Semester

Spring

Date of Graduation

2019

Document Type

Dissertation

Degree Type

PhD

College

Davis College of Agriculture, Natural Resources and Design

Department

Division of Plant and Soil Sciences

Committee Chair

Vagner A. Benedito

Committee Co-Chair

Jonathan Cumming

Committee Member

Jonathan Cumming

Committee Member

Jianbo Yao

Committee Member

Daniel Panaccione

Committee Member

Nicole Waterland

Abstract

Legumes play a crucial role in sustainable agriculture because of their intrinsic ability to reduce atmospheric N2 into NH3 via symbiotic nitrogen fixation (SNF). SNF is carried out in the symbiosome, a quasi-organelle containing endosymbiotic rhizobial bacteria in the cytoplasm of infected cells of the nodule. Therefore, the bacteria are surrounded by a symbiosome membrane (SM), which is derived from the plant plasma membrane during infection. SNF requires constant nutritional exchanges between symbionts, including reduced carbon (dicarboxylates) from the plant for reduced nitrogen (NH4+) from the bacteroids. This exchange of nutrients and signals is fundamental to SNF and occurs through various transporters in several membranes, and critically through the SM. However, despite the fact that many nodule-specific or high-expressed putative transport genes have been identified in nodules at the genetic level, little is still known about their biochemical and physiological roles for SNF. Thus, we functionally characterized two transporter genes exclusively or highly expressed in the nodules of Medicago truncatula, a well-established legume model species: MtSLAH1 (Medtr4g049640, TCDB 2.A.16.5) of the Tellurite-resistance/Dicarboxylate transporter family, and MtMATE30 (Medtr7g082810, TCDB 2.A.66.1) of the Multidrug and toxic compound Extrusion family. There are seven SLAC (Slow Anion Channel associated) and 70 MATE (Multidrug and Toxic compound Extrusion) family members in the M. truncatula genome. MtSLAH1 is the SLAC transporter with highest expression value in nodules among seven members. Its expression is reduced 5-fold in 2 days after application of nitrate, a known SNF repressor. MtSLAH1 is highly expressed in infected cells and requires bacteroid differentiation for induction. MtSLAH1 channel permeability to dicarboxylates was tested by patch-clamp of Xenopus oocytes, but no current was detected. MtSLAH1 either requires a cofactor for activation or facilitates the efflux a different anion (e.g., nitrate, chloride). On the other hand, MtMATE30 is the MATE gene with highest expression in nodules. It is induced 100-fold by low nitrogen while strongly repressed by nitrate, suggesting a role in symbiotic nitrogen fixation. It starts to express in immature nodules (6 days post-inoculation, dpi), reaches its peak in young, mature nodules (10 dpi) and maintains consistent expression in older nodules. It is expressed in all nodule zones, except the meristem. MtMATE30 canonically belongs to a phylogenetic clade that includes transporters with affinity to alkaloids. Trigonelline is a widely distributed alkaloid and commonly found in legumes. Previous research showed that rhizobia are able to catabolize trigonelline by the trc gene located in the rhizobial pSym megaplasmid. We confirmed the presence of trigonelline in nodules of M. truncatula and showed MtMATE30 affinity to trigonelline in a heterologous bacteria system. Altogether, MtMATE30 may be participating in alkaloid metabolism of nodule cells, although its precise physiological role in symbiotic nitrogen fixation still requires further investigation. In the big picture, these two membrane transporters studied here are only two examples among thousands of transporters exclusively expressed in nodule. The legume research community needs to focus more efforts to understand how legumes and rhizobia communicate and cooperate to fix nitrogen in order to enhance SNF efficiency in legume crops, and possibly to eventually extend it into non-legume crops.

Download

Share

COinS