Development of specialized transport pathways in Pisum sativum root nodules
Date
2018
Authors
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Publisher
University of Delaware
Abstract
Legumes, from the family Fabaceae, represent 30% of global primary crop produced, with grain legumes contributing approximately one third of humans’ dietary protein needs. Legumes have the unique ability to form a symbiotic relationship with nitrogen-fixing soil bacteria called rhizobia, to obtain one of the most limiting nutrients for plant growth, nitrogen. Of particular importance is their ability to replenish nitrogen in the soil with an estimated nitrogen sequestration greater than 40 million tons annually. The study of the Legume-Rhizobium symbiosis could help restore the Earth’s carbon-nitrogen balance and potentially allow us to induce the symbiosis in non-legume crops. ☐ Legume-rhizobia symbioses result in formation of a novel plant organ called a root nodule which houses the nitrogen-fixing bacteria. Components necessary for the formation, maintenance, and persistence of this organ must be transported to and from the nodule with great efficiency. Mature nodules are considered sink organs and require large amounts of imported sugar derived from photosynthesis to support the endosymbionts. In addition, the plant effectively manages export of assimilated organic nitrogen compounds from the organ. ☐ Few studies have examined transport within nitrogen-fixing nodules formed during the L-R symbiosis, particularly with regard to symplasmic transport, which is short-distance transport from cell-to-cell through intercellular channels called plasmodesmata (PD). The transport capacity of a functional mature nodule must support phloem loading and unloading of materials into and out of the organ. Paths of transport to and from infected cells must also be established for a productive symbiotic relationship. The recognized importance of symplasmic connectivity in sink organ formation and function suggests indeterminate nodules must continue to develop PD beyond nodule initiation to further support the symbiosis. ☐ Symplasmic connectivity of cells within the nodules’ central tissue was evaluated to further elucidate how the organ handles the fluxes of nutrient transport taking place. Connectivity of infected and uninfected cells was determined by quantification of the symplasts’ functional unit, plasmodesmata. TEM analysis of the frequency and distribution of these channels showed increased connections between adjacent uninfected cells and between uninfected and infected cells. These connections increased in frequency and complexity through the developmental gradient of an indeterminate nodule. The connections between two adjacent infected cells did not follow the same trend of increased PD connections nor channel complexity, even though large amounts of nitrogen are exported and sugar requirements are higher in these cell types. Interestingly, the greatest change in PD complexity was seen between infected-uninfected cell types and not adjacent uninfected cells, which suggests these cells become more interconnected as the organ matures. ☐ To determine if uninfected cells are active in symplasmic transport, a fluorescent tracer molecule was used to map the movement of water. Assessment of symplasmic flow using HPTS (8-Hydroxypyrene-1,3,6-Trisulfonic Acid, Trisodium Salt) revealed transport from the nodule vascular phloem into the parenchyma cell layer adjacent to the bundles. Dye then entered the vascular endodermal layer and radiated into the central nodule tissue, primarily through bands of uninfected cells. HPTS fluorescence was shown entering directly into each developmental zone and not exclusively into the nodule meristem. This suggests an additional symplasmic route is established during organ maturation. ☐ To map the nodule vasculature and uninfected cell networks, a novel clearing technique was developed to image deep into plant material. The goal was to develop and fine tune a method to facilitate deep-tissue imaging of intact plant organs or whole plants. Specifically, we aimed to develop an approach to retain fine cellular features in specimens, closely match the refractive indices of plant tissues, enhance light transmission through the sample, and preserve the ability to use common fluorescent stains and proteins. The plant-tissue clearing agent presented here overcomes all the major obstacles that have limited plant imaging to date, and it is, therefore, of broad use to the plant scientific community. ☐ The application of the clearing technique called ScaleP revealed a novel view of nodule vasculature branching. Imaging of cleared nodules showed uniform branching along the periphery of the nodule. In addition, rays of uninfected cells seen radiating from the periphery to the central tissue occurred in all developmental zones of the organ. Further studies using this clearing technique could reveal a developmental link with timing of vascular branching and would provide insight into the role uninfected tissue in the indeterminate root nodule.