Why so stubborn? MtKNOX4-regulated MtKCS12 manifests hardseededness

Legume seeds have evolved diverse dormancy traits to inhibit germination until favorable conditions appear. One trait is physical dormancy or hardseededness caused by a specialized hard seed coat that prevents water and oxygen entering from the surrounding environment (Finkelstein et al., 2008). Because of its essential function in legume evolution, mechanistic understanding of hard seed coat development holds enormous promise for future application in agriculture.

Legume seed coat consists of a water-impermeable palisade cell layer covered by an intact cuticle (Rolston, 1978; Chai et al., 2016). The fate of the cuticular layer in various plant tissues and organs is coordinated by heterogeneous lipid polyesters, including very long-chain fatty acids (VLCFs), cutin, and solvent-extractable waxes. The core of the fatty acid elongase complex that generates VLCFs includes β-ketoacyl-CoA synthase (KCS), a rate-limiting enzyme specifying different carbon chain lengths and cuticular composition in plants (Haslam and Kunst, 2013). While certain KCS enzymes catalyze the elongation of multiple fatty acids in various plant tissues, their role in seed coat palisade cuticle composition and hardseededness remains elusive.

In this issue of Plant Physiology, Chai et al. (2021) provide molecular and genetic insights into a regulatory network linking the composition integrity of the seed coat cuticle with physical dormancy using Medicago truncatula as a legume model. Previous work revealed a class II KNOTTED-LIKE HOMEOBOX transcription factor, KNOX4, controls physical dormancy by regulating seed-coat cuticle development in M. truncatula (Chai et al., 2016). Mutation of MtKNOX4 causes dysfunctional palisade cuticles by differentially regulating the expression of cuticle pathway genes in the mutant seeds, causing seeds to absorb water easily. Chai et al. (2021) identify KCS12 in M. truncatula as a determining factor in elongation of VLCFAs and link it with KNOX4-mediated physical dormancy. They show MtKCS12 expression downregulates in the seed coat of knox4 and hence investigated the impact of KCS12 deficiency on seed coat architecture and seed development.

The authors used quantitative PCR and promoter-β-glucuronidase assays to show KCS12 is specifically expressed in seed coat and palisade cells. Next, they created deleterious mutations in KCS12 using retrotransposons and found the mutants are defective in seed coat morphology and seed weight. Seed permeability assays further confirmed kcs12 seeds were able to imbibe and germinate, unlike wild-type seeds. These results, together with complementation experiments, showed KCS12 is required in the seed coat for physical dormancy as is KNOX4.

Chai et al. (2021) also examined the palisade cuticle structure and composition in kcs12 to assess its role in Medicago. They found decreased palisade cuticle in kcs12 using histological staining, indicative of a KCS12 role in seed coat development. They further confirmed through biochemical analyses that the kcs12 mutation impacts biosynthesis of seed coat VLCF polyester components and their derivatives associated with physical dormancy, including ester-bonded 24:0 fatty acids, fatty diols, and coumaric acid.

As KSC12 expression dramatically decreased in knox4 mutants, the authors hypothesized KNOX4 may directly regulate KCS12 expression. Chromatin immunoprecipitation assays revealed KNOX4 directly binds to the KCS12 promoter in vivo. Thus the authors suggest a model in which the KNOX4-KCS12 module determines biosynthesis of the VLCFAs in the seed coat and is critical for seed physical dormancy in Medicago. While it remains to be determined whether KCS12-like genes play conserved roles in other species that have hard seed coats, Chai et al. (2021) expand our knowledge on the mechanistic understanding of seed coat composition by identifying additional factors of the KNOX4-mediated regulatory network involved in physical dormancy.