Molecular identification and functional characterization of GhAMT1.3 in ammonium transport with a high affinity from cotton ( Gossypium hirsutum L.)

Ammonium (NH 4 + ) represents a primary nitrogen source for many plants, its effective transport into and between tissues and further assimilation in cells determine greatly plant nitrogen use efficiency. However, biological components involved in NH 4 + movement in woody plants are unclear. Here, we report kinetic evidence for cotton NH 4 + uptake and molecular identification of certain NH 4 + transporters ( AMT s) from cotton ( Gossypium hirustum ). A substrate‐influx assay using 15 N‐isotope revealed that cotton possessed a high‐affinity transport system with a K m of 58 μM for NH 4 + . Sequence analysis showed that GhAMT1.1–1.3 encoded respectively a membrane protein containing 485, 509 or 499 amino acids. Heterologous functionality test demonstrated that GhAMT1.1–1.3 expression mediated NH 4 + permeation across the plasma membrane (PM) of yeast and/or Arabidopsis qko ‐mutant cells, allowing a growth restoration of both mutants on NH 4 + . Quantitative PCR measurement showed that GhAMT1.3 was expressed in roots and leaves and markedly up‐regulated by N‐starvation, repressed by NH 4 + resupply and regulated diurnally and age‐dependently, suggesting that GhAMT1.3 should be a N‐responsive gene. Importantly, GhAMT1.3 expression in Arabidopsis improved plant growth on NH 4 + and enhanced total nitrogen accumulation (∼50% more), conforming with the observation of 2‐fold more NH 4 + absorption by GhAMT1.3 ‐transformed qko plant roots during a 1‐h root influx period. Together with its targeting to the PM and saturated transport kinetics with a K m of 72 μM for NH 4 + , GhAMT1.3 is suggested to be a high‐affinity NH 4 + permease that may play a significant role in cotton NH 4 + acquisition and utilization, adding a new member in the plant AMT family.