Research

Primary Areas of Interest

We are interested in the molecular factors regulating plant hormones transport mechanisms. We study the first events of cellular signaling - all the way to patterning of the whole plant, primarily focusing on the plant hormones auxin and gibberellin (GA). To facilitate this, we combine state-of-the-art genetics, molecular biology, chemistry and imaging approaches.

Plant Hormone Transport

Plant growth, development, and response to the environment are mediated by a group of small signaling molecules named hormones. Plants regulate hormone response pathways at multiple levels, including biosynthesis, metabolism, perception, and signaling. In addition, plants exhibit the unique ability to spatially control hormone distribution. In recent years, multiple transporters have been identified for most of the plant hormones.

Our group recently identified several novel hormone transporters:

 

ABCB6,20 auxin transporters (Zhang et al., 2018 Nature Comm).

 

ABCB15-22  auxin transporters (Chen al., 2020 BioRxiv).

ABCG17,18 ABA transporters (Zhang et al., 2021 Science Advances).

 

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Genome-scale, multi-targeted, CRISPR- and amiRNA based, forward-genetics approach to uncover inaccessible genetic variation in plants

Plant genomes are highly redundant. For example, 75% of Arabidopsis genes (22,020 of the ~25,500 total genes) belong to families with at least two members. As a result, most single null mutants do not present an evident phenotype as the overlapping function of one or more paralogs mask any effects. During the past two decades, genetic variation and forward genetics screen have been expanded by creating random mutagenized lines using chemical or radiation treatments leading to the identification of novel genetic processes. However, these approaches cannot overcome the genetic redundancy problem a large fraction of the potential phenotypic plasticity is “hidden”.

Our group addresses the problem of masked genetic variation due to functional redundancy by developing a “second generation” genetic approach in plants, combining forward-genetics with dynamically targeted genome-scale tools. The new genetic system uses genome-scale artificial microRNAs and CRISPR technologies to simultaneously targets several genes within the same family in plants. 

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Single-cell kinetics of hormone transport and activity

We are developing toolboxes to study hormone-mediated root response and movement that features: (i) the ability to control the hormone synthesis with high spatio-temporal resolution and (ii) single-cell nucleus tracking and morphokinetic analysis infrastructure.

Integration of these two features enables cutting-edge analysis of root development at a single-cell resolution based on morphokinetic parameters under normal growth conditions and during cell-type-specific induction of the hormone biosynthesis.

Using this approach we recently mapped directional auxin flow in the root and refined the contributions of key players in this process.

 

In addition, single-cell tracking allowed us to determine the quantitative kinetics of Arabidopsis root meristem skewing  (Hu et al., 2021, Nature Comm)

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ABA homeostasis and long-distance translocation

Abscisic acid (ABA) is a plant hormone that regulates growth and responses to the changing environment. For example, seed dormancy, germination, drought tolerance, stomatal closure and lateral root emergence are modulated by this important hormone under normal conditions and in response to stimuli.

We recently exploited a genome-scale amiRNA screen targeting the Arabidopsis transportome to identify novel ABCG ABA transporters. ABCG17 and ABCG18 are localized to the plasma membrane and import ABA. ABCG17- and ABCG18-knockdown lines displayed enhanced ABA accumulation, induced ABA response, and reduced stomatal aperture size, conductance, and transpiration with increased water-use efficiency compared to WT. In addition, seedlings with double-loss-of-function of ABCG17 and ABCG18 showed enhanced long-distance ABA translocation from the shoot to the root, which led to lateral root outgrowth inhibition. ABCG17 and ABCG18 are primarily expressed in the shoot mesophyll and stem cortex cells, where they drive ABA import, allowing ABA-GE formation. In turn, these ABA-GE sinks prevent active ABA accumulation in guard cells and limit ABA long-distance travel to lateral root formation sites. Under abiotic stress conditions, ABCG17 and ABCG18 are transcriptionally repressed, promoting active ABA movement, accumulation, and response  (Zhang et al., 2021, Sciense Advances)

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Gibberellin localization and transport in plants

Distribution patterns and finely tuned concentration gradients of plant hormones govern plant growth and development. Gibberellin (GA) is a plant hormone that regulates key processes in plants, many of them are of significant agricultural importance, such as seed germination, root and shoot elongation, flowering, and fruit patterning. Although studies have demonstrated that GA movement is essential for multiple developmental aspects, how GAs are transported throughout the plant and where exactly does it accumulates remain largely unknown.

Binenbaum et al., 2018, Trends in Plant Science.

Our lab has identified NPF3 is a GA and ABA transporter. NPF3 knockout plants are defective in the accumulation of both GA3-Fl and GA4-Fl in the root endodermis elongation zone. Transport assays in Xenopus oocytes demonstrated that NPF3 can import GAs across membranes.

Tal et al., 2016, Nature Comm

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Auxin transport

Auxin (IAA) is a small organic molecule that coordinates many of the key processes in plant development and adaptive growth. Plants regulate the auxin response pathways at multiple levels including biosynthesis, metabolism, and perception. 

In addition, plants exhibit the unique ability to spatially regulate hormone distribution. This ability is illustrated most clearly in the case of auxin (IAA). The combined activity of auxin influx and efflux carrier proteins generates auxin maxima and local gradients that inform developmental patterning. The regulation of the cellular localization of PIN-FORMED (PIN) efflux transporters determines the direction of auxin flow from one cell to another.

Using a transportome, multi-targeted forward-genetic screen artificial microRNAs (amiRNAs) approach we reviled ABCB6 and ABCB20 genes as auxin transport required for shoot growth (Zhang et al., 2018, Nature Comm) and ABCB15-22 as

auxin transport in the outer tissues of the root meristem to regulate lateral root spacing (Chen et al., 2020, BioRxiv).

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