Characterisation of the subunit genes of pyrophosphate-dependent phosphofructokinase from loquat (Eriobotrya japonica Lindl.)

Abstract

Pyrophosphate-dependent phosphofructokinase (PFP) catalyses the reversible phosphorylation of fructose-6-phosphate to fructose-1,6-bisphosphate in the glycolysis pathway. Two full-length complementary DNAs encoding putative PFP α-and β-subunits, named EjPFPa and EjPFPb1, were isolated by reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of the cDNA ends (RACE) from Eriobotrya japonica Lindl. (loquat). The other β-subunit gene was identified from transcriptome data obtained by Illumina sequencing, designated as EjPFPb2. They share up to 88 % identity with other plant PFP α/β-subunits. EjPFPa and EjPFPb clustered separately in clades of plant PFP α-or β-subunits, respectively. EjPFPa and EjPFPb were both shown to be mainly localised in the cell membrane using confocal microscopy of GFP fusion proteins 35S:EjPFPa/b-GFP. Monitoring the dynamic changes of transcripts and proteins demonstrated that EjPFPa and EjPFPb did not show coordinated expression during fruit development in loquat. The transcript levels of EjPFPa and EjPFPb in the leaves of loquat seedlings were significantly enhanced after 3 h of treatment with 0.5, 1.0 and 1.5 M fructose or glucose, which indicates that EjPFPa and EjPFPb are modulated by fructose and glucose in vivo. Transgenic tobacco plants overexpressed EjPFPa did not show visible phenotype changes, while plants overexpressing EjPFPb1 grew faster at the cost of reduced leaf size and sucrose content. Meanwhile, fructose content increased in growing plants compared with wild-type plants.The results confirm that genes of the two subunits from loquat PFP have different transcriptional regulation systems and play different roles in carbohydrate metabolism. Elevation of the PFP β-subunit gene expression can partly impact the glycolytic carbon allocation in growing plants.

Genome-wide identification of the PFK gene family and their expression analysis in Quercus rubra

“…PFP has been well characterized at the molecular and biochemical levels in all three domains of life since the discovery of the first PFK in 1980 (Dunaway, 1983;Siebers et al, 1998;Mustroph et al, 2007;Darikova and Sherbakov, 2009;Zhu et al, 2013). PFP contains two distinct subunits, α and β (Cawood et al, 1988;Zhu et al, 2013;Qin et al, 2014). PFP has been the subject of extensive research, but PFK has received relatively little attention due to its instability when isolated during purification (Turner and Plaxton, 2003).…”