The role of alanine synthesis and nitrate-induced nitric oxide production during hypoxia stress in Cucurbita pepo nectaries

Authors: SOLHAUG, ERIK - University Of Minnesota; ROY, RAHUL - University Of Minnesota; Venterea, Rodney - Rod; CARTER, CLAY - University Of Minnesota

Submitted to: Plant Journal
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/22/2020
Publication Date: 10/29/2020
Citation: Solhaug, E., Roy, R., Venterea, R.T., Carter, C. 2020. The role of alanine synthesis and nitrate-induced nitric oxide production during hypoxia stress in Cucurbita pepo nectaries. The Plant Journal. 105(3):580-599. https://doi.org/10.1111/tpj.15055.
DOI: https://doi.org/10.1111/tpj.15055

Interpretive Summary: Floral nectar is a sugary solution produced in the nectaries of flowering plants to reward pollinators for visitation. Nectar production requires significant energy to synthesize nectar from basic compounds including sugars and amino acids. Although amino acids have been reported in nectars, little is known about how nectar amino acids are synthesized and secreted, and what potential roles they play in nectary biology. In this study, we investigated nitrogen metabolism in the nectaries of Cucurbita pepo (squash) plants in order to better understand amino acid synthesis. The increased activity of key enzymes involved in nitrogen metabolism, including nitrate reductase, suggested that amino acids were being produced within the nectaries during nectar secretion. The amino acid alanine accounted for approximately 25% of the total amino acids during nectar secretion and was the predominant amino acid during peak secretion. However, alanine accumulation was not significantly altered when nitrate reductase activity was inhibited or when exogenous nitrate was applied. Thus, synthesis of alanine and other amino acids appears to occur independently of vasculature-derived nitrate during nectar secretion. To further probe the possible reasons for the stimulation of nitrogen metabolism in actively secreting nectaries, we demonstrated that nitric oxide (NO) is evolved from nitrate in squash nectaries during secretion and may play an important role in sustaining metabolism under low-oxygen (hypoxic) conditions. Finally, inhibition of alanine synthesis and treatment with nitrate impacted the production of hypoxic metabolites, suggesting that nitrogen metabolism plays a role in the hypoxia response in C. pepo nectaries. Cumulatively, these data improve our understanding of nitrogen metabolism in squash nectaries and may help gain further insights into nectary nitrogen metabolism in other plants. These results will be useful to scientists and others interested in better understanding plant nitrogen metabolism and nitrogen gas emissions to the atmosphere.

Technical Abstract: Floral nectar is a sugary solution produced by nectaries to reward pollinators for visitation. Nectar secretion is an energetically demanding process that relies on the synthesis of nectar components within the nectary from phloem-derived precursors. In addition to sugars, nectars contain amino acids, which impact plant-pollinator interactions. Although amino acids have been reported in nectars, very little is known about how nectar amino acids are synthesized and secreted, and what potential roles they play in nectary biology. In this study, we investigated nitrogen metabolism in Cucurbita pepo (squash) floral nectaries in order to better understand amino acid synthesis and partitioning between the nectar and nectary. The expression and activity of key enzymes involved in primary nitrogen reduction and assimilation, including nitrate reductase and nitrate transporter NRT1.5, were significantly up-regulated during secretion in C. pepo nectaries, suggesting de novo production of amino acids. Alanine accumulated to ~25% of total amino acids in nectaries during secretion and is the predominant amino acid in C. pepo nectar during peak nectar secretion; however, its accumulation was not significantly altered when either nitrate reductase activity was inhibited or when exogenous nitrate was applied. Thus, alanine and amino acid synthesis in general appears to occur independently of de novo reduction and assimilation of vasculature-derived nitrate during nectar secretion. To further probe the possible reason for the upregulation of nitrogen metabolism genes in actively secreting nectaries, we demonstrated that nitric oxide (NO) is evolved from nitrate in squash nectaries during secretion, and may play an important role in sustaining metabolism under hypoxic conditions. Finally, inhibition of alanine synthesis and treatment with nitrate impacted the production of hypoxic metabolites, suggesting nitrogen metabolism plays a role in hypoxia response in C. pepo nectaries. Cumulatively, these data improve our understanding of nitrogen metabolism squash nectaries, with the potential to help gain further insights into the role of nectary nitrogen metabolism in other plants.