The ABCB family belongs to the larger superfamily of ATP-binding cassette (ABC) transporters, membrane proteins found from bacteria to humans that burn cellular energy in the form of ATP to pump molecules across membranes. In plants, ABC transporters ferry an astonishing variety of cargo: defense compounds, heavy-metal chelators, waxes and, critically, phytohormones. Within this superfamily, the B subgroup—full-length transporters containing both nucleotide-binding domains and transmembrane domains—has attracted particular attention because several of its members in the reference plant Arabidopsis thaliana are proven hormone movers. AtABCB1 and AtABCB19, for instance, are celebrated auxin efflux carriers that help establish the localized auxin gradients sculpting virtually every organ of the plant, and recent structural work has revealed that AtABCB19 also exports brassinosteroids, the steroid hormones that drive stem elongation, vascular development and stress resilience.

What happens to these functions in crop plants with larger, more complex genomes has been far less clear. The Brassica genus, which includes cabbage, broccoli, oilseed rape and choy sum, descended from a common ancestor with Arabidopsis through extensive whole-genome triplication followed by diploidization and rearrangements, meaning that for every Arabidopsis ABCB gene there may be several Brassica relatives whose functions have diverged, specialized or been lost. Floting Chinese cabbage is an economically important vegetable in southern China, and its tall, succulent stalk is the product of vigorous cell elongation intimately tied to brassinosteroid and auxin action—making it an ideal system in which to ask what the ABCB repertoire is doing.

To answer that question, the team carried out a genome-wide survey of the B. rapa var. parachinensis genome, an effort made feasible by a recent high-continuity genome assembly of the species. Using rigorous bioinformatic screening, they identified 36 BraABCB genes and constructed phylogenetic trees that placed them into four evolutionary groups, consistent with the canonical architecture of ABCB families described in other angiosperms. Conserved domain analysis confirmed that the predicted proteins carry the hallmarks expected of functional transporters:Walker A and Walker B motifs and the signature C-loop within the nucleotide-binding domains, together with the membrane-spanning α-helices that form the translocation pathway. When Arabidopsis ABCB proteins were included in the phylogeny, the BraABCB27 and BraABCB28 proteins clustered tightly with AtABCB1 and AtABCB19, immediately flagging them as the closest functional relatives of the best-characterized hormone transporters in any plant.

Evolutionary forensics added context to this inventory. The researchers mapped each BraABCB gene to its position on the ten chromosomes of the species and examined collinearity—the synteny between genomic regions—to trace how the family expanded. Synonymous and nonsynonymous substitution rates (Ka/Ks) calculated for duplicated gene pairs told a story of constraint: with Ka/Ks values well below one, most BraABCB paralogs have been maintained under purifying selection, meaning that the protein-coding sequences have been preserved largely intact since their duplication. In other words, the family did not balloon through sloppy replication but rather through ancient genome duplication events whose products the plant has carefully conserved—an evolutionary signature typically associated with genes that matter.

But conservation at the sequence level is only a hypothesis about function; the real test lies in where the genes are switched on and what the proteins do. The group focused its experimental attention on Group IV, the clade containing the AtABCB1/19 relatives. Mining the promoter regions upstream of these genes revealed a rich catalog of cis-regulatory elements implicated in hormone responsiveness and abiotic stress, including motifs associated with drought, heat, and both auxin and brassinosteroid signaling. To put these predictions to the test, the researchers grew choy sum seedlings under several perturbations—drought stress, elevated temperature, exogenous application of the bioactive brassinosteroid brassinolide, and treatment with the primary natural auxin, indole-3-acetic acid—and quantified the expression of selected Group IV BraABCB genes by reverse-transcription quantitative PCR using the standard 2^-ΔΔCT method. The results were striking in their diversity: individual family members responded differently and often in opposite directions to the same stimulus, indicating that the Brassica expansion of this family is not mere redundancy but a differentiated toolkit, with distinct transporters likely deployed in different tissues, developmental stages and environmental contexts.

Subcellular localization supplied a further piece of the puzzle. Because ABCB transporters must sit in a membrane to move hormones across it, the team fused Group IV BraABCB proteins to fluorescent reporters and expressed the constructs to determine where the fusion proteins accumulated in living cells. The analyses showed predominant localization to the plasma membrane, exactly where an efflux carrier engaged with extracellular signaling and long-distance hormone movement would be expected to reside. This placement also matters for a second reason: the brassinosteroid receptor itself, BRI1 (BRASSINOSTEROID-INSENSITIVE 1), is a leucine-rich repeat receptor kinase embedded in the plasma membrane, discovered in the 1990s as the cell-surface sensor for the steroid hormones. If ABCB transporters share membrane real estate with BRI1, opportunities for physical and functional crosstalk multiply.

That possibility is precisely where the new study makes its most intriguing contribution. In Arabidopsis, a regulatory protein called TWISTED DWARF1—an immunophilin-like co-chaperone—has been shown to associate physically with BRI1 and to be required for the full activity of the ABCB1- and ABCB19-mediated auxin transport machinery; mutations in the corresponding gene produce pleiotropic, hormone-defective growth phenotypes, and the interplay between ABCB transporters and BRI1-related membrane complexes has become a model of how transport and signaling are coordinated at the cell surface. Using bimolecular fluorescence complementation (BiFC), a technique in which two proteins are fused to halves of a fluorescent protein so that a physical interaction brings the halves together and restores fluorescence, together with a split-ubiquitin yeast two-hybrid assay that is better suited to membrane proteins, the Guangzhou University team tested whether the choy sum homologs could engage BRI1-related proteins. The answer was yes: BraABCB27 and BraABCB28, the two closest relatives of AtABCB1 and AtABCB19, showed detectable physical associations with BRI1-related proteins in both systems.

The authors are careful in their claims—and appropriately so. Detecting an association is not the same as demonstrating a transport function, and the study stops short of showing that BraABCB27 and BraABCB28 actually pump brassinosteroids or auxin in planta. What the work provides instead is a complete, experimentally grounded framework: a full inventory of 36 genes with their evolutionary history, a demonstration that Group IV members are stress- and hormone-responsive, confirmation of plasma-membrane targeting, and two strong candidates whose interaction with BRI1-related proteins now invites direct functional interrogation. The logical next steps—CRISPR knockout or overexpression of BraABCB27 and BraABCB28, followed by measurements of stalk elongation, brassinosteroid distribution and auxin gradients—would test whether these transporters are genuinely part of the machinery that regulates the trait for which choy sum is grown.

The broader implications reach beyond a single vegetable. Brassinosteroids have become a major focus of crop engineering because of their capacity to enhance yield, stress tolerance and architectural traits, and recent structural biology has revealed the atomic details of how the Arabidopsis ABCB1 and ABCB19 transporters export these steroids. Whether the same transport-signaling coupling exists in Brassica crops—and how the triplicated genome has diversified it—remains open, but this study supplies the map on which such questions can now be asked. For breeders seeking taller stalks, denser flowering or improved drought resilience in flowering Chinese cabbage, the BraABCB family just moved from anonymous genomic baggage to a shortlist of actionable targets. And for plant biologists more generally, the finding that ABCB-BRI1 associations are conserved in a distantly related crop reinforces an emerging picture: in plants, hormone transport and hormone perception are not separate layers of regulation but physically intertwined systems working at the same membrane, in the same cells, at the same time.

At the core of plant development, auxin orchestrates various physiological processes such as cell elongation, tissue patterning, and adaptive responses to environmental cues. It achieves these feats by modulating gene expression and cellular behaviors in a tightly regulated manner. However, auxin is a charged molecule at physiological pH, rendering its passive diffusion across the hydrophobic lipid bilayers of the plasma membrane highly inefficient. This limitation pointed researchers toward specialized carriers facilitating auxin transport—a hypothesis validated through years of genetic and biochemical studies implicating the AUX/LAX family as essential auxin influx facilitators.

Despite their recognized importance, the precise molecular architecture and transport modality of AUX/LAX proteins have been largely speculative due to the intrinsic challenges in resolving membrane protein structures. The team led by Ung et al. surmounted these barriers by employing cutting-edge cryo-electron microscopy (cryo-EM) combined with functional assays, culminating in high-resolution structural models of these transporters. These models illuminate the conformational dynamics and substrate interactions that underpin auxin recognition and translocation across the cell membrane.

The revealed structures demonstrate that AUX/LAX transporters share a canonical fold characteristic of the Amino acid-Polyamine-Organocation (APC) transporter superfamily, embodying a sophisticated mechanism of ligand binding coupled to alternating access conformational changes. This alternating access model posits that the transporter cycles between inward-facing and outward-facing states, enabling directional auxin movement driven by electrochemical gradients. Intriguingly, Snapshots along the transport cycle captured through structural analyses detail the transition states, highlighting key residues involved in auxin affinity and specificity.

Mechanistic dissection unveiled that AUX/LAX proteins operate via symport, coupling auxin import with protons (H+), exploiting the proton motive force characteristic of plant cell membranes. This proton-coupled transport is critical for maintaining directional auxin influx and cellular homeostasis. The study meticulously maps out the H+ binding sites and reveals how protonation events trigger conformational shifts facilitating substrate translocation. The elucidation of this proton-coupled mechanism refines previous models and reconciles biochemical data accumulated over the last several decades.

From an evolutionary perspective, comparative structural and sequence analyses disclosed that the AUX/LAX family has diverged in angiosperms to finely tune auxin uptake efficiency and regulation. The study identifies conserved motifs indispensable for function across species alongside variable regions potentially linked to differential regulation or tissue-specific expression patterns. This evolutionary insight bolsters the functional relevance of the transporter family and their role in adapting plant growth strategies to diverse ecological niches.

Functional assays incorporating site-directed mutagenesis further corroborate the structural findings. Mutation of several residues lining the auxin binding pocket or proton translocation pathway results in marked transport impairment, validating the predicted mechanistic roles. Additionally, heterologous expression in yeast and plant protoplasts coupled with auxin uptake measurements quantitatively demonstrate the impact of these mutations on transport kinetics, underscoring their physiological significance.

The authors also explore the dynamic interplay between AUX/LAX transporters and other auxin transport systems, particularly the PIN-FORMED (PIN) efflux carriers. The coordinated activity of influx and efflux carriers establishes directional auxin fluxes essential for morphogenesis and environmental responses. Insights into the structural basis of AUX/LAX function enrich models of auxin distribution in planta and suggest potential avenues to modulate transport activity via targeted genetic or chemical interventions.

Beyond fundamental biology, this work carries profound implications for agriculture and biotechnology. Modulating AUX/LAX transporter activity could enable the engineering of crops with optimized growth patterns, improved stress resilience, or altered architecture to enhance yield. Additionally, the structural blueprint offers a platform for rational design of small molecules capable of fine-tuning auxin transport, presenting novel agrochemical strategies for crop management.

The research exemplifies the power of integrative structural biology in deciphering complex biological systems. Leveraging advances in cryo-EM technology to resolve plant membrane transporters at near-atomic resolution represents a significant technical triumph, setting the stage for comprehensive studies of other elusive plant transporter families. It also illustrates the synergy achieved by integrating structural, biochemical, and functional methodologies, enabling multifaceted insights that transcend the limitations of individual approaches.

Looking forward, the study opens several exciting research directions. Unraveling how AUX/LAX transporters are regulated post-translationally, how they interact with cellular signaling networks, and how their expression is modulated during development or stress responses will be crucial next steps. Moreover, structural snapshots of transporter complexes with auxiliary proteins or regulators could shed light on the higher-order regulatory machinery controlling auxin import.

In conclusion, the structural elucidation of AUX/LAX transporters by Ung and colleagues constitutes a landmark achievement in plant biology. It demystifies the molecular gymnastics underpinning auxin import, a cornerstone process steering plant growth and adaptation. This work not only enriches our understanding of plant physiology but also ushers in new possibilities for harnessing auxin transport mechanisms to engineer future crops and address pressing agricultural challenges amid global environmental change.

Subject of Research: AUX/LAX transporters involved in auxin import in plants

Article Title: Structures and mechanism of the AUX/LAX transporters involved in auxin import

Article References:
Ung, K.L., Schulz, L., Zuzic, L. et al. Structures and mechanism of the AUX/LAX transporters involved in auxin import. Nat. Plants (2025). https://doi.org/10.1038/s41477-025-02056-z

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