Understanding and engineering the molecular regulation of nectar production in field pennycress (Thlaspi arvense).

Abstract

Anthropogenic climate change and the growing world population are putting pressure on our agroecosystems. Sustainable farming efforts are needed when intensive agriculture systems are fallow and prone to erosion and nutrient leaching. We can mitigate these issues while increasing farmer income by planting field pennycress (Thlaspi arvense). Pennycress forms penny shaped pods containing oil-rich seeds with diverse uses from jet fuel to cooking oil. As an overwintering cover crop, pennycress grows from fall to late spring, avoiding land-use competition with summer annual cash crops. Pennycress provides an ecosystem service to pollinator populations, which are needed for fruit and vegetable production. Pollinators are suffering due partially to losses of habitat and floral resources. Fortunately, pennycress flowers provide nectar as a floral resource when most agricultural landscapes are barren. Additionally, pollinator visitation increases pennycress seed yield. Therefore, the purpose of the study was to understand the genetics behind nectar production and develop pennycress plants with altered floral traits. Microscopy was performed on pennycress flowers to characterize the structure of the nectar-producing glands called nectaries. Additionally, pennycress nectary transcriptomes were determined using transcriptomic sequencing which led to the identification of genes and metabolic pathways. In both cases, the strong similarity in nectar production was confirmed between pennycress and Arabidopsis thaliana, a model plant and close pennycress relative. Because of the close relationship, it was possible to characterize Arabidopsis genes that can later be used to find orthologs in pennycress. The Arabidopsis gene At5g60760 annotated as ‘a P-LOOP containing nucleoside triphosphate hydrolases superfamily protein’ (hereafter AT5G60760) is a putative inositol kinase highly expressed in nectaries. Through assaying mutant AT5G60760 gene expression and nectar production, it was determined that AT5G60760 negatively regulates nectar production. By using findings from nectary genetics, such as the function of AT5G60760 and the pennycress nectary transcriptome, 13 genes were mutated using CRISPR/Cas9 to alter traits relating to pollinator attraction in field pennycress. We have identified two homozygous mutant lines and conducted phenotyping. The auxin response factor 8 (arf8) mutant flowers and petals are larger and produce more nectar than wild type. The cell wall invertase 4 (cwinv4) mutants do not produce nectar and have greatly reduced invertase activity in nectaries. In the future, these plants can be grown in field settings to test pollinator attraction, assay pollinator health, and measure pennycress seed yield.