MORE AXILLARY GROWTH Research Articles

Unraveling the MAX2 Protein Network in Arabidopsis thaliana: Identification of the Protein Phosphatase PAPP5 as a Novel MAX2 Interactor

AbstractThe F-box protein MORE AXILLARY GROWTH 2 (MAX2) is a central component in the signaling cascade of strigolactones (SLs) as well as of the smoke-derived karrikins (KARs) and the so far unknown endogenous KAI2 ligand (KL). The two groups of molecules are involved in overlapping and unique developmental processes, and signal-specific outcomes are attributed to perception by the paralogous α/β-hydrolases DWARF14 (D14) for SL and KARRIKIN INSENSITIVE 2/HYPOSENSITIVE TO LIGHT (KAI2/HTL) for KAR/KL. In addition, depending on which receptor is activated, specific members of the SUPPRESSOR OF MAX2 1 (SMAX1)-LIKE (SMXL) family control KAR/KL and SL responses. As proteins that function in the same signal transduction pathway often occur in large protein complexes, we aimed at discovering new players of the MAX2, D14, and KAI2 protein network by tandem affinity purification in Arabidopsis cell cultures. When using MAX2 as a bait, various proteins were copurified, among which were general components of the Skp1-Cullin-F-box complex and members of the CONSTITUTIVE PHOTOMORPHOGENIC 9 signalosome. Here, we report the identification of a novel interactor of MAX2, a type 5 serine/threonine protein phosphatase, designated PHYTOCHROME-ASSOCIATED PROTEIN PHOSPHATASE 5 (PAPP5). Quantitative affinity purification pointed at PAPP5 as being more present in KAI2 rather than in D14 protein complexes. In agreement, mutant analysis suggests that PAPP5 modulates KAR/KL-dependent seed germination under suboptimal conditions and seedling development. In addition, a phosphopeptide enrichment experiment revealed that PAPP5 might dephosphorylate MAX2 in vivo independently of the synthetic SL analog, rac-GR24. Together, by analyzing the protein complexes to which MAX2, D14, and KAI2 belong, we revealed a new MAX2 interactor, PAPP5, that might act through dephosphorylation of MAX2 to control mainly KAR/KL-related phenotypes and, hence, provide another link with the light pathway.

Strigolactone signaling regulates specialized metabolism in tobacco stems and interactions with stem-feeding herbivores.

Plants are attacked by herbivores, which often specialize on different tissues, and in response, have evolved sophisticated resistance strategies that involve different types of chemical defenses frequently targeted to different tissues. Most known phytohormones have been implicated in regulating these defenses, with jasmonates (JAs) playing a pivotal role in complex regulatory networks of signaling interactions, often generically referred to as “cross talk.” The newly identified class of phytohormones, strigolactones (SLs), known to regulate the shoot architecture, remain unstudied with regard to plant–herbivore interactions. We explored the role of SL signaling in resistance to a specialist weevil (Trichobaris mucorea) herbivore of the native tobacco, Nicotiana attenuata, that attacks the root–shoot junction (RSJ), the part of the plant most strongly influenced by alterations in SL signaling (increased branching). As SL signaling shares molecular components, such as the core F-box protein MORE AXILLARY GROWTH 2 (MAX2), with another new class of phytohormones, the karrikins (KARs), which promote seed germination and seedling growth, we generated transformed lines, individually silenced in the expression of NaMAX2, DWARF 14 (NaD14: the receptor for SL) and CAROTENOID CLEAVAGE DIOXYGENASE 7 (NaCCD7: a key enzyme in SL biosynthesis), and KARRIKIN INSENSITIVE 2 (NaKAI2: the KAR receptor). The mature stems of all transgenic lines impaired in the SL, but not the KAR signaling pathway, overaccumulated anthocyanins, as did the stems of plants attacked by the larvae of weevil, which burrow into the RSJs to feed on the pith of N. attenuata stems. T. mucorea larvae grew larger in the plants silenced in the SL pathway, but again, not in the KAI2-silenced plants. These phenotypes were associated with elevated JA and auxin (indole-3-acetic acid [IAA]) levels and significant changes in the accumulation of defensive compounds, including phenolamides and nicotine. The overaccumulation of phenolamides and anthocyanins in the SL pathway–silenced plants likely resulted from antagonism between the SL and JA pathway in N. attenuata. We show that the repressors of SL signaling, suppressor of max2-like (NaSMXL6/7), and JA signaling, jasmonate zim-domain (NaJAZs), physically interact, promoting NaJAZb degradation and releasing JASMONATE INSENSITIVE 1 (JIN1/MYC2) (NaMYC2), a critical transcription factor promoting JA responses. However, the increased performance of T. mucorea larvae resulted from lower pith nicotine levels, which were inhibited by increased IAA levels in SL pathway–silenced plants. This inference was confirmed by decapitation and auxin transport inhibitor treatments that decreased pith IAA and increased nicotine levels. In summary, SL signaling tunes specific sectors of specialized metabolism in stems, such as phenylpropanoid and nicotine biosynthesis, by tailoring the cross talk among phytohormones, including JA and IAA, to mediate herbivore resistance of stems. The metabolic consequences of the interplay of SL, JA, and IAA signaling revealed here could provide a mechanism for the commonly observed pattern of herbivore tolerance/resistance trade-offs.

The karrikin signaling regulator SMAX1 controls Lotus japonicus root and root hair development by suppressing ethylene biosynthesis

An evolutionarily ancient plant hormone receptor complex comprising the α/β-fold hydrolase receptor KARRIKIN INSENSITIVE 2 (KAI2) and the F-box protein MORE AXILLARY GROWTH 2 (MAX2) mediates a range of developmental responses to smoke-derived butenolides called karrikins (KARs) and to yet elusive endogenous KAI2 ligands (KLs). Degradation of SUPPRESSOR OF MAX2 1 (SMAX1) after ligand perception is considered to be a key step in KAR/KL signaling. However, molecular events which regulate plant development downstream of SMAX1 removal have not been identified. Here we show that Lotus japonicus SMAX1 is specifically degraded in the presence of KAI2 and MAX2 and plays an important role in regulating root and root hair development. smax1 mutants display very short primary roots and elongated root hairs. Their root transcriptome reveals elevated ethylene responses and expression of ACC Synthase 7 (ACS7), which encodes a rate-limiting enzyme in ethylene biosynthesis. smax1 mutants release increased amounts of ethylene and their root phenotype is rescued by treatment with ethylene biosynthesis and signaling inhibitors. KAR treatment induces ACS7 expression in a KAI2-dependent manner and root developmental responses to KAR treatment depend on ethylene signaling. Furthermore, in Arabidopsis, KAR-induced root hair elongation depends on ACS7 Thus, we reveal a connection between KAR/KL and ethylene signaling in which the KAR/KL signaling module (KAI2-MAX2-SMAX1) regulates the biosynthesis of ethylene to fine-tune root and root hair development, which are important for seedling establishment at the beginning of the plant life cycle.

The SUPPRESSOR of MAX2 1 (SMAX1)-Like SMXL6, SMXL7 and SMXL8 Act as Negative Regulators in Response to Drought Stress in Arabidopsis.

Drought represents a major threat to crop growth and yields. Strigolactones (SLs) contribute to regulating shoot branching by targeting the SUPPRESSOR OF MORE AXILLARY GROWTH2 (MAX2)-LIKE6 (SMXL6), SMXL7 and SMXL8 for degradation in a MAX2-dependent manner in Arabidopsis. Although SLs are implicated in plant drought response, the functions of the SMXL6, 7 and 8 in the SL-regulated plant response to drought stress have remained unclear. Here, we performed transcriptomic, physiological and biochemical analyses of smxl6, 7, 8 and max2 plants to understand the basis for SMXL6/7/8-regulated drought response. We found that three D53 (DWARF53)-Like SMXL members, SMXL6, 7 and 8, are involved in drought response as the smxl6smxl7smxl8 triple mutants showed markedly enhanced drought tolerance compared to wild type (WT). The smxl6smxl7smxl8 plants exhibited decreased leaf stomatal index, cuticular permeability and water loss, and increased anthocyanin biosynthesis during dehydration. Moreover, smxl6smxl7smxl8 were hypersensitive to ABA-induced stomatal closure and ABA responsiveness during and after germination. In addition, RNA-sequencing analysis of the leaves of the D53-like smxl mutants, SL-response max2 mutant and WT plants under normal and dehydration conditions revealed an SMXL6/7/8-mediated network controlling plant adaptation to drought stress via many stress- and/or ABA-responsive and SL-related genes. These data further provide evidence for crosstalk between ABA- and SL-dependent signaling pathways in regulating plant responses to drought. Our results demonstrate that SMXL6, 7 and 8 are vital components of SL signaling and are negatively involved in drought responses, suggesting that genetic manipulation of SMXL6/7/8-dependent SL signaling may provide novel ways to improve drought resistance.

Transcriptional regulation of strigolactone signalling in Arabidopsis.

Plant hormones known as strigolactones control plant development and interactions between host plants and symbiotic fungi or parasitic weeds1-4. In Arabidopsis thaliana and rice, the proteins DWARF14 (D14), MORE AXILLARY GROWTH 2 (MAX2), SUPPRESSOR OF MAX2-LIKE 6, 7 and 8 (SMXL6,SMXL7 andSMXL8) and their orthologues form a complex upon strigolactone perception and play a central part in strigolactone signalling5-10. However, whether and how strigolactones activate downstream transcription remains largely unknown. Here we use a synthetic strigolactone to identify 401 strigolactone-responsive genes in Arabidopsis, and show that these plant hormones regulate shoot branching, leaf shape and anthocyanin accumulation mainly through transcriptional activation of the BRANCHED 1, TCP DOMAIN PROTEIN 1 and PRODUCTION OF ANTHOCYANIN PIGMENT 1 genes. We find that SMXL6 targets 729 genes in the Arabidopsis genome and represses the transcription of SMXL6, SMXL7 and SMXL8 by binding directly to their promoters, showing that SMXL6 serves as an autoregulated transcription factor to maintain the homeostasis of strigolactone signalling. These findings reveal an unanticipated mechanism through which a transcriptional repressor of hormone signalling can directly recognize DNA and regulate transcription in higher plants.

Structure-Function Analysis of SMAX1 Reveals Domains That Mediate Its Karrikin-Induced Proteolysis and Interaction with the Receptor KAI2.

Karrikins (KARs) are butenolides found in smoke that can influence germination and seedling development of many plants. The KAR signaling mechanism is hypothesized to be very similar to that of the plant hormone strigolactone (SL). Both pathways require the F-box protein MORE AXILLARY GROWTH2 (MAX2), and other core signaling components have shared ancestry. Putatively, KAR activates the receptor KARRIKIN INSENSITIVE2 (KAI2), triggering its association with the E3 ubiquitin ligase complex SCFMAX2 and downstream targets SUPPRESSOR OF MAX2 1 (SMAX1) and SMAX1-LIKE2 (SMXL2). Polyubiquitination and proteolysis of SMAX1 and SMXL2 then enable growth responses to KAR. However, many of the assumptions of this model have not been demonstrated. Therefore, we investigated the posttranslational regulation of SMAX1 from the model plant Arabidopsis (Arabidopsis thaliana). We find evidence that SMAX1 is degraded by KAI2-SCFMAX2 but is also subject to MAX2-independent turnover. We identify SMAX1 domains that are responsible for its nuclear localization, KAR-induced degradation, association with KAI2, and ability to interact with other SMXL proteins. KAI2 undergoes MAX2-independent degradation after KAR treatment, which we propose results from its association with SMAX1 and SMXL2. Finally, we discover an SMXL domain that mediates receptor-target interaction preferences in KAR and SL signaling, laying the foundation for understanding how these highly similar pathways evolved to fulfill different roles.

Strigolactone and Karrikin Signaling Pathways Elicit Ubiquitination and Proteolysis of SMXL2 to Regulate Hypocotyl Elongation in Arabidopsis.

Strigolactones (SLs) and karrikins (KARs) are related butenolide signaling molecules that control plant development. In Arabidopsis (Arabidopsis thaliana), they are recognized separately by two closely related receptors but use the same F-box protein MORE AXILLARY GROWTH2 (MAX2) for signal transduction, targeting different members of the SMAX1-LIKE (SMXL) family of transcriptional repressors for degradation. Both signals inhibit hypocotyl elongation in seedlings, raising the question of whether signaling is convergent or parallel. Here, we show that synthetic SL analog GR244DO enhanced the interaction between the SL receptor DWARF14 (D14) and SMXL2, while the KAR surrogate GR24 ent-5DS induced association of the KAR receptor KARRIKIN INSENSITIVE2 (KAI2) with SMAX1 and SMXL2. Both signals trigger polyubiquitination and degradation of SMXL2, with GR244DO dependent on D14 and GR24 ent-5DS dependent mainly on KAI2. SMXL2 is critical for hypocotyl responses to GR244DO and functions redundantly with SMAX1 in hypocotyl response to GR24 ent-5DS Furthermore, GR244DO induced response of D14-LIKE2 and KAR-UP F-BOX1 through SMXL2, whereas GR24 ent-5DS induced expression of these genes via both SMAX1 and SMXL2. These findings demonstrate that both SLs and KARs could trigger polyubiquitination and degradation of SMXL2, thus uncovering an unexpected but important convergent pathway in SL- and KAR-regulated gene expression and hypocotyl elongation.

Genes involved in strigolactone biosyntheses and their expression analyses in columnar apple and standard apple

Columnar apple is a valuable resource for genetic improvement of cultivated apples due to its special tree architecture. Strigolactones (SLs) are a novel class of plant hormones controlling shoot branching. The content of SLs is higher in columnar apple than in standard apples. In this study, the members of major gene families involved in SLs biosynthesis and signaling were identified from apple genomic sequences and their expression profiles were characterized in columnar and standard apples using reverse transcription quantitative polymerase chain reactions. In comparison with standard apple, the higher expressions of MORE AXILLARY GROWTH genes MdMAX3-1 and MdMAX4-4 were detected in both buds and shoots of columnar apple but the expression of DWARF gene MdD53-4 showed a lower expression in columnar apple. Overexpression of Columnar gene MdCo31 in tobacco increased SLs content and weakened the inhibition of SLs signal transduction by increasing expression of MAX3 and down-regulating the transcription of D53. Thus MdCo31 could be a strong candidate gene for the control of columnar habit.

Comparing and Contrasting the Multiple Roles of Butenolide Plant Growth Regulators: Strigolactones and Karrikins in Plant Development and Adaptation to Abiotic Stresses.

Strigolactones (SLs) and karrikins (KARs) are both butenolide molecules that play essential roles in plant growth and development. SLs are phytohormones, with SLs having known functions within the plant they are produced in, while KARs are found in smoke emitted from burning plant matter and affect seeds and seedlings in areas of wildfire. It has been suggested that SL and KAR signaling may share similar mechanisms. The α/β hydrolases DWARF14 (D14) and KARRIKIN INSENSITIVE 2 (KAI2), which act as receptors of SL and KAR, respectively, both interact with the F-box protein MORE AXILLARY GROWTH 2 (MAX2) in order to target SUPPRESSOR OF MAX2 1 (SMAX1)-LIKE/D53 family members for degradation via the 26S proteasome. Recent reports suggest that SLs and/or KARs are also involved in regulating plant responses and adaptation to various abiotic stresses, particularly nutrient deficiency, drought, salinity, and chilling. There is also crosstalk with other hormone signaling pathways, including auxin, gibberellic acid (GA), abscisic acid (ABA), cytokinin (CK), and ethylene (ET), under normal and abiotic stress conditions. This review briefly covers the biosynthetic and signaling pathways of SLs and KARs, compares their functions in plant growth and development, and reviews the effects of any crosstalk between SLs or KARs and other plant hormones at various stages of plant development. We also focus on the distinct responses, adaptations, and regulatory mechanisms related to SLs and/or KARs in response to various abiotic stresses. The review closes with discussion on ways to gain additional insights into the SL and KAR pathways and the crosstalk between these related phytohormones.

Strigolactone Signaling Genes Showing Differential Expression Patterns in Arabidopsis max Mutants.

Strigolactone (SL) is a recently discovered class of phytohormone that inhibits shoot branching. The molecular mechanism underlying SL biosynthesis, perception, and signal transduction is vital to the plant branching phenotype. Some aspects of their biosynthesis, perception, and signaling include the role of four MORE AXILLARY GROWTH genes, MAX3, MAX4, MAX1, and MAX2. It is important to identify downstream genes that are involved in SL signaling. To achieve this, we studied the genomic aspects of the strigolactone biosynthesis pathway using microarray analysis of four max mutants. We identified SL signaling candidate genes that showed differential expression patterns in max mutants. More specifically, 1-AMINOCYCLOPROPANE-1-CARBOXYLATE SYNTHASE 4 (ACC4) and PROTEIN KINASE 3 (PKS3) displayed contrasting expression patterns, indicating a regulatory mechanism in SL signaling pathway to control different phenotypes apart from branching phenotype.

Genetic dissection of cell wall defects and the strigolactone pathway in Arabidopsis.

Defects in the biosynthesis and/or deposition of secondary plant cell wall polymers result in the collapse of xylem vessels causing a dwarfed plant stature and an altered plant architecture termed irregular xylem (irx) syndrome. For example, reduced xylan O‐acetylation causes strong developmental defects and increased freezing tolerance. Recently, we demonstrated that the irx syndrome in the trichome birefringence‐like 29/eskimo1 (tbl29/esk1) mutant is dependent on MORE AXILLARY GROWTH 4 (MAX4), a key enzyme in the biosynthesis of the phytohormone strigolactone (SL). In this report, we show that other xylan‐ and cellulose‐deficient secondary wall mutants exhibit increased freezing tolerance correlated with the irx syndrome. In addition, these phenotypes are also dependent on MAX4, suggesting a more general interaction between secondary wall defects and SL biosynthesis. In contrast, MAX4 does not play a role in developmental defects triggered by primary wall deficiencies, suggesting that the interaction is restricted to vascular tissue. Through a reverse genetics approach, the requirement of different components of the SL pathway impacting the irx syndrome in tbl29 was evaluated. Our results show that the tbl29‐associated irx phenotypes are dependent on the MAX3 and MAX4 enzymes, involved in the early steps of SL biosynthesis. In contrast, this signaling is independent on downstream enzymes in the biosynthesis and perception of SL such as MAX1 and MAX2.

Impairment in karrikin but not strigolactone sensing enhances root skewing in Arabidopsis thaliana.

SummaryRoots form highly complex systems varying in growth direction and branching pattern to forage for nutrients efficiently. Here mutations in the KAI2 (KARRIKIN INSENSITIVE) α/β‐fold hydrolase and the MAX2 (MORE AXILLARY GROWTH 2) F‐box leucine‐rich protein, which together perceive karrikins (smoke‐derived butenolides), caused alteration in root skewing in Arabidopsis thaliana. This phenotype was independent of endogenous strigolactones perception by the D14 α/β‐fold hydrolase and MAX2. Thus, KAI2/MAX2 effect on root growth may be through the perception of endogenous KAI2‐ligands (KLs), which have yet to be identified. Upon perception of a ligand, a KAI2/MAX2 complex is formed together with additional target proteins before ubiquitination and degradation through the 26S proteasome. Using a genetic approach, we show that SMAX1 (SUPPRESSOR OF MAX2‐1)/SMXL2 and SMXL6,7,8 (SUPPRESSOR OF MAX2‐1‐LIKE) are also likely degradation targets for the KAI2/MAX2 complex in the context of root skewing. In A. thaliana therefore, KAI2 and MAX2 act to limit root skewing, while kai2's gravitropic and mechano‐sensing responses remained largely unaffected. Many proteins are involved in root skewing, and we investigated the link between MAX2 and two members of the SKS/SKU family. Though KLs are yet to be identified in plants, our data support the hypothesis that they are present and can affect root skewing.

Crosstalk between the cytokinin and MAX2 signaling pathways in growth and callus formation of Arabidopsis thaliana

Cytokinin (CK) signaling has been shown to play important roles in callus formation and various developmental processes by analyzing different CK-responsive mutants, including the ahk2 ahk3 (AHK, Arabidopsis histidine kinase) double mutant. Recently, an F-box protein, called MAX2 (more axillary growth 2) was identified as a key component regulating many growth and developmental processes through the strigolactone and/or karrikin pathways. However, the function of MAX2 signaling in callus formation, seed size and yield, as well as the effects of its crosstalk with CK signaling on plant growth and development remain elusive. Here, we constructed the triple mutant ahk2 ahk3max2 and analyzed the callus formation and various phenotypic traits of all three max2, ahk2 ahk3 and ahk2 ahk3 max2 mutants along with wild-type (WT) during plant growth and development. We showed that MAX2 acted as a negative regulator of seed size, but positive regulator of callus formation and seed yield albeit at lower degree, as the CK receptor kinases. Importantly, our comparative analyses revealed interactive effects of CK and MAX2 pathways on primary root growth, hypocotyl elongation and shoot branching. However, these two pathways might independently regulate root hair growth, leaf development, leaf senescence, plant height, seed size, seed yield and callus formation. Our findings provide not only evidence for the involvement of MAX2 in regulating callus formation, seed size and seed yield, but also a better understanding of the relationship between CK and MAX2 signaling pathways in many key developmental processes across a plant's life.

The ability of plants to produce strigolactones affects rhizosphere community composition of fungi but not bacteria

Strigolactones are an important group of plant hormones. When released from roots, they act as signalling molecules that induce branching of arbuscular mycorrhizal hyphae. However, the extent to which they affect the rhizosphere microbiome is unknown. Filling this knowledge gap is important because the diversity and composition of the root-associated microbiome influence plant fitness. In this study, we hypothesised that strigolactone-producing plants harbour a different community of rhizosphere bacteria and fungi compared to plants whose strigolactone synthesis is impaired. To test this hypothesis, we compared the diversity of rhizosphere bacterial and fungal communities associated with wild-type Arabidopsis thaliana and a mutant impaired in the production of strigolactones due to a disruption of the MORE AXILLARY GROWTH 4 (MAX4) gene. Our results indicate that the plant's ability to produce strigolactone is significantly correlated with changes in the composition (beta diversity) of rhizosphere fungal but not bacterial communities. No differences in alpha diversity (richness and evenness) were observed for either bacterial or fungal communities between the rhizospheres of max4 and wild-type. Epicoccum nigrum, Penicillium, Fibulochlamys chilensis, Herpotrichiellaceae, Mycosphaerella and Mycosphaerellaceae were among the fungal taxa possibly attracted to or mostly influenced by strigolactones given that they were present at higher abundances in the rhizosphere of the wild-type compared to the mutant. Our study provides evidence that rhizosphere fungal diversity are more strongly affected than bacterial diversity by the plant's ability to produce strigolactones.

Functional Analysis of MAX2 in Phototropins-Mediated Cotyledon Flattening in Arabidopsis.

Phototropins (phot1 and phot2) are blue-light receptors that control cotyledon flattening and positioning under strong light; however, their functional redundancy restricts our understanding of the specific roles of phot2. To identify the factors responsible for phot2-dependent cotyledon flattening and growth, we screened for light-insensitive mutants among mutagenized phot1 mutants in Arabidopsis thaliana. The double mutant phot1 lea1 (leaf expansion associated 1), which is defective in cotyledon flattening and positioning but not the phototropic response was selected. This mutant phenotype could be alleviated by constitutively expressing MORE AXILLARY GROWTH 2 (MAX2), indicating that LEA1 was allelic to MAX2. The max2 mutants (max2-2 and max2-3) are defective in cotyledon flattening, which is similar to that of the phot1 phot2 mutants. Moreover, the amounts of MAX2 transcripts are inhibited in leaves of phot1 mutant. However, the additional disruption of PHOT1 gene in max2-2 or max2-3 did not affect their phenotype, including MAX2-mediated inhibition of hypocotyl elongation. By contrast, phototropins-mediated hypocotyl phototropism was not regulated by MAX2. Together, these results suggest that cotyledon flattening was mediated by both phototropins and MAX2 signaling, but the relationship between two pathways need further study.

Physcomitrella patens MAX2 characterization suggests an ancient role for this F-box protein in photomorphogenesis rather than strigolactone signalling.

Strigolactones (SLs) are key hormonal regulators of flowering plant development and are widely distributed amongst streptophytes. In Arabidopsis, SLs signal via the F-box protein MORE AXILLARY GROWTH2 (MAX2), affecting multiple aspects of development including shoot branching, root architecture and drought tolerance. Previous characterization of a Physcomitrella patens moss mutant with defective SL synthesis supports an ancient role for SLs in land plants, but the origin and evolution of signalling pathway components are unknown. Here we investigate the function of a moss homologue of MAX2, PpMAX2, and characterize its role in SL signalling pathway evolution by genetic analysis. We report that the moss Ppmax2 mutant shows very distinct phenotypes from the moss SL-deficient mutant. In addition, the Ppmax2 mutant remains sensitive to SLs, showing a clear transcriptional SL response in dark conditions, and the response to red light is also altered. These data suggest divergent evolutionary trajectories for SL signalling pathway evolution in mosses and vascular plants. In P.patens, the primary roles for MAX2 are in photomorphogenesis and moss early development rather than in SL response, which may require other, as yet unidentified, factors.

Identification and functional characterization of a MAX2 ortholog from switchgrass (Panicum virgatum L.)

Switchgrass (Panicum virgatum L.) is a sustainable cellulosic energy crop with high biomass yield on marginal soils. Tillering, an important agronomic characteristic related to biomass production in gramineous plants, is regulated by complex interacting factors, such as plant hormones. Strigolactones (SLs) comprise a novel class of plant hormones that inhibit shoot branching. The MORE AXILLARY GROWTH2 (MAX2)/DWARF 3 (D3)/RAMOSUS (RMS4) genes encode proteins involved in the SL signaling pathway in various plants. The switchgrass tetraploid genome likely contains two high-similarity MAX2 homologs, one of which is 6 bp longer than the other. The longest is named PvMAX2 and is the ortholog of MAX2 in Arabidopsis, D3 in rice, and RMS4 in petunia. PvMAX2 is expressed ubiquitously in switchgrass tissues, with higher expression levels observed in the stem and shoot. PvMAX2 gene expression is upregulated by GR24, a synthetic SL analog. Ectopic expression of PvMAX2 in the Arabidopsis max2 mutant rescued the dwarf and bushy phenotypes and small leaf size in the mutant, suggesting that functions of AtMAX2 in Arabidopsis are conserved in PvMAX2. Ectopic PvMAX2 expression also restored the wild-type primary root and hypocotyl length phenotypes and restored the response to GR24. These results indicate that PvMAX2 may play an important role in switchgrass tillering through the SL pathway.

A missense allele of KARRIKIN-INSENSITIVE2 impairs ligand-binding and downstream signaling in Arabidopsis thaliana

A smoke-derived compound, karrikin (KAR), and an endogenous but as yet unidentified KARRIKIN INSENSITIVE2 (KAI2) ligand (KL) have been identified as chemical cues in higher plants that impact on multiple aspects of growth and development. Genetic screening of light-signaling mutants in Arabidopsis thaliana has identified a mutant designated as ply2 (pleiotropic long hypocotyl2) that has pleiotropic light-response defects. In this study, we used positional cloning to identify the molecular lesion of ply2 as a missense mutation of KAI2/HYPOSENSITIVE TO LIGHT, which causes a single amino acid substitution, Ala219Val. Physiological analysis and genetic epistasis analysis with the KL-signaling components MORE AXILLARY GROWTH2 (MAX2) and SUPPRESSOR OF MAX2 1 suggested that the pleiotropic phenotypes of the ply2 mutant can be ascribed to a defect in KL-signaling. Molecular and biochemical analyses revealed that the mutant KAI2ply2 protein is impaired in its ligand-binding activity. In support of this conclusion, X-ray crystallography studies suggested that the KAI2ply2 mutation not only results in a narrowed entrance gate for the ligand but also alters the structural flexibility of the helical lid domains. We discuss the structural implications of the Ala219 residue with regard to ligand-specific binding and signaling of KAI2, together with potential functions of KL-signaling in the context of the light-regulatory network in Arabidopsis thaliana.

The tomato MAX1 homolog, SlMAX1, is involved in the biosynthesis of tomato strigolactones from carlactone.

Strigolactones (SLs) are rhizosphere signalling molecules exuded by plants that induce seed germination of root parasitic weeds and hyphal branching of arbuscular mycorrhiza. They are also phytohormones regulating plant architecture. MORE AXILLARY GROWTH 1 (MAX1) and its homologs encode cytochrome P450 (CYP) enzymes that catalyse the conversion of the strigolactone precursor carlactone to canonical strigolactones in rice (Oryza sativa), and to an SL-like compound in Arabidopsis. Here, we characterized the tomato (Solanum lycopersicum) MAX1 homolog, SlMAX1. The targeting induced local lesions in genomes method was used to obtain Slmax1 mutants that exhibit strongly reduced production of orobanchol, solanacol and didehydro-orobanchol (DDH) isomers. This results in a severe strigolactone mutant phenotype in vegetative and reproductive development. Transient expression of SlMAX1 - together with SlD27, SlCCD7 and SlCCD8 - in Nicotiana benthamiana showed that SlMAX1 catalyses the formation of carlactonoic acid from carlactone. Plant feeding assays showed that carlactone, but not 4-deoxy-orobanchol, is the precursor of orobanchol, which in turn is the precursor of solanacol and two of the three DDH isomers. Inhibitor studies suggest that a 2-oxoglutarate-dependent dioxygenase is involved in orobanchol biosynthesis from carlactone and that the formation of solanacol and DDH isomers from orobanchol is catalysed by CYPs.

Structural Basis of the Strigolactone Signaling through Plant SCF E3 complex

Strigolactones (SL) are plant hormones that play an essential role in the control of plant branching, and act as rhizospheric signals for communication with symbiotic fungi and parasitic plants. MORE AXILLARY GROWTH2 (MAX2/D3) and DWARF14 (D14), are crucial signalling components that regulate SL‐repressed plant branching. D14 is a α/β hydrolase that hydrolyses SL into an inactive intermediate. MAX2/D3 is an ubiquitin ligase that has a leucine‐rich (LRR) F‐box region, which interacts with D14 in a SL‐dependent fashion. Once formed the D14‐D3 complex can bind to the repressor DWARF53 (D53) for subsequent ubiquitination and proteasomal degradation. The D3‐D14‐D53 complex is proposed to be involved in SL signaling. However, the recruitment of D53 by the D3‐D14 complex and the role of SL perception and hydrolysis remains largely unclear. In this study, we determine the structures of rice MAX2/D3 and D14‐SL complexes. In addition we measure the hydrolytic activity of D14 with and with out its binding partners D3 and D53. We also measured the binding of these three components using ALPHA technology. Our structural and biochemical studies shed light on SL sensing mechanism that is largely regulated by MAX2/D3 ubiquitin ligase.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.