The origin of a terrestrial flora in the Ordovician required adaptation to novel biotic and abiotic stressors. Oil bodies, a synapomorphy of liverworts, accumulate secondary metabolites, but their function and development are poorly understood. Oil bodies of Marchantia polymorpha develop within specialized cells as one single large organelle. Here, we show that a CLASS I HOMEODOMAIN LEUCINE-ZIPPER (C1HDZ) transcription factor controls the differentiation of oil body cells in two different ecotypes of the liverwort M. polymorpha, a model genetic system for early divergent land plants. In flowering plants, these transcription factors primarily modulate responses to abiotic stresss including drought. However, loss-of-function alleles of the single ortholog gene, MpC1HDZ, in M. polymorpha did not exhibit phenotypes associated with abiotic stress. Rather Mpc1hdz mutant plants were more susceptible to herbivory and total plant extracts of the mutant exhibited reduced antibacterial activity. Transcriptomic analysis of the mutant revealed a reduction in expression of genes related to secondary metabolism that was accompanied by a specific depletion of oil body terpenoid compounds. Through time lapse imaging we observed that MpC1HDZ expression maxima precede oil body formation indicating that MpC1HDZ mediates differentiation of oil body cells. Our results indicate that M. polymorpha oil bodies, and MpC1HDZ, are critical for defense against herbivory but not for abiotic stress-tolerance. Thus, C1HDZ genes were co-opted to regulate separate responses to biotic and abiotic stressors in two distinct land plant lineages..

26 27 28 SUMMARY 29 The origin of a terrestrial flora in the Ordovician required adaptation to novel biotic and abiotic 30 stressors. Oil bodies, a synapomorphy of liverworts, accumulate secondary metabolites, but their 31 function and development are poorly understood. Oil bodies of Marchantia polymorpha develop 32 within specialized cells as one single large organelle. Here, we show that a CLASS I 33 HOMEODOMAIN LEUCINE-ZIPPER (C1HDZ) transcription factor controls the differentiation of 34 oil body cells in two different ecotypes of the liverwort M. polymorpha, a model genetic system for 35 early divergent land plants. In flowering plants, these transcription factors primarily modulate 36 responses to abiotic stresss including drought. However, loss-of-function alleles of the single ortholog 37 gene, MpC1HDZ, in M. polymorpha did not exhibit phenotypes associated with abiotic stress. Rather 38 Mpc1hdz mutant plants were more susceptible to herbivory and total plant extracts of the mutant 39 exhibited reduced antibacterial activity. Transcriptomic analysis of the mutant revealed a reduction 40 in expression of genes related to secondary metabolism that was accompanied by a specific depletion 41 of oil body terpenoid compounds. Through time lapse imaging we observed that MpC1HDZ 42 expression maxima precede oil body formation indicating that MpC1HDZ mediates differentiation 43 of oil body cells. Our results indicate that M. polymorpha oil bodies, and MpC1HDZ, are critical for 44 defense against herbivory but not for abiotic stress-tolerance. Thus, C1HDZ genes were co-opted to 45 regulate separate responses to biotic and abiotic stressors in two distinct land plant lineages.46 47 48 49 The emergence of land flora in the Ordovician and the subsequent radiation of plants into terrestrial 50 environments were major evolutionary events in the history of Earth. This radical breakthrough for 51 life on the planet entailed the evolution of both new developmental programs and biochemical 52 pathways [1]. Early land plants were faced with novel biotic and abiotic stressors, including a 53 desiccating aerial environment, increased solar radiation, and greater interspecific competition, 54 including herbivory. This transition was accompanied by a dramatic increase in the diversity of 55 secondary metabolites and the complexity of transcriptional regulation, to protect plants from 56 herbivores and pathogens, and also abiotic stressors such as desiccation and light intensity [2-4]. 57 Liverworts represent one of the earliest diverging land plant lineages, with a predicted Ordovician-58 Silurian origin of all three major extant liverwort clades [5, 6]. One synapomorphy of this lineage is 59 the presence of oil bodies, specialized organelles containing an array of lipophilic secondary 60 compounds [7]. In some liverworts, multiple oil bodies are found in nearly all differentiated cells of 61 both gametophyte and sporophyte generations. Whilst in other liverworts, such as Marchantia 62 polymorpha (M. polymorpha), oil bodies are ...