shrunken4 is a mutant allele of ZmYSL2 that affects aleurone development and starch synthesis in maize.

Minerals are stored in the aleurone layer and embryo during maize seed development, but how they affect endosperm development and activity is unclear. Here, we cloned the gene underlying the classic maize kernel mutant shrunken4 (sh4) and found that it encodes the YELLOW STRIPE-LIKE oligopeptide metal transporter ZmYSL2. sh4 kernels had a shrunken phenotype with developmental defects in the aleurone layer and starchy endosperm cells. ZmYSL2 showed iron and zinc transporter activity in Xenopus laevis oocytes. Analysis using a specific antibody indicated that ZmYSL2 predominately accumulated in the aleurone and sub-aleurone layers in endosperm and the scutellum in embryos. Specific iron deposition was observed in the aleurone layer in wild-type kernels. In sh4, however, the outermost monolayer of endosperm cells failed to accumulate iron and lost aleurone cell characteristics, indicating that proper functioning of ZmYSL2 and iron accumulation are essential for aleurone cell development. Transcriptome analysis of sh4 endosperm revealed that loss of ZmYSL2 function affects the expression of genes involved in starch synthesis and degradation processes, which is consistent with the delayed development and premature degradation of starch grains in sh4 kernels. Therefore, ZmYSL2 is critical for aleurone cell development and starchy endosperm cell activity during maize seed development.