Functional characterization of polyol/monosaccharide transporter 1 in Lotus japonicus.

Polyol/Monosaccharide Transporters (PLTs/PMTs) localized in the plasma membrane have previously been identified in plants. The physiological role and the functional properties of these proteins in legume plants are, however, unclear. Here we describe the functional analysis of LjPLT1, a plasma membrane-localized PLT protein from Lotus japonicus. The LjPLT1 gene was strongly expressed in the vascular tissue of roots, stems and leaves. Expression of the LjPLT1 cDNAs in yeast revealed that the protein functions as a broad-spectrum H+ -symporter for both linear polyols of sorbitol and mannitol, and cyclic polyol myo-inositol. It also catalyzes the transport of different hexoses, including fructose, glucose, galactose and mannose. Overexpression of LjPLT1 (OELjPLT1) results in inhibition of plant growth and a decrease in nodule nitrogenase activity in L. japonicus. The soluble sugars were increased in newly expanded leaves, roots and nodules but decreased in mature leaves in OELjPLT1 plants. In addition, the OELjPLT1 seedlings displayed an increased sensitivity to high content mannitol and boron toxicity, but neither drought nor salinity stresses. Taken together, the present study indicates that the LjPLT1 protein may participate in the translocation of hexoses/polyols to regulate multiple physiological and growth processes in L. japonicus.

Overexpression of LjPLT3 Enhances Salt Tolerance in Lotus japonicus.

Intracellular polyols are used as osmoprotectants by many plants under environmental stress. However, few studies have shown the role of polyol transporters in the tolerance of plants to abiotic stresses. Here, we describe the expression characteristics and potential functions of Lotus japonicus polyol transporter LjPLT3 under salt stress. Using LjPLT3 promoter-reporter gene plants showed that LjPLT3 was expressed in the vascular tissue of L. japonicus leaf, stem, root, and nodule. The expression was also induced by NaCl treatment. Overexpression of LjPLT3 in L. japonicus modified the growth rate and saline tolerance of the transgenic plants. The OELjPLT3 seedlings displayed reduced plant height under both nitrogen-sufficient and symbiotic nitrogen fixation conditions when 4 weeks old. The nodule number of OELjPLT3 plants was reduced by 6.7-27.4% when 4 weeks old. After exposure to a NaCl treatment in Petri dishes for 10 days, OELjPLT3 seedlings had a higher chlorophyll concentration, fresh weight, and survival rate than those in the wild type. For symbiotic nitrogen fixation conditions, the decrease in nitrogenase activity of OELjPLT3 plants was slower than that of the wild type after salt treatment. Compared to the wild type, both the accumulation of small organic molecules and the activity of antioxidant enzymes were higher under salt stress. Considering the concentration of lower reactive oxygen species (ROS) in transgenic lines, we speculate that overexpression of LjPLT3 in L. japonicus might improve the ROS scavenging system to alleviate the oxidative damage caused by salt stress, thereby increasing plant salinity tolerance. Our results will direct the breeding of forage legumes in saline land and also provide an opportunity for the improvement of poor and saline soils.

A d-pinitol transporter, LjPLT11, regulates plant growth and nodule development in Lotus japonicus.

Polyol transporters have been functionally characterized in yeast and Xenopus laevis oocytes as H+-symporters with broad substrate specificity, but little is known about their physiological roles in planta. To extend this knowledge, we investigated the role of LjPLT11 in Lotus japonicus-Mesorhizobium symbiosis. Functional analyses of LjPLT11 in yeast characterized it as an energy-independent transporter of xylitol, two O-methyl inositols, xylose, and galactose. We showed that LjPLT11 is located on peribacteroid membranes and functions as a facilitative transporter of d-pinitol within infected cells of L. japonicus nodules. Knock-down of LjPLT11 (LjPLT11i) in L. japonicus accelerated plant growth under nitrogen sufficiency, but resulted in abnormal bacteroids with corresponding reductions in nitrogenase activity in nodules and plant growth in the nitrogen-fixing symbiosis. LjPLT11i nodules had higher osmotic pressure in cytosol, and lower osmotic pressure in bacteroids, than wild-type nodules both 3 and 4 weeks after inoculation of Mesorhizobium loti. Levels and distributions of reactive oxygen species were also perturbed in infected cells of 4-week-old nodules in LjPLT11i plants. The results indicate that LjPLT11 plays a key role in adjustment of the levels of its substrate pinitol, and thus maintenance of osmotic balance in infected cells and peribacteroid membrane stability during nodule development.

LAZY3 plays a pivotal role in positive root gravitropism in Lotus japonicus.

LAZY1 family genes play important roles in both shoot and root gravitropism in plants. Here we report a Lotus japonicus mutant that displays negative gravitropic response in primary and lateral roots. Map-based cloning identified the mutant gene LAZY3 as a functional ortholog of the LAZY1 gene. Mutation of the LAZY3 gene reduced rootward polar auxin transport (PAT) in the primary root, which was also insensitive to the PAT inhibitor N-1-naphthylphthalamic acid. Moreover, immunolocalization of enhanced green fluorescent protein-tagged LAZY3 in L. japonicus exhibited polar localization of LAZY3 on the plasma membrane in root stele cells. We therefore suggest that the polar localization of LAZY3 in stele cells might be required for PAT in L. japonicus root. LAZY3 transcripts displayed asymmetric distribution at the root tip within hours of gravistimulation, while overexpression of LAZY3 under a constitutive promoter in lazy3 plants rescued the gravitropic response in roots. These data indicate that root gravitropism depends on the presence of LAZY3 but not on its asymmetric expression in root tips. Expression of other LAZY genes in a lazy3 background did not rescue the growth direction of roots, suggesting that the LAZY3 gene plays a distinct role in root gravitropism in L. japonicus.

The role of endogenous thiamine produced via THIC in root nodule symbiosis in Lotus japonicus.

Thiamine is a pivotal primary metabolite which is indispensable to all organisms. Although its biosynthetic pathway has been well documented, the mechanism by which thiamine influences the legume-rhizobium symbiosis remains uncertain. Here, we used overexpressing transgenic plants, mutants and grafting experiments to investigate the roles played by thiamine in Lotus japonicus nodulation. ljthic mutants displayed lethal phenotypes and the defect could be overcome by supplementation of thiamine or by overexpression of LjTHIC. Reciprocal grafting between L. japonicus wild-type Gifu B-129 and ljthic showed that the photosynthetic products of the aerial part made a major contribution to overcoming the nodulation defect in ljthic. Overexpression of LjTHIC in Lotus japonicus (OE-LjTHIC) decreased shoot growth and increased the activity of the enzymes 2-oxoglutarate dehydrogenase and pyruvate dehydrogenase. OE-LjTHIC plants exhibited an increase in the number of infection threads and also developed more nodules, which were of smaller size but unchanged nitrogenase activity compared to the wildtype. Taken together, our results suggest that endogenous thiamine produced via LjTHIC acts as an essential nutrient provided by the host plant for rhizobial infection and nodule growth in the Lotus japonicus - rhizobium interaction.

Heterogeneity in the expression and subcellular localization of POLYOL/MONOSACCHARIDE TRANSPORTER genes in Lotus japonicus.

Polyols can serve as a means for the translocation of carbon skeletons and energy between source and sink organs as well as being osmoprotective solutes and antioxidants which may be involved in the resistance of some plants to biotic and abiotic stresses. Polyol/Monosaccharide transporter (PLT) proteins previously identified in plants are involved in the loading of polyols into the phloem and are reported to be located in the plasma membrane. The functions of PLT proteins in leguminous plants are not yet clear. In this study, a total of 14 putative PLT genes (LjPLT1-14) were identified in the genome of Lotus japonicus and divided into 4 clades based on phylogenetic analysis. Different patterns of expression of LjPLT genes in various tissues were validated by qRT-PCR analysis. Four genes (LjPLT3, 4, 11, and 14) from clade II were expressed at much higher levels in nodule than in other tissues. Moreover, three of these genes (LjPLT3, 4, and 14) showed significantly increased expression in roots after inoculation with Mesorhizobium loti. Three genes (LjPLT1, 3, and 9) responded when salinity and/or osmotic stresses were applied to L. japonicus. Transient expression of GFP-LjPLT fusion constructs in Arabidopsis and Nicotiana benthamiana protoplasts indicated that the LjPLT1, LjPLT6 and LjPLT7 proteins are localized to the plasma membrane, but LjPLT2 (clade IV), LjPLT3, 4, 5 (clade II) and LjPLT8 (clade III) proteins possibly reside in the Golgi apparatus. The results suggest that members of the LjPLT gene family may be involved in different biological processes, several of which may potentially play roles in nodulation in this nitrogen-fixing legume.