Class II KNOX and BELL TALE-class HD genes give insights into the evolution of secondary cell wall development and pectin biosynthesis during land plant evolution

Land plants evolved from an ancestral alga around 470 million years ago, evolving complex multicellularity in both haploid gametophyte and diploid sporophyte generations. The evolution of water conducting tissues in the sporophyte generation was crucial for the success of land plants, paving the way for the colonization of a variety of terrestrial habitats. Class II KNOX (KNOX2) genes are major regulators of secondary cell wall formation and seed mucilage (pectin) deposition in flowering plants. Here we show that in the liverwort Marchantia polymorpha loss-of-function alleles of the KNOX2 ortholog, MpKNOX2, or its dimerization partner MpBELL1, have defects in capsule wall secondary cell wall and spore pectin biosynthesis. Both genes are expressed in the gametophytic calyptra surrounding the sporophyte and exhibits maternal effects, suggesting an intergenerational regulation from the maternal gametophyte to the sporophyte generation. These findings also suggest the presence of a “vascular-like” program in the non-vascular liverwort capsule wall. Overall design: We generated inducible co-overexpression alleles of MpKNOX2 and MpBELL1. M. polymorpha spores were doubly transformed using one construct in cis and one in trans, resulting in lines constitutively expressing MpKNOX2 coding sequence with an estrogen inducible MpBELL1 coding sequence (proEF1:MpKNOX2; proEF1:XVE>>MpBELL1). Gemmae were grown under normal growth conditions (23°C, 24h white light) on ½ B5 Gamborg's media for 10 days, and then transferred to plates containing 5µM 17-ß-estradiol or mock. Plants were sampled at 0h (not induced) and 72h.