Two cDNAs were isolated from the model legume Lotus japonicus that encode two proteins of the MIP (Major Intrinsic Protein) family: LIMP1 and LIMP2. Based on sequence comparisons, LIMP1 falls into the TIP (Tonoplast membrane Intrinsic Protein) subfamily of plant MIPs. LIMP1 mRNA is expressed predominately in nodules and roots, but the highest LIMP1 protein levels are found in roots. LIMP1 appears to be regulated by diurnal cycling conditions, with expression increasing during the day hours, but falling off during the night cycle. Functional analysis of LIMP1 shows the ability to flux water but not other solutes, indicating that it is a water-specific aquaporin. LIMP2 shares the highest sequence identity (68%) with soybean nodulin 26 and falls into the NIP (Nodulin-like Intrinsic Protein) subfamily. Nodulin 26 and LIMP2 share the ability to facilitate the transport of water as well as small-uncharged solutes (e.g. glycerol). Analysis of the tissue distribution of LIMP2 by Northern blot and immunolocalization reveal that the LIMP2 transcript is expressed only in nodule tissue and that this protein, like nodulin 26, is present on the symbiosome membrane.; Nodulin 26 and LIMP2 share the ability to be phosphorylated by a calcium dependent protein kinase (CDPK), suggesting that these proteins are targets of calcium-dependent regulation by phosphorylation. Using an antibody, which specifically recognizes the phosphorylated form of nodulin 26, phosphorylation was observed only in mature nodules with well differentiated infected zones and fully developed symbiosomes. Phosphorylation of nodulin 26 was low or undetectable in young immature nodules and in very old nodules in plants after flowering (100 days) even though there is no apparent change in protein levels. Using this phosphorylation-specific antibody it was also determined that in mature soybean nodules the inherent level of phosphorylation is stimulated in response to osmotic stress (drought and salinity), suggesting that this protein may be regulated as part of the adaptive response of nodules to water deprivation.; Taken together, these results show that Lotus japonicus nodules have two discreet MIP proteins, which likely play divergent roles in water homeostasis and stress adaptation within the nodule.
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