The importance of auxin in plant organ development, including root nodule formation, is well known. The spatiotemporal distribution pattern of auxin during nodule development has been illustrated using auxin reporter constructs. However, our understanding of how this pattern is established and maintained remains elusive. Here, we studied how the auxin gradient is associated with the spatiotemporal expression patterns of known auxin biosynthesis and transport genes at different stages of nodule development in Medicago (Medicago truncatula). In addition, we examined the Medicago PIN-FORMED10 (MtPIN10) expression pattern and polar positioning on the cell membrane during nodule primordium development to investigate auxin flux. RNA interference and the application of auxin biosynthesis inhibitors were used to demonstrate the importance of auxin biosynthesis and transport at the initial stages of nodulation. Our results show that upon rhizobium inoculation before the first cell divisions, a specific subset of Medicago YUCCA (MtYUC) and MtPIN genes, as well as Medicago LIKE AUXIN RESISTANT2 (MtLAX2), are expressed in the pericycle and contribute to the creation of an auxin maximum. Overall, we demonstrate that the dynamic spatiotemporal expression of both MtYUC and MtPIN genes results in specific auxin outputs during the different stages of nodule primordia and nodule meristem formation.

Early sprouting is a main cause of onion spoilage during storage. However, limited knowledge is available on which factors trigger sprouting. Here, this was studied in the Hyfive and Exhibition cultivars, which largely differ in sprouting time. Sprouting progress was compared to the fructan and abscisic acid (ABA) profiles in the bulb scales and basal plates. Fructan concentrations decreased in the scales from harvest time onwards in the late-sprouting cultivar Hyfive, while remaining constant in the cultivar Exhibition until internal sprouting. In the basal plates, fructan concentrations increased in both cultivars from approximately one month after harvest, but reached maximum concentrations at moments that could not be related to the difference in internal sprouting. ABA levels generally decreased in the scales of both cultivars, while increasing in their basal plates. Nevertheless, for fructans, the measured variation in ABA concentrations was not consistently associated with differences in internal sprouting. A subsequent perturbation of internal sprouting by Maleic Hydrazide treatment in the cultivar Hyfive confirmed a lack of correlation. Altogether, this indicates that fructan and ABA levels in the scales and basal plate tissue change independent of internal sprouting and cannot be regarded as predictive markers for sprouting and storability.

Ethylene is a well-established negative regulator of nodulation, yet how ethylene biosynthesis and perception are spatially coordinated during early symbiotic signaling remains unresolved. Here, we investigate the dynamics of ethylene responses in Medicago (Medicago truncatula) using transcriptomics, promoter–reporter analyses, loss-of-function approaches and a synthetic reporter. We show that the activity of the ethylene-responsive EBSn reporter shifts from inner root tissues under nonsymbiotic conditions to the outer cortex and epidermis following rhizobial inoculation, revealing a spatial reprogramming of ethylene signaling. Among the 8 Medicago 1-AMINOCYCLOPROPANE-1-CARBOXYLIC ACID SYNTHASE (ACS) genes, MtACS3 is induced in outer root cell layers upon rhizobia application, while MtACS10 is repressed in the inner cortex and pericycle, mirroring the shift in ethylene perception. Functional analysis demonstrates that MtACS10 restricts nodule initiation, whereas MtACS3 modulates the number of infection threads, prevents nodule clustering, and contributes to the radial positioning of nodule primordia. Rhizobial induced ectopic ACS expression in the root interior counteracts MtACS10 repression and blocks nodulation, highlighting the requirement for spatially confined downregulation of ethylene biosynthesis. Together, these findings establish a framework in which localized shifts in ethylene biosynthesis, mediated by distinct Medicago ACS genes, balance infection and organogenesis while co-defining the spatial limits of the root susceptible zone.

The importance of auxin in plant organ development including root nodule formation is well established. Using auxin reporter constructs the spatiotemporal auxin distribution pattern during nodule development has previously been illustrated. However, our understanding of how this pattern is built-up and maintained still remains elusive. To this end, we studied how the auxin gradient visualized by DR5 expression patterns at different stages of nodule development in Medicago truncatula (Medicago), is correlated with the spatiotemporal expression patterns of known auxin biosynthesis and auxin transport genes. In addition, we record the MtPIN10-GFP expression pattern and polar positioning on the cell plasma membranes during nodule primordium development to investigate the auxin flux. RNA interference and the application of auxin synthesis blockers were used to demonstrate the relevance of biosynthesis and transport at the initial stages of the nodulation process. Our results show that upon rhizobium inoculation, preceding the first mitotic activity, a specific set of MtYUCs and MtPINs as well as MtLAX2 are expressed in the pericycle contributing to the creation of an auxin maximum. Overall, we demonstrate that dynamic spatiotemporal expression of both, MtYUCs and MtPINs, result in specific auxin outputs in subsequent stages of nodule primordia and nodule meristem formation.