More Like this

1. A KARRIKIN INSENSITIVE2 paralog in lettuce mediates highly sensitive germination responses to karrikinolide

   https://doi.org/https://doi.org/10.1101/2021.10.13.464162  

   Martinez, Stephanie E.; Conn, Caitlin E.; Guercio, Angelica M.; Sepulveda, Claudia; Fiscus, Christopher J.; Koenig, Daniel; Shabek, Nitzan; Nelson, David C. (October 2021, bioRxiv)

   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. 

   more » « less
2. KARRIKIN UP-REGULATED F-BOX 1 (KUF1) imposes negative feedback regulation of karrikin and KAI2 ligand metabolism in Arabidopsis thaliana

   https://doi.org/10.1073/pnas.2112820119  

   Sepulveda, Claudia; Guzmán, Michael A.; Li, Qingtian; Villaécija-Aguilar, José Antonio; Martinez, Stephanie E.; Kamran, Muhammad; Khosla, Aashima; Liu, Wei; Gendron, Joshua M.; Gutjahr, Caroline; et al (March 2022, Proceedings of the National Academy of Sciences)

   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). 

   more » « less
3. Rapid analysis of strigolactone receptor activity in a Nicotiana benthamiana dwarf14 mutant

   https://doi.org/10.1002/pld3.389  

   White, Alexandra R. F.; Mendez, Jose A.; Khosla, Aashima; Nelson, David C. (March 2022, Plant Direct)

   Abstract DWARF14 (D14) is an ɑ/β‐hydrolase and receptor for the plant hormone strigolactone (SL) in angiosperms. Upon SL perception, D14 works with MORE AXILLARY GROWTH2 (MAX2) to trigger polyubiquitination and degradation of DWARF53(D53)‐type proteins in the SUPPRESSOR OF MAX2 1‐LIKE (SMXL) family. We used CRISPR‐Cas9 to generate knockout alleles of the two homoeologousD14genes in theNicotiana benthamianagenome. TheNbd14a,bdouble mutant had several phenotypes that are consistent with the loss of SL perception in other plants, including increased axillary bud outgrowth, reduced height, shortened petioles, and smaller leaves. A ratiometric fluorescent reporter system was used to monitor degradation of SMXL7 fromArabidopsis thaliana(AtSMXL7) after transient expression inN. benthamianaand treatment with the strigolactone analog GR24. AtSMXL7 was degraded after treatment with GR24^5DS, which has the stereochemical configuration of natural SLs, as well as its enantiomer GR24^ent‐5DS. InNbd14a,bleaves, AtSMXL7 abundance was unaffected byrac‐GR24 or either GR24 stereoisomer. Transient coexpression of AtD14 with the AtSMXL7 reporter inNbd14a,brestored the degradation response torac‐GR24, but required an active catalytic triad. We used this platform to evaluate the ability of several AtD14 mutants that had not been characterized in plants to target AtSMXL7 for degradation. 

   more » « less
4. Desmethyl butenolides are optimal ligands for karrikin receptor proteins

   https://doi.org/10.1111/nph.17224  

   Yao, Jiaren; Scaffidi, Adrian; Meng, Yongjie; Melville, Kim T.; Komatsu, Aino; Khosla, Aashima; Nelson, David C.; Kyozuka, Junko; Flematti, Gavin R.; Waters, Mark T. (February 2021, New Phytologist)

   Summary Strigolactones and karrikins are butenolide molecules that regulate plant growth. They are perceived by the α/β‐hydrolase DWARF14 (D14) and its homologue KARRIKIN INSENSITIVE2 (KAI2), respectively. Plant‐derived strigolactones have a butenolide ring with a methyl group that is essential for bioactivity. By contrast, karrikins are abiotic in origin, and the butenolide methyl group is nonessential. KAI2 is probably a receptor for an endogenous butenolide, but the identity of this compound remains unknown.Here we characterise the specificity of KAI2 towards differing butenolide ligands using genetic and biochemical approaches.We find that KAI2 proteins from multiple species are most sensitive to desmethyl butenolides that lack a methyl group. Desmethyl‐GR24 and desmethyl‐CN‐debranone are active by KAI2 but not D14. They are more potent KAI2 agonists compared with their methyl‐substituted reference compounds bothin vitroand in plants. The preference of KAI2 for desmethyl butenolides is conserved inSelaginella moellendorffiiandMarchantia polymorpha, suggesting that it is an ancient trait in land plant evolution.Our findings provide insight into the mechanistic basis for differential ligand perception by KAI2 and D14, and support the view that the endogenous substrates for KAI2 and D14 have distinct chemical structures and biosynthetic origins. 

   more » « less
5. Structural insights into rice KAI2 receptor provide functional implications for perception and signal transduction

   https://doi.org/10.1016/j.jbc.2024.107593  

   Guercio, Angelica M; Gilio, Amelia K; Pawlak, Jacob; Shabek, Nitzan (July 2024, Journal of Biological Chemistry)

   KAI2 receptors, classified as plant α/β hydrolase enzymes, are capable of perceiving smoke-derived butenolide signals and endogenous, yet unidentified KAI2-ligands (KLs). While the number of functional KAI2 receptors varies among land plant species, rice has only one KAI2 gene. Rice, a significant crop and representative of grasses, relies on KAI2-mediated Arbuscular mycorrhiza (AM) symbioses to flourish in traditionally arid and nutrient-poor environments. This study presents the first crystal structure of an active rice (Oryza sativa, Os) KAI2 hydrolase receptor. Our structural and biochemical analyses uncover grass-unique pocket residues influencing ligand sensitivity and hydrolytic activity. Through structure-guided analysis, we identify a specific residue whose mutation enables the increase or decrease of ligand perception, catalytic activity, and signal transduction. Furthermore, we investigate OsKAI2-mediated signaling by examining its ability to form a complex with its binding partner, the F-box protein DWARF3 (D3) ubiquitin ligase and subsequent degradation of the target substrate OsSMAX1, demonstrating the significant role of hydrophobic interactions in the OsKAI2-D3 interface. This study provides new insights into the diverse and pivotal roles of the OsKAI2 signaling pathway in the plant kingdom, particularly in grasses. 

   more » « less