Quantitative tandem affinity purification, an effective tool to investigate protein complex composition in plant hormone signaling : strigolactones in the spotlight

Phytohormones tightly regulate plant growth by integrating changing environmental and developmental cues. Although the key players have been identified in many plant hormonal pathways, the molecular mechanisms and mode of action of perception and signaling remain incompletely resolved. Characterization of protein partners of known signaling components provides insight into the formed protein complexes, but, unless quantification is involved, does not deliver much, if any, information about the dynamics of the induced or disrupted protein complexes. Therefore, in proteomics research, the discovery of what actually triggers, regulates or interrupts the composition of protein complexes is gaining importance. Here, tandem affinity purification coupled to mass spectrometry (TAP-MS) is combined with label-free quantification (LFQ) to a highly valuable tool to detect physiologically relevant, dynamic protein-protein interactions in Arabidopsis thaliana cell cultures. To demonstrate its potential, we focus on the signaling pathway of one of the most recently discovered phytohormones, strigolactones

Unraveling the MAX2 protein network in Arabidopsis thaliana : identification of the protein phosphatase PAPP5 as a novel MAX2 interactor

The 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

MAPs: cellular navigators for microtubule array orientations in Arabidopsis

Microtubules are subcellular nanotubes composed of alpha- and beta-tubulin that arise from microtubule nucleation sites and are mainly composed of gamma-tubulin complexes. Cell wall encased plant cells have evolved four distinct microtubule arrays that regulate cell division and expansion. Microtubule-associated proteins, the so called MAPs, construct, destruct and reorganize microtubule arrays thus regulating their spatiotemporal transitions during the cell cycle. By physically binding to microtubules and/or modulating their functions, MAPs control microtubule dynamic instability and/or interfilament cross talk. We survey the recent analyses of Arabidopsis MAPs such as MAP65, MOR1, CLASP, katanin, TON1, FASS, TRM, TAN1 and kinesins in terms of their effects on microtubule array organizations and plant development

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

Bioassays for the effects of strigolactones and other small molecules on root and root hair development

Growth and development of plant roots are highly dynamic and adaptable to environmental conditions. They are under the control of several plant hormone signaling pathways, and therefore root developmental responses can be used as bioassays to study the action of plant hormones and other small molecules. In this chapter, we present different procedures to measure root traits of the model plant Arabidopsis thaliana. We explain methods for phenotypic analysis of lateral root development, primary root length, root skewing and straightness, and root hair density and length. We describe optimal growth conditions for Arabidopsis seedlings for reproducible root and root hair developmental outputs; and how to acquire images and measure the different traits using image analysis with relatively low-tech equipment. We provide guidelines for a semiautomatic image analysis of primary root length, root skewing, and root straightness in Fiji and a script to automate the calculation of root angle deviation from the vertical and root straightness. By including mutants defective in strigolactone (SL) or KAI2 ligand (KL) synthesis and/or signaling, these methods can be used as bioassays for different SLs or SL-like molecules. In addition, the techniques described here can be used for studying seedling root system architecture, root skewing, and root hair development in any context

Histone deacetylases regulate MORE AXILLARY BRANCHED 2-dependent germination of Arabidopsis thaliana

Under specific conditions, the germination of Arabidopsis thaliana is dependent on the activation of the KARRIKIN INSENSITIVE 2 (KAI2) signalling pathway by the KAI2-dependent perception of karrikin (KAR) or the artificial strigolactone analogue rac-GR24. To regulate induction of germination, the KAI2 signalling pathway relies on MORE AXILLARY BRANCHED 2 (MAX2)-dependent ubiquitination and proteasomal degradation of the repressor protein SUPPRESSOR OF MAX2 1 (SMAX1). It is not yet known how degradation of SMAX1 proteins eventually results in the regulation of seed germination, but it has been hypothesized that SMAX1-LIKE (SMXL) generally function as transcriptional repressors through the recruitment of co-repressors TOPLESS (TPL) and TPL-related, which in turn interact with histone deacetylases (HDACs). Here we show the involvement of histone deacetylases HDA6, HDA9, HDA19, and HDT1 in MAX2-dependent germination of Arabidopsis, and more specifically, that HDA6 is required for the induction of DLK2 expression in response to rac-GR24 treatment

Exploring the protein-protein interaction landscape in plants

Protein-protein interactions (PPIs) represent an essential aspect of plant systems biology. Identification of key protein players and their interaction networks provide crucial insights into the regulation of plant developmental processes and into interactions of plants with their environment. Despite the great advance in the methods for the discovery and validation of PPIs, still several challenges remain. First, the PPI networks are usually highly dynamic, and the in vivo interactions are often transient and difficult to detect. Therefore, the properties of the PPIs under study need to be considered to select the most suitable technique, because each has its own advantages and limitations. Second, besides knowledge on the interacting partners of a protein of interest, characteristics of the interaction, such as the spatial or temporal dynamics, are highly important. Hence, multiple approaches have to be combined to obtain a comprehensive view on the PPI network present in a cell. Here, we present the progress in commonly used methods to detect and validate PPIs in plants with a special emphasis on the PPI features assessed in each approach and how they were or can be used for the study of plant interactions with their environment

The whats, the wheres and the hows of strigolactone action in the roots

Strigolactones control various aspects of plant development, including root architecture. Here, we review how strigolactones act in the root and survey the strigolactone specificity of signaling components that affect root development. Strigolactones are a group of secondary metabolites produced in plants that have been assigned multiple roles, of which the most recent is hormonal activity. Over the last decade, these compounds have been shown to regulate various aspects of plant development, such as shoot branching and leaf senescence, but a growing body of literature suggests that these hormones play an equally important role in the root. In this review, we present all known root phenotypes linked to strigolactones. We examine the expression and presence of the main players in biosynthesis and signaling of these hormones and bring together the available information that allows us to explain how strigolactones act to modulate the root system architecture

Design and visualization of second generation cyanoisoindole based fluorescent strigolactone analogs

Strigolactones (SLs) are a family of terpenoid allelochemicals that were recognized as plant hormones only a decade ago. They influence a myriad of both above- and belowground developmental processes, and are an important survival strategy for plants in nutrient-deprived soils. A rapidly emerging approach to gain knowledge on hormone signaling is the use of traceable analogs. A unique class of labeled SL analogs was constructed, where the original tricyclic lactone moiety of natural SLs is replaced by a fluorescent cyanoisoindole ring system. Biological evaluation as parasitic seed germination stimulant and hypocotyl elongation repressor proved the potency of the cyanoisoindole strigolactone analogs (CISA) to be comparable to the commonly accepted standard GR24. Additionally, via a SMXL6 protein degradation assay, we provided molecular evidence that the compounds elicit SL-like responses through the natural signaling cascade. All CISA analogs were shown to exhibit fluorescent properties, and the high quantum yield and Stokes shift of the pyrroloindole derivative CISA-7 also enabled in vivo visualization in plants. In contrast to the previously reported fluorescent analogs, CISA-7 displays a large similarity in shape and structure with natural SLs, which renders the analog a promising tracer to investigate the spatiotemporal distribution of SLs in plants and fungi