Integration of rice apocarotenoid profile and expression pattern of Carotenoid Cleavage Dioxygenases reveals a positive effect of ß-ionone on mycorrhization.

Carotenoids are susceptible to degrading processes initiated by oxidative cleavage reactions mediated by Carotenoid Cleavage Dioxygenases that break their backbone, leading to products called apocarotenoids. These carotenoid-derived metabolites include the phytohormones abscisic acid and strigolactones, and different signaling molecules and growth regulators, which are utilized by plants to coordinate many aspects of their life. Several apocarotenoids have been recruited for the communication between plants and arbuscular mycorrhizal (AM) fungi and as regulators of the establishment of AM symbiosis. However, our knowledge on their biosynthetic pathways and the regulation of their pattern during AM symbiosis is still limited. In this study, we generated a qualitative and quantitative profile of apocarotenoids in roots and shoots of rice plants exposed to high/low phosphate concentrations, and upon AM symbiosis in a time course experiment covering different stages of growth and AM development. To get deeper insights in the biology of apocarotenoids during this plant-fungal symbiosis, we complemented the metabolic profiles by determining the expression pattern of CCD genes, taking advantage of chemometric tools. This analysis revealed the specific profiles of CCD genes and apocarotenoids across different stages of AM symbiosis and phosphate supply conditions, identifying novel reliable markers at both local and systemic levels and indicating a promoting role of ß-ionone in AM symbiosis establishment.

Disruption of the rice 4-DEOXYOROBANCHOL HYDROXYLASE unravels specific functions of canonical strigolactones.

Strigolactones (SLs) regulate many developmental processes, including shoot-branching/tillering, and mediate rhizospheric interactions. SLs originate from carlactone (CL) and are structurally diverse, divided into a canonical and a noncanonical subfamily. Rice contains two canonical SLs, 4-deoxyorobanchol (4DO) and orobanchol (Oro), which are common in different plant species. The cytochrome P450 OsMAX1-900 forms 4DO from CL through repeated oxygenation and ring closure, while the homologous enzyme OsMAX1-1400 hydroxylates 4DO into Oro. To better understand the biological function of 4DO and Oro, we generated CRISPR/Cas9 mutants disrupted in OsMAX1-1400 or in both OsMAX1-900 and OsMAX1-1400. The loss of OsMAX1-1400 activity led to a complete lack of Oro and an accumulation of its precursor 4DO. Moreover, Os1400 mutants showed shorter plant height, panicle and panicle base length, but no tillering phenotype. Hormone quantification and transcriptome analysis of Os1400 mutants revealed elevated auxin levels and changes in the expression of auxin-related, as well as of SL biosynthetic genes. Interestingly, the Os900/1400 double mutant lacking both Oro and 4DO did not show the observed Os1400 architectural phenotypes, indicating their being a result of 4DO accumulation. Treatment of wild-type plants with 4DO confirmed this assumption. A comparison of the Striga seed germinating activity and the mycorrhization of Os900, Os900/1400, and Os1400 loss-of-function mutants demonstrated that the germination activity positively correlates with 4DO content while disrupting OsMAX1-1400 has a negative impact on mycorrhizal symbiosis. Taken together, our paper deciphers the biological function of canonical SLs in rice and reveals their particular contributions to establishing architecture and rhizospheric communications.

ZAXINONE SYNTHASE 2 regulates growth and arbuscular mycorrhizal symbiosis in rice.

Carotenoid cleavage, catalyzed by CAROTENOID CLEAVAGE DIOXYGENASEs (CCDs), provides signaling molecules and precursors of plant hormones. Recently, we showed that zaxinone, a apocarotenoid metabolite formed by the CCD ZAXINONE SYNTHASE (ZAS), is a growth regulator required for normal rice (Oryza sativa) growth and development. The rice genome encodes three OsZAS homologs, called here OsZAS1b, OsZAS1c, and OsZAS2, with unknown functions. Here, we investigated the enzymatic activity, expression pattern, and subcellular localization of OsZAS2 and generated and characterized loss-of-function CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats and associated protein 9)-Oszas2 mutants. We show that OsZAS2 formed zaxinone in vitro. OsZAS2 was predominantly localized in plastids and mainly expressed under phosphate starvation. Moreover, OsZAS2 expression increased during mycorrhization, specifically in arbuscule-containing cells. Oszas2 mutants contained lower zaxinone content in roots and exhibited reduced root and shoot biomass, fewer tillers, and higher strigolactone (SL) levels. Exogenous zaxinone application repressed SL biosynthesis and partially rescued the growth retardation of the Oszas2 mutant. Consistent with the OsZAS2 expression pattern, Oszas2 mutants displayed a lower frequency of arbuscular mycorrhizal colonization. In conclusion, OsZAS2 is a zaxinone-forming enzyme that, similar to the previously reported OsZAS, determines rice growth, architecture, and SL content, and is required for optimal mycorrhization.

Zaxinone synthase controls arbuscular mycorrhizal colonization level in rice.

The Oryza sativa (rice) carotenoid cleavage dioxygenase OsZAS was described to produce zaxinone, a plant growth-promoting apocarotenoid. A zas mutant line showed reduced arbuscular mycorrhizal (AM) colonization, but the mechanisms underlying this behavior are unknown. Here, we investigated how OsZAS and exogenous zaxinone treatment regulate mycorrhization. Micromolar exogenous supply of zaxinone rescued root growth but not the mycorrhizal defects of the zas mutant, and even reduced mycorrhization in wild-type and zas genotypes. The zas line did not display the increase in the level of strigolactones (SLs) that was observed in wild-type plants at 7 days post-inoculation with AM fungus. Moreover, exogenous treatment with the synthetic SL analog GR24 rescued the zas mutant mycorrhizal phenotype, indicating that the lower AM colonization rate of zas is caused by a deficiency in SLs at the early stages of the interaction, and indicating that during this phase OsZAS activity is required to induce SL production, possibly mediated by the Dwarf14-Like (D14L) signaling pathway. OsZAS is expressed in arbuscule-containing cells, and OsPT11prom::OsZAS transgenic lines, where OsZAS expression is driven by the OsPT11 promoter active in arbusculated cells, exhibit increased mycorrhization compared with the wild type. Overall, our results show that the genetic manipulation of OsZAS activity in planta leads to a different effect on AM symbiosis from that of exogenous zaxinone treatment, and demonstrate that OsZAS influences the extent of AM colonization, acting as a component of a regulatory network that involves SLs.

Arbuscular Mycorrhizal Symbiosis Differentially Affects the Nutritional Status of Two Durum Wheat Genotypes under Drought Conditions.

Durum wheat is one of the most important agricultural crops, currently providing 18% of the daily intake of calories and 20% of daily protein intake for humans. However, being wheat that is cultivated in arid and semiarid areas, its productivity is threatened by drought stress, which is being exacerbated by climate change. Therefore, the identification of drought tolerant wheat genotypes is critical for increasing grain yield and also improving the capability of crops to uptake and assimilate nutrients, which are seriously affected by drought. This work aimed to determine the effect of arbuscular mycorrhizal fungi (AMF) on plant growth under normal and limited water availability in two durum wheat genotypes (Svevo and Etrusco). Furthermore, we investigated how the plant nutritional status responds to drought stress. We found that the response of Svevo and Etrusco to drought stress was differentially affected by AMF. Interestingly, we revealed that AMF positively affected sulfur homeostasis under drought conditions, mainly in the Svevo cultivar. The results provide a valuable indication that the identification of drought tolerant plants cannot ignore their nutrient use efficiency or the impact of other biotic soil components (i.e., AMF).

Multi-omics approaches explain the growth-promoting effect of the apocarotenoid growth regulator zaxinone in rice.

The apocarotenoid zaxinone promotes growth and suppresses strigolactone biosynthesis in rice. To shed light on the mechanisms underlying its growth-promoting effect, we employed a combined omics approach integrating transcriptomics and metabolomics analysis of rice seedlings treated with zaxinone, and determined the resulting changes at the cellular and hormonal levels. Metabolites as well as transcripts analysis demonstrate that zaxinone application increased sugar content and triggered glycolysis, the tricarboxylic acid cycle and other sugar-related metabolic processes in rice roots. In addition, zaxinone treatment led to an increased root starch content and induced glycosylation of cytokinins. The transcriptomic, metabolic and hormonal changes were accompanied by striking alterations of roots at cellular level, which showed an increase in apex length, diameter, and the number of cells and cortex cell layers. Remarkably, zaxinone did not affect the metabolism of roots in a strigolactone deficient mutant, suggesting an essential role of strigolactone in the zaxinone growth-promoting activity. Taken together, our results unravel zaxinone as a global regulator of the transcriptome and metabolome, as well as of hormonal and cellular composition of rice roots. Moreover, they suggest that zaxinone promotes rice growth most likely by increasing sugar uptake and metabolism, and reinforce the potential of this compound in increasing rice performance.