# A pennycress transparent testa 8 knockout mutant has drastic changes in seed coat anatomy and chemical compositions

## TL;DR

A CRISPR-edited pennycress mutant has altered seed coat chemistry and anatomy, affecting seed permeability and aging.

## Contribution

Identification of TT8 gene's role in seed coat composition and permeability in pennycress through CRISPR knockout.

## Key findings

- tt8-2bp seeds have drastically reduced proanthocyanidins and increased permeability.
- tt8-2bp seeds show altered nutrient partitioning with increased embryo dry weight.
- Seed coat cuticle layer is present but may have structural defects in tt8-2bp.

## Abstract

Pennycress is a winter annual intermediate crop with ∼30% seed oil content suitable for producing biofuels. Here, we evaluated seed development, anatomy, and agronomically relevant traits of a transparent testa 8 knockout mutant (tt8-2bp) generated by CRISPR genome editing to improve seed quality. We performed histochemical analyses on wild-type and tt8-2bp seeds at different developmental stages. No visible anatomical defects were observed in embryos and endosperm of tt8-2bp seeds. However, tt8-2bp seed coats had drastically reduced proanthocyanidins and proanthocyanidin monomers, which correlated with increased seed coat permeability, increased imbibition rates, and altered seed aging of tt8-2bp seeds. A cuticle layer was detected in tt8-2bp and wild-type seed coats. Further analysis is required to assess possible quantitative and structural defects in the tt8-2bp seed cuticle. Based on metabolomic and solid-state NMR analyses, tt8-2bp seed coats had decreased aromatic compounds and cell wall polysaccharides compared with wild-type seed coats. Consistently, tt8-2bp seeds also had reduced non-embryonic tissue dry weights, increased embryo dry weights, and unchanged total seed weights compared with wild-type seeds. This indicated altered nutrient partitioning during tt8-2bp seed development. The agronomic implications of tt8-2bp altered seed traits on pennycress domestication are discussed in depth.

Histochemical analyses of pennycress seeds revealed proanthocyanidin deficiency in tt8-2bp seed coats accompanied by increased seed coat permeability, increased imbibition rates, and altered seed aging compared with wild-type seeds.