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Functional study of plant hormone transporters using a heterologous system
Release time:
2025-06-04
Plant hormones are key signaling molecules that regulate plant growth, development, morphogenesis, and responses to environmental stresses. These hormones are often synthesized in specific tissues and transported via the vascular system to other organs where they exert their functions. In some cases, plant hormones are synthesized intracellularly and subsequently transported to neighboring cells.
In recent years, numerous plant hormone transporters have been identified and functionally characterized. However, due to the complexity of hormone transport mechanisms, studying these transporters in native plant systems remains challenging. Heterologous systems have long played an important role in biological research. Owing to limitations such as the lack of a stable genetic transformation system and long life cycles in some species, it is often difficult to carry out functional studies in the native host. By introducing target genes into non-native hosts, heterologous systems enable the investigation of specific protein functions in a simplified background.
Currently, a variety of systems have been used to study the function of plant hormone transporters in a heterologous context, with yeast and Xenopus oocytes being the most common. After expressing transporter genes in Xenopus oocytes or mutant yeast strains, hormone levels inside and outside the cells can be quantified using gas or liquid chromatography coupled with mass spectrometry (GC-MS or LC-MS), allowing researchers to assess the transport activity of these proteins toward their substrates.
Yeast system
In plants, members of transporter protein families often exhibit functional redundancy. As a result, gene knockout mutants frequently fail to display obvious phenotypes due to compensatory effects, making functional analysis challenging.
Similar to the Xenopus oocyte system, the traditional approach for studying transporters in yeast involves functional complementation using mutant yeast strains deficient in specific transporters. The transport activity of the introduced plant transporter can then be assessed by quantifying intra- and extracellular hormone levels using gas or liquid chromatography coupled with mass spectrometry (GC-MS or LC-MS).
A study published in Plant Physiology in 2024, titled "Abscisic acid root-to-shoot translocation by transporter AtABCG25 mediates stomatal movements in Arabidopsis", employed a yeast heterologous expression system to investigate the function of the AtABCG25 transporter. Yeast cells expressing AtABCG25 were cultured in a medium containing deuterium-labeled abscisic acid (²H₆-ABA) and ABA-glucose ester (ABA-GE). The extracellular concentrations of ABA were then quantified using HPLC-MS/MS. Compared to control yeast carrying an empty vector, AtABCG25-expressing yeast showed significantly higher levels of ABA and ABA-GE in the culture medium. These results demonstrate that AtABCG25 functions as an efflux transporter for both ABA and ABA-GE.
Figure . Analysis of ABA and ABA-GE efflux mediated by AtABCG25 in yeast
However, with the development of a specialized yeast two-hybrid system, it has become possible to investigate the function of plant hormone transporters more indirectly and elegantly.
In 2012, a study published in Proceedings of the National Academy of Sciences (PNAS), titled "Identification of an abscisic acid transporter by functional screening using the receptor complex as a sensor", developed a yeast two-hybrid system to indirectly identify abscisic acid (ABA) transporters. The system was based on ABA-dependent interactions between ABA receptors (PYR/PYL/RCAR) and clade A PP2C-type protein phosphatases. In this setup, the PP2C protein was fused to the GAL4 activation domain (AD), while the ABA receptor was fused to the GAL4 DNA-binding domain (BD), with HIS3 used as a reporter gene. A cDNA library from Arabidopsis thaliana was co-transformed into yeast cells along with the sensor system. If an ABA transporter was present in the cDNA library, it would mediate ABA uptake into the yeast cell, triggering an ABA-dependent interaction between the receptor and the PP2C, thereby activating reporter gene expression and allowing yeast growth on histidine-deficient medium.
Using this screening system, four candidate ABA transporter genes (AITs) were identified: At1g69850 (NRT1.2), At1g27040, At3g25260, and At3g25280.
Figure . Influence of NRT1/PTR family members on ABA-dependent interaction between AD-ABI1 and BD-PYR1
Subsequent studies measured the intracellular ABA levels in yeast strains transformed with an empty vector, AIT1, and AIT3, confirming that both AIT1 and AIT3 mediate ABA uptake into yeast cells.
Figure . Intracellular ABA content in yeast treated with varying ABA concentrations
The yeast system can be employed not only to study hormone uptake transporters but also to investigate hormone efflux transporters.
In 2024, a study published in Molecular Plant, titled "Endomembrane-biased dimerization of ABCG16 and ABCG25 transporters determines their substrate selectivity in ABA-regulated plant growth and stress responses," utilized a yeast two-hybrid system based on the interaction between two proteins, PYR1 and ABI1, which is promoted by low concentrations of ABA and inhibited by high concentrations of ABA.
Under high ABA treatment, yeast cells transformed with the ABCG16 gene exhibited better growth compared to the empty vector (M52) control. This result indicates that ABCG16 likely functions as an ABA efflux transporter, reducing intracellular ABA concentration by exporting ABA out of the yeast cells. Consequently, this alleviates the growth inhibition caused by high ABA concentrations interfering with the PYR1-ABI1 interaction, supporting the role of ABCG16 as a mediator of ABA efflux.
Figure .Yeast two-hybrid system validation of ABCG16-mediated cellular ABA efflux
Xenopus laevis oocytes
Functional studies of transport proteins using Xenopus laevis oocytes involve expressing the transporter gene in the oocytes, followed by tracer analysis of plant hormone molecules inside and outside the cells. The transport activity of the protein is assessed by measuring the hormone concentrations using gas/liquid chromatography coupled with mass spectrometry (GC/LC-MS).
A study published in Nature Plants in 2023, titled "Cryo-EM structure and molecular mechanism of abscisic acid transporter ABCG25," validated the ABA transport function of AtABCG25 using Xenopus laevis oocytes. The researchers injected complementary RNA (cRNA) encoding GFP-AtABCG25 into the oocytes, incubated the oocytes in ABA-containing buffer, and subsequently measured the intracellular ABA content using liquid chromatography-mass spectrometry (LC-MS). The results demonstrated that expression of AtABCG25 promoted substantial export of ABA from the oocytes.
Figure . Assessment of ABA transport activity of AtABCG25
This review introduces the role of heterologous systems in studying the function of plant hormone transporters. The underlying principle involves the movement of plant hormones across membranes mediated by transport proteins. By expressing the target transporter protein in a heterologous system, researchers can determine whether a protein functions as a hormone transporter by analyzing changes in hormone concentrations on either side of the membrane. This approach simplifies the research background and shortens the experimental cycle for studying plant hormone transporters.
Furthermore, combined with the yeast two-hybrid screening method, this system not only allows functional validation of candidate transporters but also enables the identification of potential novel transporters. This ingenious strategy has greatly expanded the scope of plant hormone transporter research. It is hoped that such innovative ideas will provide useful insights and aid future studies in this field.
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