Transcriptional Regulation of Genes Involved in Zinc Uptake, Sequestration and Redistribution Following Foliar Zinc Application to Medicago sativa.

Zinc (Zn) is an essential micronutrient for plants and animals, and Zn deficiency is a widespread problem for agricultural production. Although many studies have been performed on biofortification of staple crops with Zn, few studies have focused on forages. Here, the molecular mechanisms of Zn transport in alfalfa (Medicago sativa L.) were investigated following foliar Zn applications. Zinc uptake and redistribution between shoot and root were determined following application of six Zn doses to leaves. Twelve putative genes encoding proteins involved in Zn transport (MsZIP1-7, MsZIF1, MsMTP1, MsYSL1, MsHMA4, and MsNAS1) were identified and changes in their expression following Zn application were quantified using newly designed RT-qPCR assays. These assays are the first designed specifically for alfalfa and resulted in being more efficient than the ones already available for Medicago truncatula (i.e., MtZIP1-7 and MtMTP1). Shoot and root Zn concentration was increased following foliar Zn applications ≥ 0.1 mg plant-1. Increased expression of MsZIP2, MsHMA4, and MsNAS1 in shoots, and of MsZIP2 and MsHMA4 in roots was observed with the largest Zn dose (10 mg Zn plant-1). By contrast, MsZIP3 was downregulated in shoots at Zn doses ≥ 0.1 mg plant-1. Three functional gene modules, involved in Zn uptake by cells, vacuolar Zn sequestration, and Zn redistribution within the plant, were identified. These results will inform genetic engineering strategies aimed at increasing the efficiency of crop Zn biofortification.

Direct transfer of zinc between plants is channelled by common mycorrhizal network of arbuscular mycorrhizal fungi and evidenced by changes in expression of zinc transporter genes in fungus and plant.

The role that common mycorrhizal networks (CMNs) play in plant-to-plant transfer of zinc (Zn) has not yet been investigated, despite the proved functions of arbuscular mycorrhizal fungi (AMF) in crop Zn acquisition. Here, two autotrophic Medicago truncatula plants were linked by a CMN formed by Rhizophagus irregularis. Plants were grown in vitro in physically separated compartments (Donor-C and Receiver-C) and their connection ensured only by CMN. A symbiosis-defective mutant of M. truncatula was used as control in Receiver-C. Plants in both compartments were grown on Zn-free medium, and only the leaves of the donor plants were Zn fertilized. A direct transfer of Zn was demonstrated from donor leaves to receiver shoots mediated by CMN. Direct transfer of Zn was supported by changes in the expression of fungal genes, RiZRT1 and RiZnT1, and plant gene MtZIP2 in roots and MtNAS1 in roots and shoots of the receiver plants. Moreover, Zn transfer was supported by the change in expression of MtZIP14 gene in AM fungal colonized roots. This work is the first evidence of a direct Zn transfer from a donor to a receiver plant via CMN, and of a triggering of transcriptional regulation of fungal-plant genes involved in Zn transport-related processes.

HyLength: a semi-automated digital image analysis tool for measuring the length of roots and fungal hyphae of dense mycelia.

In plant-fungus phenotyping, determining fungal hyphal and plant root lengths by digital image analysis can reduce labour and increase data reproducibility. However, the degree of software sophistication is often prohibitive and manual measuring is still used, despite being very time-consuming. We developed the HyLength tool for measuring the lengths of hyphae and roots in in vivo and in vitro systems. The HyLength was successfully validated against manual measures of roots and fungal hyphae obtained from all systems. Compared with manual methods, the HyLength underestimated Medicago sativa roots in the in vivo system and Rhizophagus irregularis hyphae in the in vitro system by about 12 cm per m and allowed to save about 1 h for a single experimental unit. As regards hyphae of R. irregularis in the in vivo system, the HyLength overestimated the length by about 21 cm per m compared with manual measures, but time saving was up to 20.5 h per single experimental unit. Finally, with hyphae of Aspergillus oryzae, the underestimation was about 8 cm per m with a time saving of about 10 min for a single germinating spore. By benchmarking the HyLength against the AnaMorf plugin of the ImageJ/Fiji, we found that the HyLength performed better for dense fungal hyphae, also strongly reducing the measuring time. The HyLength can allow measuring the length over a whole experimental unit, eliminating the error due to sub-area selection by the user and allowing processing a high number of samples. Therefore, we propose the HyLength as a useful freeware tool for measuring fungal hyphae of dense mycelia.