Polar auxin transport is unique to plants and coordinates their growth and development^1,2. The PIN-FORMED (PIN) auxin transporters exhibit highly asymmetrical localizations at the plasma membrane and drive polar auxin transport^3,4; however, their structures and transport mechanisms remain largely unknown. Here, we report three inward-facing conformation structures of Arabidopsis thaliana PIN1: the apo state, bound to the natural auxin indole-3-acetic acid (IAA), and in complex with the polar auxin transport inhibitor N-1-naphthylphthalamic acid (NPA). The transmembrane domain of PIN1 shares a conserved NhaA fold⁵. In the substrate-bound structure, IAA is coordinated by both hydrophobic stacking and hydrogen bonding. NPA competes with IAA for the same site at the intracellular pocket, but with a much higher affinity. These findings inform our understanding of the substrate recognition and transport mechanisms of PINs and set up a framework for future research on directional auxin transport, one of the most crucial processes underlying plant development.

Brassinosteroids are steroidal phytohormones that regulate plant development and physiology, including adaptation to environmental stresses. Brassinosteroids are synthesized in the cell interior but bind receptors at the cell surface, necessitating a yet to be identified export mechanism. Here, we show that a member of the ATP-binding cassette (ABC) transporter superfamily, ABCB19, functions as a brassinosteroid exporter. We present its structure in both the substrate-unbound and the brassinosteroid-bound states. Bioactive brassinosteroids are potent activators of ABCB19 ATP hydrolysis activity, and transport assays showed that ABCB19 transports brassinosteroids. In Arabidopsis thaliana , ABCB19 and its close homolog, ABCB1, positively regulate brassinosteroid responses. Our results uncover an elusive export mechanism for bioactive brassinosteroids that is tightly coordinated with brassinosteroid signaling.

Abscisic acid (ABA) is a phytohormone essential to the regulation of numerous aspects of plant growth and development. The cellular level of ABA is critical to its signalling and is determined by its rate of biosynthesis, catabolism and the rates of ABA transport. ABCG25 in Arabidopsis thaliana has been identified to be an ABA exporter and play roles in regulating stomatal closure and seed germination. However, its ABA transport mechanism remains unknown. Here we report the structures of ABCG25 under different states using cryo-electron microscopy single particle analysis: the apo state and ABA-bound state of the wild-type ABCG25 and the ATP-bound state of the ATPase catalytic mutant. ABCG25 forms a homodimer. ABA binds to a cone-shaped, cytosolic-facing cavity formed in the middle of the transmembrane domains. Key residues in ABA binding are identified and verified by a cell-based ABA transport assay. ATP binding leads to closing of the nucleotide-binding domains of opposing monomers and conformational transitions of the transmembrane domains. Together, these results provide insights into the substrate recognition and transport mechanisms of ABCG25 in Arabidopsis, and facilitate our understanding of the ABA transport and signalling pathway in plants.

Brassinosteroids (BRs) are steroidal phytohormones indispensable for plant growth, development, and responses to environmental stresses. The export of bioactive BRs to the apoplast is essential for BR signaling initiation, which requires binding of a BR molecule to the extracellular domains of the plasma membrane-localized receptor complex. We have previously shown that the Arabidopsis thaliana ATP-binding cassette (ABC) transporter ABCB19 functions as a BR exporter and, together with its close homolog ABCB1, positively regulates BR signaling. Here, we demonstrate that ABCB1 is another BR transporter. The ATP hydrolysis activity of ABCB1 can be stimulated by bioactive BRs, and its transport activity was confirmed in proteoliposomes and protoplasts. Structures of ABCB1 were determined in substrate-unbound (apo), brassinolide (BL)-bound, and ATP plus BL-bound states. In the BL-bound structure, BL is bound to the hydrophobic cavity formed by the transmembrane domain and triggers local conformational changes. Together, our data provide additional insights into ABC transporter-mediated BR export.

Cytokinins are adenine derivatives serving as phytohormones, which are essential regulators of plant growth, development, and response to environmental factors. The transport process of cytokinins determines their spatial distributions and is critical to their functions. The AZA-GUANINE RESISTANT (AZG) family member AZG2 in Arabidopsis thaliana has been identified as a cytokinin and purine transporter. Here, we characterize the binding and transport of AZG2 towards the natural cytokinin, trans-zeatin (tZ), as well as adenine. AZG2 structures are determined in both the substrate-unbound, adenine-bound, and tZ-bound states, under both acidic and neutral pH. Key residues involved in substrate binding are identified. Two distinct conformations are observed in the tZ-bound state of AZG2 in the neutral pH. Structural analysis reveals the structural dynamics of AZG2 during cytokinin transport, which fit into the elevator-type transport model. These results provide insights into the molecular mechanism of cytokinin transport in plants.

Efficient ammonium uptake is essential for plant growth and nitrogen-use efficiency, yet the structural basis of ammonium transport and regulation in plant AMTs remains unclear. Here, we determined multiple cryo-electron microscopy structures of the Arabidopsis thaliana ammonium transporter AMT1;1 in apo, ammonium-bound, methylammonium-bound, low-pH and phosphomimetic states. These structures reveal a conserved ammonium conduction pathway comprising an aromatic recruitment vestibule, a Phe-gate/Twin-His-containing pore, and a polar intracellular exit region. We further identified a conserved extracellular N-terminal disulfide bond required for oligomer stability and transport activity, suggesting potential redox-dependent regulation. On the cytoplasmic side, phosphorylation modulates C-terminal interactions with intracellular loops, revealing an allosteric regulatory mechanism. Comparison with the bacterial ammonium-sensing protein Sd -Amt1 uncovers a conserved ammonium-recognition region in At AMT1;1 that contributes to transport activity but lacks the signaling module and ligand-dependent conformational switching observed in Sd -Amt1. Together, these findings provide structural insight into ammonium transport and coordinated regulation in plant AMT transporters.