Transcriptional Analysis in the Arabidopsis Roots Reveals New Regulators that Link rac-GR24 Treatment with Changes in Flavonol Accumulation, Root Hair Elongation and Lateral Root Density.

The synthetic strigolactone (SL) analog, rac-GR24, has been instrumental in studying the role of SLs as well as karrikins because it activates the receptors DWARF14 (D14) and KARRIKIN INSENSITIVE 2 (KAI2) of their signaling pathways, respectively. Treatment with rac-GR24 modifies the root architecture at different levels, such as decreasing the lateral root density (LRD), while promoting root hair elongation or flavonol accumulation. Previously, we have shown that the flavonol biosynthesis is transcriptionally activated in the root by rac-GR24 treatment, but, thus far, the molecular players involved in that response have remained unknown. To get an in-depth insight into the changes that occur after the compound is perceived by the roots, we compared the root transcriptomes of the wild type and the more axillary growth2 (max2) mutant, affected in both SL and karrikin signaling pathways, with and without rac-GR24 treatment. Quantitative reverse transcription (qRT)-PCR, reporter line analysis and mutant phenotyping indicated that the flavonol response and the root hair elongation are controlled by the ELONGATED HYPOCOTYL 5 (HY5) and MYB12 transcription factors, but HY5, in contrast to MYB12, affects the LRD as well. Furthermore, we identified the transcription factors TARGET OF MONOPTEROS 5 (TMO5) and TMO5 LIKE1 as negative and the Mediator complex as positive regulators of the rac-GR24 effect on LRD. Altogether, hereby, we get closer toward understanding the molecular mechanisms that underlay the rac-GR24 responses in the root.

The Response of the Root Proteome to the Synthetic Strigolactone GR24 in Arabidopsis.

Strigolactones are plant metabolites that act as phytohormones and rhizosphere signals. Whereas most research on unraveling the action mechanisms of strigolactones is focused on plant shoots, we investigated proteome adaptation during strigolactone signaling in the roots of Arabidopsis thaliana. Through large-scale, time-resolved, and quantitative proteomics, the impact of the strigolactone analog rac-GR24 was elucidated on the root proteome of the wild type and the signaling mutant more axillary growth 2 (max2). Our study revealed a clear MAX2-dependent rac-GR24 response: an increase in abundance of enzymes involved in flavonol biosynthesis, which was reduced in the max2-1 mutant. Mass spectrometry-driven metabolite profiling and thin-layer chromatography experiments demonstrated that these changes in protein expression lead to the accumulation of specific flavonols. Moreover, quantitative RT-PCR revealed that the flavonol-related protein expression profile was caused by rac-GR24-induced changes in transcript levels of the corresponding genes. This induction of flavonol production was shown to be activated by the two pure enantiomers that together make up rac-GR24. Finally, our data provide much needed clues concerning the multiple roles played by MAX2 in the roots and a comprehensive view of the rac-GR24-induced response in the root proteome.

The Whats, the Wheres and the Hows of strigolactone action in the roots.

MAIN CONCLUSION: 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.

Strigolactones spatially influence lateral root development through the cytokinin signaling network.

Strigolactones are important rhizosphere signals that act as phytohormones and have multiple functions, including modulation of lateral root (LR) development. Here, we show that treatment with the strigolactone analog GR24 did not affect LR initiation, but negatively influenced LR priming and emergence, the latter especially near the root-shoot junction. The cytokinin module ARABIDOPSIS HISTIDINE KINASE3 (AHK3)/ARABIDOPSIS RESPONSE REGULATOR1 (ARR1)/ARR12 was found to interact with the GR24-dependent reduction in LR development, because mutants in this pathway rendered LR development insensitive to GR24. Additionally, pharmacological analyses, mutant analyses, and gene expression analyses indicated that the affected polar auxin transport stream in mutants of the AHK3/ARR1/ARR12 module could be the underlying cause. Altogether, the data reveal that the GR24 effect on LR development depends on the hormonal landscape that results from the intimate connection with auxins and cytokinins, two main players in LR development.

Strigolactones as an auxiliary hormonal defence mechanism against leafy gall syndrome in Arabidopsis thaliana.

Leafy gall syndrome is the consequence of modified plant development in response to a mixture of cytokinins secreted by the biotrophic actinomycete Rhodococcus fascians. The similarity of the induced symptoms with the phenotype of plant mutants defective in strigolactone biosynthesis and signalling prompted an evaluation of the involvement of strigolactones in this pathology. All tested strigolactone-related Arabidopsis thaliana mutants were hypersensitive to R. fascians. Moreover, treatment with the synthetic strigolactone mixture GR24 and with the carotenoid cleavage dioxygenase inhibitor D2 illustrated that strigolactones acted as antagonistic compounds that restricted the morphogenic activity of R. fascians. Transcript profiling of the MORE AXILLARY GROWTH1 (MAX1), MAX2, MAX3, MAX4, and BRANCHED1 (BRC1) genes in the wild-type Columbia-0 accession and in different mutant backgrounds revealed that upregulation of strigolactone biosynthesis genes was triggered indirectly by the bacterial cytokinins via host-derived auxin and led to the activation of BRC1 expression, inhibiting the outgrowth of the newly developing shoots, a typical hallmark of leafy gall syndrome. Taken together, these data support the emerging insight that balances are critical for optimal leafy gall development: the long-lasting biotrophic interaction is possible only because the host activates a set of countermeasures-including the strigolactone response-in reaction to bacterial cytokinins to constrain the activity of R. fascians.

A fluorescent alternative to the synthetic strigolactone GR24.

Strigolactones have recently been implicated in both above- and below-ground developmental pathways in higher plants. To facilitate the molecular and chemical properties of strigolactones in vitro and in vivo, we have developed a fluorescent strigolactone molecule, CISA-1, synthesized via a novel method which was robust, high-yielding, and used simple starting materials. We demonstrate that CISA-1 has a broad range of known strigolactone activities and further report on an adventitious rooting assay in Arabidopsis which is a highly sensitive and rapid method for testing biological activity of strigolactone analogs. In this rooting assay and the widely used Orobanche germination assay, CISA-1 showed stronger biological activity than the commonly tested GR24. CISA-1 and GR24 were equally effective at inhibiting branching in Arabidopsis inflorescence stems. In both the branching and adventitious rooting assay, we also demonstrated that CISA-1 activity is dependent on the max strigolactone signaling pathway. In water methanol solutions, CISA-1 was about threefold more stable than GR24, which may contribute to the increased activity observed in the various biological tests.