MNC1 Negatively Regulates Nectar Production through Auxin and Jasmonic Acid Response Pathways in Arabidopsis thaliana

Abstract

Many flowering plants offer a reward for pollinators in the form of nectar. Despite the central role of pollination in reproduction of plants and the considerable amount of energy a plant devotes to produce nectar, little is known of the molecular mechanism of nectar production and its regulation. Previous reports have suggested a significant role for the plant hormone auxin in regulating nectar production. Recent transcriptome studies have made it possible to focus research on several nectary-specific candidate genes with putative roles in the auxin response. In Arabidopsis thaliana this includes a gene termed MEDIAN NECTARY CUPIN 1 (MNC1; At1g74820), which is highly expressed in median nectaries. MNC1 silenced mutants (mnc1) showed more nectar production and increased auxin response activity while MNC1 overexpresser mutant (MNC1 T6) showed significantly less nectar production and less auxin response activity in nectaries. A comparative sequence analysis of proteins with known function shows that MNC1 is a germin-like protein belonging to the RmlC-like cupins superfamily. MNC1 also has a conserved zinc binding domain with known Auxin Binding Protein1. Thus, we hypothesize that MNC1 negatively regulates nectar production, likely through an auxin dependent pathway. PIN6 (At1g77110), an auxin transporter family protein, has been reported to be an auxin transporter localized to the ER modulating cytoplasmic free auxin concentration in nectaries. Arabidopsis thaliana mutant lines with different combinations of crossed target genes were used to understand the feedback mechanism of auxin, nectar production, PIN6, and MNC1 protein behind nectar regulation and production. Transformed Escherichia coli and Pichia pastoris, a methylotrophic yeast species, expressing MNC1 protein were employed for studying its biochemical nature, including auxin binding activity. Jasmonic acid (JA) was also suggested to be required for nectar production, with possible crosstalk to auxin in regulation of nectar production. COI1-independent JA response pathway was found to regulate nectar production by altering the expression of nectary-specific genes such as SWEET9, a sucrose transporter required for nectar production, CWINV4, a cell wall invertase required for nectar production, and MNC1. A potential auxin-JA crosstalk mechanism was constructed based on results in this study and previous studies.