Extended: ZINC FINGER PROTEIN mediates seed loading by affecting funiculus

The plant funiculus is the anchor connecting the developing seed to the placenta within the inner dorsal pod strands of the silique wall and acts as the direct pathway for transporting nutrients to the seed. The secondary cell wall (SCW) is a crucial component of funiculus cells. However, the molecular mechanism underlying SCW biosynthesis in funiculus remains elusive. Here, we identified a previously uncharacterized C2H2-type zinc finger protein, ZINC FINGER PROTEIN (ZFP), which played an essential role in mediating funiculus function. We found that ZFP was highly expressed in the Arabidopsis non-seed tissues of siliques, including funiculus. The ZFP loss-of-function mutants exhibited various morphological changes in siliques, such as crinkled and shorter siliques, as well as reduced seed size and seed oil content. Comparative transcriptomic analysis indicated that various genes involved in SCW biosynthesis, including NAC SECONDARY WALL THICKENING PROMOTING FACTOR 1 (NST1), were upregulated in the zfp mutant. Dual-Luciferase assays in Nicotiana benthamiana leaves revealed that ZFP significantly repressed the activity of the NST1 promoter (proNST1). Chromatin immunoprecipitation (ChIP)-qPCR and electrophoretic mobility shift (EMSA) assays showed ZFP directly bound to proNST1. The in situ analysis of SCW distribution revealed that zfp mutants exhibited ectopic lignified SCW in funiculus. We found the glucosinolate contents were substantially lower in zfp mutants compared to wild-type (WT), suggesting ZFP affected seed loading, possibly through repressing NST1. Collectively, our findings suggest that ZFP fine-tunes NST1 expression, thus controlling the SCW biosynthesis in the funiculus to assure proper seed loading.