DWARF14 Research Articles

Regulatory mechanisms of strigolactone perception in rice.

Strigolactones (SLs) are hormones essential for plant development and environmental responses. SL perception requires the formation of the complex composed of an SL receptor DWARF14 (D14), F-box protein D3, and transcriptional repressor D53, triggering ubiquitination and degradation of D53 to activate signal transduction. However, mechanisms of SL perception and their influence on plant architecture and environmental responses remain elusive and controversial. Here, we report that key residues at interfaces of the AtD14-D3-ASK1 complex are essential for the activation of SL perception, discover that overexpression of the D3-CTH motif negatively regulates SL perception to enhance tillering, and reveal the importance of phosphorylation and N-terminal disordered (NTD) domain in mediating ubiquitination and degradation of D14. Importantly, low nitrogen promotes phosphorylation and stabilization of D14 to repress rice tillering. These findings reveal a panorama of the activation, termination, and regulation of SL perception, which determines the plasticity of plant architecture in complex environments.

Novel Mechanisms of Strigolactone-Induced DWARF14 Degradation in Arabidopsis thaliana.

In angiosperms, the strigolactone (SL) receptor is the α/β hydrolase DWARF14 (D14) that, upon SL binding, undergoes conformational changes, triggers SL-dependent responses and hydrolyses SLs. SL signalling involves the formation of a complex between SL-bound D14, the E3-ubiquitin ligase SCFMAX2 and the transcriptional corepressors SMXL6/7/8, which become ubiquitinated and degraded by the proteasome. SL also destabilises the D14 receptor. The current model proposes that D14 degradation occurs after SMXLs ubiquitination via SCFMAX2 and proteasomal degradation. Using fluorescence and luminescence assays on transgenic lines expressing D14 fused to GREEN FLUORESCENT PROTEIN or LUCIFERASE, we showed that SL-induced D14 degradation may also occur independently of SCFMAX2 and/or SMXL6/7/8 through a proteasome-independent mechanism. Furthermore, SLs hydrolysis was not essential for triggering either D14 or SMXL7 degradation. The activity of mutant D14 proteins predicted to be non-functional for SL signalling was also examined, and their capability to bind SLs in vitro was studied using Differential Scanning Fluorimetry. Finally, we found that under certain conditions, the efficiency of D14 degradation was not aligned with that of SMXL7 degradation. These findings suggest a more complex regulatory mechanism governing D14 degradation than previously anticipated and provide novel insights into the dynamics of SL signalling in Arabidopsis.

The chloroplast pentatricopeptide repeat protein RCN22 regulates tiller number in rice by affecting sugar levels via the TB1–RCN22–RbcL module

As an important yield component, rice tiller number controls panicle number and determines grain yield. Regulation of rice tiller number by chloroplast pentatricopeptide repeat (PPR) proteins has not been reported previously. Here, we report the rice reduced culm number22 (rcn22) mutant, which produces few tillers owing to suppressed tiller bud elongation. Map-based cloning revealed that RCN22 encodes a chloroplast-localized P-type PPR protein. We found that RCN22 specifically binds to the 5′ UTR of RbcL mRNA (encoding the large subunit of Rubisco) and enhances its stability. The reduced abundance of RbcL mRNA in rcn22 leads to a lower photosynthetic rate and decreased sugar levels. Consequently, transcript levels of DWARF3 (D3) and TEOSINTE BRANCHED1 (TB1) (which encode negative regulators of tiller bud elongation) are increased, whereas protein levels of the positive regulator DWARF53 (D53) are decreased. Furthermore, high concentrations of sucrose can rescue the tiller bud growth defect of the rcn22 mutant. On the other hand, TB1 directly binds to the RCN22 promoter and downregulates its expression. The tb1/rcn22 double mutant shows a tillering phenotype similar to that of rcn22. Our results suggest that the TB1–RCN22–RbcL module plays a vital role in rice tiller bud elongation by affecting sugar levels.

SMXL5 attenuates strigolactone signaling in Arabidopsis thaliana by inhibiting SMXL7 degradation

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 ethylene-responsive-element binding-factor-associated amphiphilic repression (EAR) motif, which typically mediates interactions with the TOPLESS family of transcriptional corepressors. However, we found 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.

Perception of butenolides by Bacillus subtilis via the α/β hydrolase RsbQ

The regulation of behavioral and developmental decisions by small molecules is common to all domains of life. In plants, strigolactones and karrikins are butenolide growth regulators that influence several aspects of plant growth and development, as well as interactions with symbiotic fungi.1,2,3 DWARF14 (D14) and KARRIKIN INSENSITIVE2 (KAI2) are homologous enzyme-receptors that perceive strigolactones and karrikins, respectively, and that require hydrolase activity to effect signal transduction.4,5,6,7 RsbQ, a homolog of D14 and KAI2 from the gram-positive bacterium Bacillus subtilis, regulates growth responses to nutritional stress via the alternative transcription factor SigmaB (σB).8,9 However, the molecular function of RsbQ is unknown. Here, we show that RsbQ perceives butenolide compounds that are bioactive in plants. RsbQ is thermally destabilized by the synthetic strigolactone GR24 and its desmethyl butenolide equivalent dGR24. We show that, like D14 and KAI2, RsbQ is a functional butenolide hydrolase that undergoes covalent modification of the catalytic histidine residue. Exogenous application of both GR24 and dGR24 inhibited the endogenous signaling function of RsbQ invivo, with dGR24 being 10-fold more potent. Application of dGR24 to B.subtilis phenocopied loss-of-function rsbQ mutations and led to a significant downregulation of σB-regulated transcripts. We also discovered that exogenous butenolides promoted the transition from planktonic to biofilm growth. Our results suggest that butenolides may serve as inter-kingdom signaling compounds between plants and bacteria to help shape rhizosphere communities.

Conformational Dynamics of the D53-D3-D14 Complex in Strigolactone Signaling.

Strigolactones (SLs) play fundamental roles in regulating plant architecture, which is a major factor determining crop yield. The perception and signal transduction of SLs require the formation of a complex containing the receptor DWARF14 (D14), an F-box protein D3 and a transcriptional regulator D53 in an SL-dependent manner. Structural and biochemical analyses of D14 and its orthologues DAD2 and AtD14, D3, and the complexes of ASK1-D3-AtD14 and D3CTH-D14 have made great contributions to understanding the mechanisms of SL perception. However, structural analyses of D53 and the D53-D3-D14 holo-complex are challenging, and the biochemical mechanism underlying complex assembly remains poorly understood. Here, we found that apo-D53 was rather flexible and reconstituted the holo-complex containing D53, SKP1, D3, and D14 with rac-GR24. The cryo-EM structure of SKP1-D3-D14 in the presence of D53 was analyzed and superimposed on the crystal structure of ASK1-D3-AtD14 without D53. No large conformational rearrangement was observed but a 9 Å rotation appeared between D14 and AtD14. Using hydrogen-deuterium exchange monitored by mass spectrometry, we analyzed dynamic motifs of D14, D3 and D53 in the D53-SKP1-D3-D14 complex assembly process and further identified two potential interfaces in D53 that are located in the N and D2 domains, respectively. Together, our results uncovered the dynamic conformational changes and built a model of the holo-complex D53-SKP1-D3-D14, offering valuable information for the biochemical and genetic mechanisms of SL perception and signal transduction.

Desmethyl type germinone, a specific agonist for the HTL/KAI2 receptor, induces the Arabidopsis seed germination in a gibberellin-independent manner

DWARF14 (D14) and HTL/KAI2 (KAI2) are paralogous receptors in the α/β-hydrolase superfamily. D14 is the receptor for a class of plant hormones, strigolactones (SLs), and KAI2 is the receptor for the smoke-derived seed germination inducer, Karrikin (KAR), in Arabidopsis. Germinone (Ger) was previously reported as a KAI2 agonist with germination-inducing activity for thermo-inhibited Arabidopsis seed. However, Ger was not specific to KAI2, and could also bind to D14. It was reported that SL analogs with a desmethyl-type D-ring structure are specifically recognized by KAI2. On the basis of this observation, we synthesized a desmethyl-type germinone (dMGer). We found that dMGer is highly specific to KAI2. Moreover, dMGer induced Arabidopsis seed germination more effectively than did Ger. In addition, dMGer induced the seed germination of Arabidopsis in a manner independently of GA, a well-known germination inducer in plants.

Species-specific regulatory pathways of small RNAs play sophisticated roles in flower development in Dimocarpus longan Lour.

Flower development plays vital role in horticultural plants. Post-transcriptional regulation via small RNAs is important for plant flower development. To uncover post-transcriptional regulatory networks during the flower development in Dimocarpus longan Lour. ‘Shixia’, an economically important fruit crop in subtropical regions, we collected and analyzed sRNA deep-sequencing datasets and degradome libraries Apart from identifying miRNAs and phased siRNA generating loci (PHAS loci), 120 hairpin loci, producing abundant sRNAs, were identified by in-house protocols. Our results suggested that 56 miRNA-target pairs, 22 21-nt-PHAS loci, and 111 hairpin loci are involved in post-transcriptional gene silencing during longan reproductive development. Lineage-specific or species-specific post-transcriptional regulatory modules have been unveiled, including miR482-PHAS and miRN15. miR482-PHAS might be involved in longan flower development beyond their conserved roles in plant defense, and miRN15 is a novel miRNA likely associated with a hairpin locus (HPL-056) to regulate strigolactone receptor gene DWARF14 (D14) and the biogenesis of phasiRNAs from D14. These small RNAs are enriched in flower buds, suggesting they are likely involved in post-transcriptional regulatory networks essential for longan flower development via the strigolactone signaling pathway.

PROG1 acts upstream of LAZY1 to regulate rice tiller angle as a repressor

Rice tiller angle, as a component of plant architecture, affects rice grain yield via plant density. However, the molecular mechanism underlying rice tiller angle remains elusive. We report that the key domestication gene PROSTRATE GROWTH 1 (PROG1) controls rice tiller angle by regulating shoot gravitropism and LAZY1 (LA1)-mediated asymmetric distribution of auxin. Acting as a transcriptional repressor, PROG1 negatively regulates the expression of LA1 in light-grown rice seedlings. Overexpression of LA1 partially rescued the larger tiller angle of the PROG1 complementation transgenic plant (prog1-D). Double-mutant analysis showed that PROG1 acts upstream of LA1 to regulate shoot gravitropism and tiller angle. Mutation of Suppressors of lazy1 (SOL1), encoding DWARF3 (D3) acting in the strigolactone signal pathway, suppressed the large tiller angle of prog1-D by rescuing the transcription of LA1. The discovery of a light-sensitive PROG1-LA1 transcription regulatory module controlling rice shoot gravitropism and tiller angle sheds light on the genetic control of rice tiller angle.

The strigolactone receptor SlDWARF14 plays a role in photosynthetic pigment accumulation and photosynthesis in tomato.

Tomato DWARF14 regulates the development of roots, shoot branches and leaves, and also plays a role in photosynthetic pigment accumulation and photosynthetic capacity. Strigolactones (SLs) are a novel class of plant hormones. DWARF14 (D14) is the only SL receptor identified to date, but it is not functionally analyzed in tomato (Solanum lycopersicum). In the present study, we identified the potential SL receptor in tomato by bioinformatic analysis, which was designated as SlD14. SlD14 was expressed in roots, stems, flowers and developing fruits, with the highest expression level in leaves. sld14 mutant plants produced by the CRISPR/Cas9 system displayed reduced plant height and root biomass, increased shoot branching and altered leaf shape comparing with WT plants. The cytokinin biosynthetic gene ISOPENTENYLTRANSFERASE 3 (SlIPT3), auxin biosynthetic genes FLOOZY (SlFZY) and TRYPTOPHAN AMINOTRANSFERASE RELATED 1 (SlTAR1) and several auxin transport genes SlPINs, which are involved in branch formation, showed higher expression levels in the sld14 plant stem. In addition, sld14 plants exhibited light-green leaves, reduced chlorophyll and carotenoid contents, abnormal chloroplast structure and reduced photosynthetic capacity. Transcriptomic analysis showed that the transcript levels of six chlorophyll biosynthetic genes, three carotenoid biosynthetic genes and numerous chlorophyll a/b-binding protein genes were decreased in sld14 plants. These results suggest that tomato SL receptor gene SlD14 not only regulates the development of roots, shoot branches and leaves, but also plays a role in regulating photosynthetic pigment accumulation and photosynthetic capacity.

The miR528-D3 Module Regulates Plant Height in Rice by Modulating the Gibberellin and Abscisic Acid Metabolisms

Plant height, as one of the important agronomic traits of rice, is closely related to yield. In recent years, plant height-related genes have been characterized and identified, among which the DWARF3 (D3) gene is one of the target genes of miR528, and regulates rice plant height and tillering mainly by affecting strigolactone (SL) signal transduction. However, it remains unknown whether the miR528 and D3 interaction functions in controlling plant height, and the underlying regulatory mechanism in rice. In this study, we found that the plant height, internode length, and cell length of internodes of d3 mutants and miR528-overexpressing (OE-miR528) lines were greatly shorter than WT, D3-overexpressing (OE-D3), and miR528 target mimicry (OE-MIM528) transgenic plants. Knockout of D3 gene (d3 mutants) or miR528-overexpressing (OE-miR528) triggers a substantial reduction of gibberellin (GA) content, but a significant increase of abscisic acid (ABA) accumulation than in WT. The d3 and OE-miR528 transgenic plants were much more sensitive to GA, but less sensitive to ABA than WT. Moreover, the expression level of GA biosynthesis-related key genes, including OsCPS1, OsCPS2, OsKO2 and OsKAO was remarkably higher in OE-D3 plants, while the NECD2 expression, a key gene involved in ABA biosynthesis, was significantly higher in d3 mutants than in WT and OE-D3 plants. The results indicate that the miR528-D3 module negatively regulates plant height in rice by modulating the GA and ABA homeostasis, thereby further affecting the elongation of internodes, and resulting in lower plant height, which adds a new regulatory role to the D3-mediated plant height controlling in rice.

Masks Start to Drop: Suppressor of MAX2 1-Like Proteins Reveal Their Many Faces.

Although the main players of the strigolactone (SL) signaling pathway have been characterized genetically, how they regulate plant development is still poorly understood. Of central importance are the SUPPRESSOR OF MAX2 1-LIKE (SMXL) proteins that belong to a family of eight members in Arabidopsis thaliana, of which one subclade is involved in SL signaling and another one in the pathway of the chemically related karrikins. Through proteasomal degradation of these SMXLs, triggered by either DWARF14 (D14) or KARRIKIN INSENSITIVE2 (KAI2), several physiological processes are controlled, such as, among others, shoot and root architecture, seed germination, and seedling photomorphogenesis. Yet another clade has been shown to be involved in vascular development, independently of the D14 and KAI2 actions and not relying on proteasomal degradation. Despite their role in several aspects of plant development, the exact molecular mechanisms by which SMXLs regulate them are not completely unraveled. To fill the major knowledge gap in understanding D14 and KAI2 signaling, SMXLs are intensively studied, making it challenging to combine all the insights into a coherent characterization of these important proteins. To this end, this review provides an in-depth exploration of the recent data regarding their physiological function, evolution, structure, and molecular mechanism. In addition, we propose a selection of future perspectives, focusing on the apparent localization of SMXLs in subnuclear speckles, as observed in transient expression assays, which we couple to recent advances in the field of biomolecular condensates and liquid–liquid phase separation.

A conformational switch in the SCF-D3/MAX2 ubiquitin ligase facilitates strigolactone signalling.

Strigolactones (SLs) are a class of plant hormones that regulate numerous processes of growth and development. SL perception and signal activation involves interaction between F-box E3 ubiquitin ligase D3/MAX2 and DWARF14 (D14) α/β-hydrolase in a SL-dependent manner and targeting of D53/SMXL6/7/8 transcriptional repressors (SMXLs) for proteasome-mediated degradation. D3/MAX2 has been shown to exist in multiple conformational states in which the C-terminal helix (CTH) undergoes a closed-to-open dynamics and regulates D14 binding and SL perception. Despite the multiple modes of D3-D14 interactions found in vitro, the residues that regulate the conformational switch of D3/MAX2 CTH in targeting D53/SMXLs and the subsequent effect on SL signalling remain unclear. Here we elucidate the functional dynamics of ASK1-D3/MAX2 in SL signalling by leveraging conformational switch mutants in vitro and in plants. We report the crystal structure of a dislodged CTH of the ASK1-D3 mutant and demonstrate that disruptions in CTH plasticity via either CRISPR-Cas9 genome editing or expression of point mutation mutants result in impairment of SL signalling. We show that the conformational switch in ASK1-D3/MAX2 CTH directly regulates ubiquitin-mediated protein degradation. A dislodged conformation involved in D53/SMXLs SL-dependent recruitment and ubiquitination and an engaged conformation are required for the release of polyubiquitinated D53/SMXLs and subsequently D14 for proteasomal degradation. Finally, we uncovered an organic acid metabolite that can directly trigger the D3/MAX2 CTH conformational switch. Our findings unravel a new regulatory function of a SKP1-CUL1-F-box ubiquitin ligase in plant signalling.

KAI2 regulates seedling development by mediating light-induced remodelling of auxin transport.

Summary Photomorphogenic remodelling of seedling growth is a key developmental transition in the plant life cycle. The α/β‐hydrolase signalling protein KARRIKIN‐INSENSITIVE2 (KAI2), a close homologue of the strigolactone receptor DWARF14 (D14), is involved in this process, but it is unclear how the effects of KAI2 on development are mediated.Here, using a combination of physiological, pharmacological, genetic and imaging approaches in Arabidopsis thaliana (Heynh.) we show that kai2 phenotypes arise because of a failure to downregulate auxin transport from the seedling shoot apex towards the root system, rather than a failure to respond to light per se.We demonstrate that KAI2 controls the light‐induced remodelling of the PIN‐mediated auxin transport system in seedlings, promoting a reduction in PIN7 abundance in older tissues, and an increase of PIN1/PIN2 abundance in the root meristem. We show that removing PIN3, PIN4 and PIN7 from kai2 mutants, or pharmacological inhibition of auxin transport and synthesis, is sufficient to suppress most kai2 seedling phenotypes.We conclude that KAI2 regulates seedling morphogenesis by its effects on the auxin transport system. We propose that KAI2 is not required for the light‐mediated changes in PIN gene expression but is required for the appropriate changes in PIN protein abundance within cells.

Dual Role of Strigolactone Receptor Signaling Partner in Inhibiting Substrate Hydrolysis.

Plant branch and root growth relies on metabolism of the strigolactone (SL) hormone. The interaction between the SL molecule, Oryza sativa DWARF14 (D14) SL receptor, and D3 F-box protein has been shown to play a critical role in SL perception. Previously, it was believed that D3 only interacts with the closed form of D14 to induce downstream signaling, but recent experiments indicate that D3, as well as its C-terminal helix (CTH), can interact with the open form as well to inhibit strigolactone signaling. Two hypotheses for the CTH induced inhibition are that either the CTH affects the conformational ensemble of D14 by stabilizing catalytically inactive states or the CTH interacts with SLs in a way that prevents them from entering the binding pocket. In this study, we have performed molecular dynamics (MD) simulations to assess the validity of these hypotheses. We used an apo system with only D14 and the CTH to test the active site conformational stability and a holo system with D14, the CTH, and an SL molecule to test the interaction between the SL and CTH. Our simulations show that the CTH affects both active site conformation and the ability of SLs to move into the binding pocket. In the apo system, the CTH allosterically stabilized catalytic residues into their inactive conformation. In the holo system, significant interactions between SLs and the CTH hindered the ability of SLs to enter the D14 binding pocket. These two mechanisms account for the observed decrease in SL binding to D14 and subsequent ligand hydrolysis in the presence of the CTH.

Rapid analysis of strigolactone receptor activity in a Nicotiana benthamiana dwarf14 mutant.

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 homoeologous D14 genes in the Nicotiana benthamiana genome. The Nbd14a,b double 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 from Arabidopsis thaliana (AtSMXL7) after transient expression in N. benthamiana and treatment with the strigolactone analog GR24. AtSMXL7 was degraded after treatment with GR245DS, which has the stereochemical configuration of natural SLs, as well as its enantiomer GR24 ent‐5DS. In Nbd14a,b leaves, AtSMXL7 abundance was unaffected by rac‐GR24 or either GR24 stereoisomer. Transient coexpression of AtD14 with the AtSMXL7 reporter in Nbd14a,b restored the degradation response to rac‐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.

A structural homologue of the plant receptor D14 mediates responses to strigolactones in the fungal phytopathogen Cryphonectria parasitica.

Summary Strigolactones (SLs) are plant hormones and important signalling molecules required to promote arbuscular mycorrhizal (AM) symbiosis. While in plants an α/β‐hydrolase, DWARF14 (D14), was shown to act as a receptor that binds and cleaves SLs, the fungal receptor for SLs is unknown.Since AM fungi are currently not genetically tractable, in this study, we used the fungal pathogen Cryphonectria parasitica, for which gene deletion protocols exist, as a model, as we have previously shown that it responds to SLs. By means of computational, biochemical and genetic analyses, we identified a D14 structural homologue, CpD14.Molecular homology modelling and docking support the prediction that CpD14 interacts with and hydrolyses SLs. The recombinant CpD14 protein shows α/β hydrolytic activity in vitro against the SLs synthetic analogue GR24; its enzymatic activity requires an intact Ser/His/Asp catalytic triad. CpD14 expression in the d14‐1 loss‐of‐function Arabidopsis thaliana line did not rescue the plant mutant phenotype. However, gene inactivation by knockout homologous recombination reduced fungal sensitivity to SLs.These results indicate that CpD14 is involved in SLs responses in C. parasitica and strengthen the role of SLs as multifunctional molecules acting in plant–microbe interactions.

The strigolactone receptor D14 targets SMAX1 for degradation in response to GR24 treatment and osmotic stress

The effects of the phytohormone strigolactone (SL) and smoke-derived karrikins (KARs) on plants are generally distinct, despite the fact that they are perceived through very similar mechanisms. The homologous receptors DWARF14 (D14) and KARRIKIN-INSENSITIVE2 (KAI2), together with the F-box protein MORE AXILLARY GROWTH2 (MAX2), mediate SL and KAR responses, respectively, by targeting different SMAX1-LIKE (SMXL) family proteins for degradation. These mechanisms are putatively well-insulated, with D14-MAX2 targeting SMXL6, SMXL7, and SMXL8 and KAI2-MAX2 targeting SMAX1 and SMXL2 in Arabidopsis thaliana. Recent evidence challenges this model. We investigated whether D14 can target SMAX1 and whether this occurs naturally. Genetic analysis indicates that the SL analog GR24 promotes D14-SMAX1 crosstalk. Although D14 shows weaker interactions with SMAX1 than with SMXL2 or SMXL7, D14 mediates GR24-induced degradation of SMAX1 in plants. Osmotic stress triggers SMAX1 degradation, which is protective, through SL biosynthesis and signaling genes. Thus, D14-SMAX1 crosstalk may be beneficial and not simply a vestige of the evolution of the SL pathway.

Localized expression of the Dwarf14-like2a gene in rice roots on infection of arbuscular mycorrhizal fungus and hydrolysis of rac-GR24 by the encoded protein

ABSTRACT Strigolactones (SLs) are plant hormones that control diverse aspects of the shoot and root growth and are exuded into the soil as recruitment signals for arbuscular mycorrhizal (AM) fungi. SL signaling in plants is transduced via the α/β-hydrolase receptor Dwarf14 (D14). The D14 family consists of D14, Dwarf14-like (D14L), and Dwarf14-like 2 (D14L2) clades in rice. The D14L receptor is known to condition pre-symbiotic perception of AM fungi. In this study, it was found that the Dwarf14-like2a (D14L2a) gene expression was significantly induced by AM fungal colonization. The transcript of D14L2a appeared not only in mature arbuscule-containing cells but also in epidermal/cortical cells at an early colonization stage and near the elongating intercellular hyphae. D14L2a transcript was detected normally in mycorrhizal roots of str1-2 mutant that form stunted arbuscules, suggesting that the gene expression is independent of arbuscule development. Moreover, the recombinant D14L2a protein exhibited hydrolase activity of synthetic SL, rac-GR24. Based on these results, we discussed the role of D14L2 in the establishment of AM symbiosis.

The Physcomitrium (Physcomitrella) patens PpKAI2L receptors for strigolactones and related compounds function via MAX2-dependent and -independent pathways.

In angiosperms, the α/β hydrolase DWARF14 (D14), along with the F-box protein MORE AXILLARY GROWTH2 (MAX2), perceives strigolactones (SL) to regulate developmental processes. The key SL biosynthetic enzyme CAROTENOID CLEAVAGE DIOXYGENASE8 (CCD8) is present in the moss Physcomitrium patens, and PpCCD8-derived compounds regulate moss extension. The PpMAX2 homolog is not involved in the SL response, but 13 PpKAI2LIKE (PpKAI2L) genes homologous to the D14 ancestral paralog KARRIKIN INSENSITIVE2 (KAI2) encode candidate SL receptors. In Arabidopsis thaliana, AtKAI2 perceives karrikins and the elusive endogenous KAI2-Ligand (KL). Here, germination assays of the parasitic plant Phelipanche ramosa suggested that PpCCD8-derived compounds are likely noncanonical SLs. (+)-GR24 SL analog is a good mimic for PpCCD8-derived compounds in P. patens, while the effects of its enantiomer (-)-GR24, a KL mimic in angiosperms, are minimal. Interaction and binding assays of seven PpKAI2L proteins pointed to the stereoselectivity toward (-)-GR24 for a single clade of PpKAI2L (eu-KAI2). Enzyme assays highlighted the peculiar behavior of PpKAI2L-H. Phenotypic characterization of Ppkai2l mutants showed that eu-KAI2 genes are not involved in the perception of PpCCD8-derived compounds but act in a PpMAX2-dependent pathway. In contrast, mutations in PpKAI2L-G, and -J genes abolished the response to the (+)-GR24 enantiomer, suggesting that PpKAI2L-G, and -J proteins are receptors for moss SLs.