Overexpression of the Starch Phosphorylase-Like Gene (PHO3) in Lotus japonicus has a Profound Effect on the Growth of Plants and Reduction of Transitory Starch Accumulation

Two isoforms of starch phosphorylase (PHO; EC 2.4.1.1), plastidic PHO1 and cytosolic PHO2, have been found in all plants studied to date. Another starch phosphorylase-like gene, PHO3, which is an ortholog of Chlamydomonas PHOB, has been detected in some plant lineages. In this study, we identified three PHO isoform (LjPHO) genes in the Lotus japonicus genome. Expression of the LjPHO3 gene was observed in all tissues tested in L. japonicus, and the LjPHO3 protein was located in the chloroplast. Overexpression of LjPHO3 in L. japonicus resulted in a drastic decline in starch granule sizes and starch content in leaves. The LjPHO3 overexpression transgenic seedlings were smaller, and showed decreased pollen fertility and seed set rate. Our results suggest that LjPHO3 may participate in transitory starch metabolism in L. japonicus leaves, but its catalytic properties remain to be studied.

Protocol publication

[…] Sequences of PHO proteins were retrieved from GenBank. Arabidopsis PHO proteins were used as queries in BLAST searches against the L. japonicus genome database. Conserved motifs in PHO proteins were analyzed using the NCBI’s CDD database (). The plastid transit peptide cleavage site of the PHO proteins was predicted using the program TargetP Server v1.01 (). For phylogenetic analysis, multiple sequence alignments of PHO amino acid sequences were performed using ClustalW. The tree was constructed using the neighbor-joining (NJ) method and 100 bootstraps in order to group putative full-length PHO amino acid sequences, and the results were displayed with Mega software version 4 (). […]