basis of selective and sensitive KAR1 perception in lettuce. The lettuce genome contains two copies of KARRIKIN INSENSITIVE2 (KAI2), a receptor that is required for KAR responses in Arabidopsis thaliana. LsKAI2b is more highly expressed than LsKAI2a in dry achenes and during early stages of seed imbibition. Through cross-species complementation assays in Arabidopsis we found that LsKAI2b confers robust responses to KAR1, but LsKAI2a does not. Therefore, LsKAI2b likely mediates KAR1 responses in lettuce. We compared homology models of the ligand-binding pockets of KAI2 proteins from lettuce and a fire follower, Emmenanthe penduliflora. This identified pocket residues 96, 124, 139, and 161 as candidates that influence the ligand-specificity of KAI2. Further support for the significance of these residues was found through a broader comparison of pocket residue conservation among 324 asterid KAI2 proteins. We tested the effects of substitutions at these four positions in Arabidopsis thaliana KAI2 and found that a broad array of responses to KAR1, KAR2, and rac-GR24 could be achieved. 

Karrikin (KAR) molecules found in smoke stimulate seed germination of many plant species that emerge after fire. Genetic studies in Arabidopsis thaliana have identified core components of the KAR signaling pathway, including an α/β-hydrolase, KARRIKIN INSENSITIVE2 (KAI2), that is required for KAR responses. Although KAI2 is often considered a KAR receptor, recent evidence suggests that KARs may require metabolism to become bioactive signals. In addition to sensing KARs or a KAR-derived signal, KAI2 is thought to recognize an unknown endogenous signal, KAI2 ligand (KL). We generated loss-of-function mutations in KARRIKIN-UP-REGULATED F-BOX1 ( KUF1 ), which is a transcriptional marker of KAR/KL signaling in A. thaliana and other plants. The kuf1 mutant in Arabidopsis shows several phenotypes that are consistent with enhanced activity of the KAI2 pathway, including reduced hypocotyl elongation, enhanced cotyledon expansion in light-grown seedlings, increased root hair density and elongation, and differential expression of KAR/KL-responsive transcriptional markers. Seedling phenotypes of kuf1 are dependent on KAI2 and its signaling partner MORE AXILLARY GROWTH2 (MAX2). Furthermore, kuf1 mutants are hypersensitive to KAR 1 , but not to other molecules that can signal through KAI2 such as GR24. This implies that kuf1 does not increase the overall responsiveness of the KAI2-dependent signaling pathway, but specifically affects the ability of KAI2 to detect certain signals. We hypothesize that KUF1 imposes feedback inhibition of KL biosynthesis and KAR 1 metabolism. As an F-box protein, KUF1 likely participates in an E3 ubiquitin ligase complex that imposes this regulation through polyubiquitylation of a protein target(s).