Daniela S. Floss

Apocarotenoids: hormones, mycorrhizal metabolites and aroma volatiles.

Apocarotenoids are tailored from carotenoids by oxidative enzymes [carotenoid cleavage oxygenases (CCOs)], cleaving specific double bonds of the polyene chain. The cleavage products can act as hormones, signaling compounds, chromophores and scent/aroma constituents. Recent advances were the identification of strigolactones as apocarotenoids and the description of their novel role as shoot branching inhibitor hormones. Strigolactones are also involved in plant signaling to both harmful (parasitic weeds) and beneficial [arbuscular mycorrhizal (AM) fungi] rhizosphere residents. This review describes the progress in the characterization of CCOs, termed CCDs and NCEDs, in plants. It highlights the importance of sequential cleavage reactions of C40 carotenoid precursors, the apocarotenoid cleavage oxygenase (ACO) nature of several CCOs and the topic of compartmentation. Work on the biosynthesis of abundant C13 cyclohexenone and C14 mycorradicin apocarotenoids in mycorrhizal roots has revealed a new role of CCD1 as an ACO of C27 apocarotenoid intermediates, following their predicted export from plastid to cytosol. Manipulation of the AM-induced apocarotenoid pathway further suggests novel roles of C13 apocarotenoids in controlling arbuscule turnover in the AM symbiosis. CCD7 has been established as a biosynthetic crosspoint, controlling both strigolactone and AM-induced C13 apocarotenoid biosynthesis. Interdependence of the two apocarotenoid pathways may thus play a role in AM-mediated reduction of parasitic weed infestations. Potential scenarios of C13 scent/aroma volatile biogenesis are discussed, including the novel mechanism revealed from mycorrhizal roots. The recent progress in apocarotenoid research opens up new perspectives for fundamental work, but has also great application potential for the horticulture, food and fragrance industries.

DELLA proteins regulate arbuscule formation in arbuscular mycorrhizal symbiosis

Significance Arbuscular mycorrhizal (AM) symbiosis is a mutualistic interaction formed between most land plants and soil fungi. During symbiosis the fungus develops branched hyphae, known as arbuscules, inside the root cortical cells. Arbuscules are critical to the symbiosis and function in phosphate delivery to the plant. Here we show that arbuscule formation is regulated by DELLA proteins. DELLA proteins are negative regulators of gibberellic acid (GA) signaling and repress plant growth and development. Our data provide insights into regulation of arbuscule formation and identify a potential mechanism by which the plant can coordinate the symbiosis with its growth and nutrient status. Most flowering plants are able to form endosymbioses with arbuscular mycorrhizal fungi. In this mutualistic association, the fungus colonizes the root cortex and establishes elaborately branched hyphae, called arbuscules, within the cortical cells. Arbuscule development requires the cellular reorganization of both symbionts, and the resulting symbiotic interface functions in nutrient exchange. A plant symbiosis signaling pathway controls the development of the symbiosis. Several components of the pathway have been identified, but transcriptional regulators that control downstream pathways for arbuscule formation are still unknown. Here we show that DELLA proteins, which are repressors of gibberellic acid (GA) signaling and function at the nexus of several signaling pathways, are required for arbuscule formation. Arbuscule formation is severely impaired in a Medicago truncatula Mtdella1/Mtdella2 double mutant; GA treatment of wild-type roots phenocopies the della double mutant, and a dominant DELLA protein (della1-Δ18) enables arbuscule formation in the presence of GA. Ectopic expression of della1-Δ18 suggests that DELLA activity in the vascular tissue and endodermis is sufficient to enable arbuscule formation in the inner cortical cells. In addition, expression of della1-Δ18 restores arbuscule formation in the symbiosis signaling pathway mutant cyclops/ipd3, indicating an intersection between DELLA and symbiosis signaling for arbuscule formation. GA signaling also influences arbuscule formation in monocots, and a Green Revolution wheat variety carrying dominant DELLA alleles shows enhanced colonization but a limited growth response to arbuscular mycorrhizal symbiosis.

RNA Interference-Mediated Repression of MtCCD1 in Mycorrhizal Roots of Medicago truncatula Causes Accumulation of C27 Apocarotenoids, Shedding Light on the Functional Role of CCD1

Tailoring carotenoids by plant carotenoid cleavage dioxygenases (CCDs) generates various bioactive apocarotenoids. Recombinant CCD1 has been shown to catalyze symmetrical cleavage of C40 carotenoid substrates at 9,10 and 9′,10′ positions. The actual substrate(s) of the enzyme in planta, however, is still unknown. In this study, we have carried out RNA interference (RNAi)-mediated repression of a Medicago truncatula CCD1 gene in hairy roots colonized by the arbuscular mycorrhizal (AM) fungus Glomus intraradices. As a consequence, the normal AM-mediated accumulation of apocarotenoids (C13 cyclohexenone and C14 mycorradicin derivatives) was differentially modified. Mycorradicin derivatives were strongly reduced to 3% to 6% of the controls, while the cyclohexenone derivatives were only reduced to 30% to 47%. Concomitantly, a yellow-orange color appeared in RNAi roots. Based on ultraviolet light spectra and mass spectrometry analyses, the new compounds are C27 apocarotenoic acid derivatives. These metabolic alterations did not lead to major changes in molecular markers of the AM symbiosis, although a moderate shift to more degenerating arbuscules was observed in RNAi roots. The unexpected outcome of the RNAi approach suggests C27 apocarotenoids as the major substrates of CCD1 in mycorrhizal root cells. Moreover, literature data implicate C27 apocarotenoid cleavage as the general functional role of CCD1 in planta. A revised scheme of plant carotenoid cleavage in two consecutive steps is proposed, in which CCD1 catalyzes only the second step in the cytosol (C27 → C14 + C13), while the first step (C40 → C27 + C13) may be catalyzed by CCD7 and/or CCD4 inside plastids.

Suppression of Arbuscule Degeneration in Medicago truncatula phosphate transporter4 Mutants Is Dependent on the Ammonium Transporter 2 Family Protein AMT2;3

Analyses of phosphate and ammonium transporter mutants reveal differential requirements for these proteins during arbuscular mycorrhizal symbiosis depending on the plant nutrient status. During arbuscular mycorrhizal (AM) symbiosis, the plant gains access to phosphate (Pi) and nitrogen delivered by its fungal symbiont. Transfer of mineral nutrients occurs at the interface between branched hyphae called arbuscules and root cortical cells. In Medicago truncatula, a Pi transporter, PT4, is required for symbiotic Pi transport, and in pt4, symbiotic Pi transport fails, arbuscules degenerate prematurely, and the symbiosis is not maintained. Premature arbuscule degeneration (PAD) is suppressed when pt4 mutants are nitrogen-deprived, possibly the result of compensation by PT8, a second AM-induced Pi transporter. However, PAD is also suppressed in nitrogen-starved pt4 pt8 double mutants, negating this hypothesis and furthermore indicating that in this condition, neither of these symbiotic Pi transporters is required for symbiosis. In M. truncatula, three AMT2 family ammonium transporters are induced during AM symbiosis. To test the hypothesis that suppression of PAD involves AMT2 transporters, we analyzed double and triple Pi and ammonium transporter mutants. ATM2;3 but not AMT2;4 was required for suppression of PAD in pt4, while AMT2;4, but not AMT2;3, complemented growth of a yeast ammonium transporter mutant. In summary, arbuscule life span is influenced by PT4 and ATM2;3, and their relative importance varies with the nitrogen status of the plant.

Role of carotenoid cleavage dioxygenase 1 (CCD1) in apocarotenoid biogenesis revisited

Oxidative tailoring of C40 carotenoids by double bond-specific cleavage enzymes (carotenoid cleavage dioxygenases, CCDs) gives rise to various apocarotenoids. AtCCD1 generating C13 and C14 apocarotenoids and orthologous enzymes in other plants are the only CCDs acting in the cytosol, while the hitherto presumed C40 substrate is localized in the plastid. A new model for CCD1 action arising from a RNAi-mediated CCD1 gene silencing study in mycorrhizal hairy roots of Medicago truncatula may solve this contradiction. This approach unexpectedly resulted in the accumulation of C27 apocarotenoids but not C40 carotenoids suggesting C27 as the main substrates for CCD1 in planta. It further implies a consecutive two-step cleavage process, in which another CCD performs the primary cleavage of C40 to C27 in the plastid followed by C27 export and further cleavage by CCD1 in the cytosol. We compare the specificities and subcellular locations of the various CCDs and propose the plastidial CCD7 to be the first player in mycorrhizal apocarotenoid biogenesis.

Hyphal Branching during Arbuscule Development Requires Reduced Arbuscular Mycorrhiza1.

A plant transcription factor regulates the expression of genes necessary to enable arbuscular mycorrhizal fungi to develop branched hyphae in root cortical cells. During arbuscular mycorrhizal symbiosis, arbuscule development in the root cortical cell and simultaneous deposition of the plant periarbuscular membrane generate the interface for symbiotic nutrient exchange. The transcriptional changes that accompany arbuscule development are extensive and well documented. By contrast, the transcriptional regulators that control these programs are largely unknown. Here, we provide a detailed characterization of an insertion allele of Medicago truncatula Reduced Arbuscular Mycorrhiza1 (RAM1), ram1-3, which reveals that RAM1 is not necessary to enable hyphopodium formation or hyphal entry into the root but is essential to support arbuscule branching. In ram1-3, arbuscules consist only of the arbuscule trunk and in some cases, a few initial thick hyphal branches. ram1-3 is also insensitive to phosphate-mediated regulation of the symbiosis. Transcript analysis of ram1-3 and ectopic expression of RAM1 indicate that RAM1 regulates expression of EXO70I and Stunted Arbuscule, two genes whose loss of function impacts arbuscule branching. Furthermore, RAM1 regulates expression of a transcription factor Required for Arbuscule Development (RAD1). RAD1 is also required for arbuscular mycorrhizal symbiosis, and rad1 mutants show reduced colonization. RAM1 itself is induced in colonized root cortical cells, and expression of RAM1 and RAD1 is modulated by DELLAs. Thus, the data suggest that DELLAs regulate arbuscule development through modulation of RAM1 and RAD1 and that the precise transcriptional control essential to place proteins in the periarbuscular membrane is controlled, at least in part, by RAM1.

DELLA proteins regulate expression of a subset of AM symbiosis-induced genes in Medicago truncatula.

ABSTRACT The majority of the vascular flowering plants form symbiotic associations with fungi from the phylum Glomeromycota through which both partners gain access to nutrients, either mineral nutrients in the case of the plant, or carbon, in the case of the fungus.1 The association develops in the roots and requires substantial remodeling of the root cortical cells where branched fungal hyphae, called arbuscules, are housed in a new membrane-bound apoplastic compartment.2 Nutrient exchange between the symbionts occurs over this interface and its development and maintenance is critical for symbiosis. Previously, we showed that DELLA proteins, which are well known as repressors of gibberellic acid signaling, also regulate development of AM symbiosis and are necessary to enable arbuscule development.3 Furthermore, constitutive overexpression of a dominant DELLA protein (della1-Δ18) is sufficient to induce transcripts of several AM symbiosis-induced genes, even in the absence of the fungal symbiont.4 Here we further extend this approach and identify AM symbiosis genes that respond transcriptionally to constitutive expression of a dominant DELLA protein and also genes that do respond to this treatment. Additionally, we demonstrate that DELLAs interact with REQUIRED FOR ARBUSCULE DEVELOPMENT 1 (RAD1) which further extends our knowledge of GRAS factor complexes that have the potential to regulate gene expression during AM symbiosis.

How grow-and-switch gravitropism generates root coiling and root waving growth responses in Medicago truncatula

Significance Root waving, a growth response previously discussed predominantly in Arabidopsis, is reported in Medicago truncatula. Analogous to bacterial chemotaxis where Escherichia coli uses a “run-and-tumble” strategy to find sources of food, our experiments reveal a “grow-and-switch” gravitropic response in these root systems. This finding offers valuable insights into the strategies used for plants as they navigate heterogeneous environments in search of water and nutrient resources. Experimental studies show that plant root morphologies can vary widely from straight gravity-aligned primary roots to fractal-like root architectures. However, the opaqueness of soil makes it difficult to observe how environmental factors modulate these patterns. Here, we combine a transparent hydrogel growth medium with a custom built 3D laser scanner to directly image the morphology of Medicago truncatula primary roots. In our experiments, root growth is obstructed by an inclined plane in the growth medium. As the tilt of this rigid barrier is varied, we find Medicago transitions between randomly directed root coiling, sinusoidal root waving, and normal gravity-aligned morphologies. Although these root phenotypes appear morphologically distinct, our analysis demonstrates the divisions are less well defined, and instead, can be viewed as a 2D biased random walk that seeks the path of steepest decent along the inclined plane. Features of this growth response are remarkably similar to the widely known run-and-tumble chemotactic behavior of Escherichia coli bacteria, where biased random walks are used as optimal strategies for nutrient uptake.

Gene Silencing in Medicago truncatula roots using RNAi

Medicago truncatula is used widely as a model system for studies of root symbioses, interactions with parasitic nematodes and fungal pathogens, as well as studies of development and secondary metabolism. In Medicago truncatula as well as other legumes, RNA interference (RNAi) coupled with Agrobacterium rhizogenes-mediated root transformation, has been used very successfully for analyses of gene function in roots. One of the major advantages of this approach is the ease and relative speed with which transgenic roots can be generated. There are several methods, both for the generation of the RNAi constructs and the root transformation. Here we provide details of an RNAi and root transformation protocol that has been used successfully in M. truncatula and which can be scaled up to enable the analysis of several hundred constructs.

Control of Plastidial Isoprenoid Precursor Supply: Divergent 1-Deoxy-d-Xylulose 5-Phosphate Synthase (DXS) Isogenes Regulate the Allocation to Primary or Secondary Metabolism

Following the description of two separate pathways for isoprenoid precursor biosynthesis in plants, a new level of complexity has been introduced by the discovery of two divergent gene classes encoding the first enzyme of the plastidial methylerythritol phosphate (MEP) pathway. These nonredundant 1-deoxy- d -xylulose 5-phosphate synthase (DXS) isogenes are differentially expressed in such a way that DXS1 appears to serve housekeeping functions, whereas DXS2 is associated with the production of specialized (secondary) isoprenoids involved in ecological functions. Examples of the latter are apocarotenoid formation in roots colonized by arbuscular mycorrhizal fungi and mono- or diterpenoid biosynthesis in trichomes. Knockdown of DXS2 genes can specifically suppress secondary isoprenoid formation without affecting basic plant functions. Analyzing DXS isogenes along the progression of land plant evolution shows separation in structure and complementary expression already at the level of gymnosperms, which is maintained in all angiosperms except Arabidopsis.