Reinvestigation of THOUSAND-GRAIN WEIGHT 6 grain weight genes in wheat and rice indicates a role in pollen development rather than regulation of auxin content in grains.

KEY MESSAGE: Phylogenetic and expression analyses of grain weight genes TaTGW6 and OsTGW6 and investigation of substrate availability indicate TGW6 does not regulate auxin content of grains but may affect pollen development. The THOUSAND-GRAIN WEIGHT 6 genes (TaTGW6 and OsTGW6) are reported to result in larger grains of wheat and rice by reducing production of indole-3-acetic acid (IAA) in developing grains. However, a critical comparison of data on TaTGW6 and OsTGW6 with other reports on IAA synthesis in cereal grains requires that this hypothesis be reinvestigated. Here, we show that TaTGW6 and OsTGW6 are members of a large gene family that has undergone major, lineage-specific gene expansion. Wheat has nine genes, and rice three genes encoding proteins with more than 80% amino acid identity with TGW6, making it difficult to envisage how a single inactive allele could have a major effect on IAA levels in grains. In our study, we show that neither TaTGW6 nor OsTGW6 is expressed in developing grains. Instead, both genes and their close homologues are exclusively expressed in pre-emergent inflorescences; TaTGW6 is expressed particularly in microspores prior to mitosis. This evidence, combined with our observation that developing wheat grains have undetectable levels of ester IAA in comparison to free IAA and do not express an IAA-glucose synthase suggests that TaTGW6 and OsTGW6 do not regulate grain size via the hydrolysis of IAA-glucose. Instead, their similarity to rice strictosidine synthase-like (OsSTRL2) suggests they play a key role in pollen development.