This work was carried with funding from Department of Biotechnology, Government of India.
FUNCTIONAL CHARACTERIZATION OF NODULIN 26 AND GAMMA TIP ORTHOLOGS FROM THE NODULES OF LOTUS JAPONICUS
J.F. Guenther, D.M. Roberts
Dept Biochem. Cell. Molecular. Biol., Univ. of Tennessee, Knoxville, TN 37996, USA
During the establishment of rhizobia/legume symbioses a subset of nodulins are targeted to the symbiosome membrane that encloses the rhizobia bacteroid. Among these proteins is nodulin 26 (Fortin et al. 1987), which constitutes 10-15% of the total symbiosome membrane protein (Weaver et al. 1991). Nodulin 26 is a member of the MIP superfamily, an ancient family of conserved integral membrane proteins. These proteins have been shown to mediate the flux of water, small uncharged solutes or both (multifunctional aquaglyceroporins) (Borgnia et al. 1999).
The fact that nodulin 26 is the major MIP in the symbiosome membrane argues for a selective role in symbiosis. Many roles have been suggested for nodulin 26 ranging from osmoregulation (via water or solute transport; Dean et al. 1999; Guenther, Roberts 2000) to metabolite transport (e.g. NH3; Niemietz, Tyerman 2000). Though much is known regarding nodulin 26 transport, its biological function remains unresolved. To address the biological role of nodulin 26 and other MIPs in the nodule, we have investigated these proteins in the genetically tractable model legume Lotus japonicus. Using RT-PCR, cDNAs encoding two MIPs were identified that are expressed in mature nitrogen-fixing nodules of L. japonicus. These proteins were designated LIMP 1 and LIMP 2 (Lotus Intrinsic Membrane Protein). LIMP 1 is a member of the TIP (Tonoplast Intrinsic Protein) subclass of plant MIPs and shows high sequence similarity to gamma TIP (78% identity) (Guenther, Roberts 2000). It is expressed at high levels in nodules and roots. Functional analysis by expression in Xenopus oocytes shows that LIMP 1 is a water-selective aquaporin.
In contrast, LIMP 2 shows high similarity to soybean nodulin 26 (68% identity). LIMP 2 shows nodule specific expression based on Northern blot analysis. Western blot with an anti-LIMP 2 antibody shows that it is localized to the L. japonicus symbiosome membrane. Further, similar to nodulin 26 (Weaver et al. 1991), LIMP 2 is phosphorylated on its carboxyl-terminal domain by a calcium-dependent protein kinase (CDPK) and is likely a regulatory target of this enzyme. Finally, LIMP 2, similar to nodulin 26 (Dean et al. 1999), is multifunctional since it is able to transport water as well as uncharged solutes such as glycerol upon expression in oocytes. Thus, two functionally distinct MIPs are expressed in L. japonicus nodules: LIMP2 which is an ortholog of soybean nodulin 26; and LIMP 1 which is a water-selective TIP present both in roots and nodules that likely plays a separate osmoregulatory role. Interestingly, TIP proteins have been localized to regions of the nodule cortex involved in the regulation of gas diffusion, raising the possibility that they serve in this role (Serraj et al. 1998). The identification of these two nodule MIPs in a genetically tractable legume will allow the investigation of their roles in nodule physiology. (Supported by USDA NRICGP 9703548 andNSF MCB-9904978.)
Borgnia M, Nielsen S, Engel A, Agre P (1999) Annu. Rev. Biochem. 68, 425-458 Dean RM, Rivers RL, Zeidel ML, Roberts DM (1999) Biochem. 38, 347-353 Fortin MG, Morrison NA, Verma DP (1987) Nucl. Acids Res. 15, 813-824 Guenther JF, Roberts DM (2000) Planta 210, 741-748 Niemietz CM, Tyerman SD (2000) FEBS Lett. 465, 110-114
Serraj R, Frange N, Maeshima M, Fleurat-Lessard P, Drevon JJ (1998) Planta 206, 681-684 Weaver CD, Crombie B, Stacey G, Roberts DM (1991) Plant Physiol. 95, 222-227
NITROGEN FIXATION ASSOCIATED WITH TUBERCULATE ECTOMYCORRHIZAE
L. Paul1, B. Chapman2, F.B. Holl1, C. Chanway1"3
'Faculty of Agricultural Sciences
2B.C. Ministry of Forests, Williams Lake, BC
3Faculty of Forestry, UBC, Vancouver, BC
Tuberculate ectomycorrhizae (TEM) are a unique form of mycorrhizae where the ectotrophic fungus forms tuber-like structures (tubercles) on roots of the host plant somewhat similar in appearance to legume root nodules. The function of tubercles has yet to be precisely determined. This study focuses on the possibility that TEM on lodgepole pine roots supply fixed nitrogen from diazotrophic bacteria known to reside within tubercles. The acetylene reduction assay (ARA) was used to quantify nitrogen fixation associated with pine TEM in situ. Nitrogen fixation ranged from 0-4.27 g/m3/yr depending on soil type and pine stand age. Three bacterial species were isolated from the inner matrix of tubercles using N-free media and identified using GC Fame and BIOLOG as Bacillus spp., Micrococcus luteus and Methylobacterium extorquens. In vitro ARA results were positive for Bacillus spp. and M. luteus, but not M. extorquens. Because TEM often have a pink cast, we looked for hemoglobin in extracts of TEM tissues, initially using Drabkin's Reagent and then by cellulose acetate electrophoresis with Ponceau S and o-dianisidine staining for protein and heme, respectively. Human hemoglobin was used as a comparative standard. Both assays indicated the presence of hemoglobin or a hemoglobin-like compound within TEM tissues.
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