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

Abstract

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.

Fig 1. Comparison of the 14 LjPLT proteins.

Schematic alignment of the deduced protein sequences (boxes) of LjPLT1 to LjPLT14 based on the positions of intron (arrows) in these genes. Grey boxes (I–XII) indicate the positions of transmembrane helices as predicted by the HMMTOP software package. Thin lines show small gaps in the sequences. Numbers of amino acids encoded by the different exons are indicated (white).

Fig 2. Phylogenetic tree of polyol transporters in different species.

The tree was constructed using the neighbor-joining method with 1000 bootstrap replications. Asterisks indicate proteins of plant that have been reported to be localized to the plasma membrane. Accession numbers for the PLT genes are listed in S3 Table.

Fig 3. Cis-element analysis of putative LjPLT promoters related to stress responses.

Different cis-elements with the same or similar functions are shown in the same color.

Fig 4. Expression patterns of LjPLT genes.

The relative expression levels of the LjPLTs in different organs were tested by qRT-PCR. Relative expression was normalized to the reference genes LjATPase and LjUBC (internal control) and the expression level in pods was defined as “1”.

Fig 5. Expression patterns of LjPLT genes in response to M. loti.

Levels of LjPLT transcripts in infected roots at 0, 1, 2, 3 and 7 d after inoculation with M. loti.

Fig 6. The response of LjPLT genes to salt and osmotic stresses applied to L. japonicus.

Expression of LjPLTs in roots and shoots at different time points after 10% PEG and 100 mM NaCl treatments.

Fig 7. Transient expression of GFP-LjPLT fusion constructs in Arabidopsis protoplasts.

(A) GFP-LjPLT1, GFP-LjPLT6 and GFP-LjPLT7 are localized in the plasma membrane. Scale bar, 5 μm. (B) GFP-LjPLT8 is localized both in the cytoplasm and at the plasma membrane. The images were recorded with a Leica TCS SP8 confocal microscope. Scale bar, 5 μm.

Fig 8. Transient expression of GFP-LjPLT fusion constructs in N. benthamiana expressing a marker of the Golgi apparatus, mCherry-tagged alpha-mannosidase II (AMAN-2).

(A) The proteins GFP-LjPLT2, GFP-LjPLT3, GFP-LjPLT4, GFP-LjPLT5 and GFP-LjPLT8 co-localized with mCherry-tagged AMAN-2. The images were recorded with a Leica TCS SP8 X confocal microscope, in the GFP region with time gating (gate on time: 0.0–12.0 ns) and in the mCherry region with time gating (gate on time: 0.3–11.9 ns). Scale bar, 5 μm. (B) GFP-LjPLT3 and GFP-LjPLT8 are localized in the Golgi apparatus as shown by 3-D reconstructions. 3-D Images were acquired using a Nikon A1 confocal microscope, with a 60× oil objective.