Structure and function of the Arabidopsis ABC transporter ABCB19 in brassinosteroid export.

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.

Plasmodesmata mediate cell-to-cell transport of brassinosteroid hormones.

Brassinosteroids (BRs) are steroidal phytohormones that are essential for plant growth, development and adaptation to environmental stresses. BRs act in a dose-dependent manner and do not travel over long distances; hence, BR homeostasis maintenance is critical for their function. Biosynthesis of bioactive BRs relies on the cell-to-cell movement of hormone precursors. However, the mechanism of the short-distance BR transport is unknown, and its contribution to the control of endogenous BR levels remains unexplored. Here we demonstrate that plasmodesmata (PD) mediate the passage of BRs between neighboring cells. Intracellular BR content, in turn, is capable of modulating PD permeability to optimize its own mobility, thereby manipulating BR biosynthesis and signaling. Our work uncovers a thus far unknown mode of steroid transport in eukaryotes and exposes an additional layer of BR homeostasis regulation in plants.

Brassinosteroid gene regulatory networks at cellular resolution in the Arabidopsis root.

Brassinosteroids are plant steroid hormones that regulate diverse processes, such as cell division and cell elongation, through gene regulatory networks that vary in space and time. By using time series single-cell RNA sequencing to profile brassinosteroid-responsive gene expression specific to different cell types and developmental stages of the Arabidopsis root, we identified the elongating cortex as a site where brassinosteroids trigger a shift from proliferation to elongation associated with increased expression of cell wall-related genes. Our analysis revealed HOMEOBOX FROM ARABIDOPSIS THALIANA 7 (HAT7) and GT-2-LIKE 1 (GTL1) as brassinosteroid-responsive transcription factors that regulate cortex cell elongation. These results establish the cortex as a site of brassinosteroid-mediated growth and unveil a brassinosteroid signaling network regulating the transition from proliferation to elongation, which illuminates aspects of spatiotemporal hormone responses.

BRASSINOSTEROID INSENSITIVE1 internalization can occur independent of ligand binding.

The brassinosteroid (BR) hormone and its plasma membrane (PM) receptor BR INSENSITIVE1 (BRI1) are one of the best-studied receptor-ligand pairs for understanding the interplay between receptor endocytosis and signaling in plants. BR signaling is mainly determined by the PM pool of BRI1, whereas BRI1 endocytosis ensures signal attenuation. As BRs are ubiquitously distributed in the plant, the tools available to study the BRI1 function without interference from endogenous BRs are limited. Here, we designed a BR binding-deficient Arabidopsis (Arabidopsis thaliana) mutant based on protein sequence-structure analysis and homology modeling of members of the BRI1 family. This tool allowed us to re-examine the BRI1 endocytosis and signal attenuation model. We showed that despite impaired phosphorylation and ubiquitination, BR binding-deficient BRI1 internalizes similarly to the wild type form. Our data indicate that BRI1 internalization relies on different endocytic machineries. In addition, the BR binding-deficient mutant provides opportunities to study non-canonical ligand-independent BRI1 functions.

Plasma membrane phospholipid signature recruits the plant exocyst complex via the EXO70A1 subunit.

Polarized exocytosis is essential for many vital processes in eukaryotic cells, where secretory vesicles are targeted to distinct plasma membrane domains characterized by their specific lipid-protein composition. Heterooctameric protein complex exocyst facilitates the vesicle tethering to a target membrane and is a principal cell polarity regulator in eukaryotes. The architecture and molecular details of plant exocyst and its membrane recruitment have remained elusive. Here, we show that the plant exocyst consists of two modules formed by SEC3-SEC5-SEC6-SEC8 and SEC10-SEC15-EXO70-EXO84 subunits, respectively, documenting the evolutionarily conserved architecture within eukaryotes. In contrast to yeast and mammals, the two modules are linked by a plant-specific SEC3-EXO70 interaction, and plant EXO70 functionally dominates over SEC3 in the exocyst recruitment to the plasma membrane. Using an interdisciplinary approach, we found that the C-terminal part of EXO70A1, the canonical EXO70 isoform in Arabidopsis, is critical for this process. In contrast to yeast and animal cells, the EXO70A1 interaction with the plasma membrane is mediated by multiple anionic phospholipids uniquely contributing to the plant plasma membrane identity. We identified several evolutionary conserved EXO70 lysine residues and experimentally proved their importance for the EXO70A1-phospholipid interactions. Collectively, our work has uncovered plant-specific features of the exocyst complex and emphasized the importance of the specific protein-lipid code for the recruitment of peripheral membrane proteins.

Local brassinosteroid biosynthesis enables optimal root growth.

Brassinosteroid (BR) hormones are indispensable for root growth and control both cell division and cell elongation through the establishment of an increasing signalling gradient along the longitudinal root axis. Because of their limited mobility, the importance of BR distribution in achieving a signalling maximum is largely overlooked. Expression pattern analysis of all known BR biosynthetic enzymes revealed that not all cells in the Arabidopsis thaliana root possess full biosynthetic machinery, and that completion of biosynthesis relies on cell-to-cell movement of hormone precursors. We demonstrate that BR biosynthesis is largely restricted to the root elongation zone, where it overlaps with BR signalling maxima. Moreover, optimal root growth requires hormone concentrations to be low in the meristem and high in the root elongation zone, attributable to increased biosynthesis. Our finding that spatiotemporal regulation of hormone synthesis results in local hormone accumulation provides a paradigm for hormone-driven organ growth in the absence of long-distance hormone transport in plants.

Brassinosteroids: Multidimensional Regulators of Plant Growth, Development, and Stress Responses.

Brassinosteroids (BRs) are a group of polyhydroxylated plant steroid hormones that are crucial for many aspects of a plant's life. BRs were originally characterized for their function in cell elongation, but it is becoming clear that they play major roles in plant growth, development, and responses to several stresses such as extreme temperatures and drought. A BR signaling pathway from cell surface receptors to central transcription factors has been well characterized. Here, we summarize recent progress toward understanding the BR pathway, including BR perception and the molecular mechanisms of BR signaling. Next, we discuss the roles of BRs in development and stress responses. Finally, we show how knowledge of the BR pathway is being applied to manipulate the growth and stress responses of crops. These studies highlight the complex regulation of BR signaling, multiple points of crosstalk between BRs and other hormones or stress responses, and the finely tuned spatiotemporal regulation of BR signaling.

BRexit: Possible Brassinosteroid Export and Transport Routes.

The movement and differential distribution of endogenous plant hormones are the determining factors for many developmental processes. Brassinosteroids (BRs) are a group of plant steroidal hormones that promote growth and development. Although synthesis and signalling of BRs are well described and characterized, the exit mechanism of these compounds from the cell remains uncharacterizd. Whether BRs are able to move within the apoplast and whether the BR synthesis in one cell can trigger the signalling in its neighbours is also unknown. Here, we draw the attention to these aspects of the BR biology, propose several BR cell export routes and discuss possible consequences of local BR hormonal gradients, resulting from localized biosynthesis and a short-distance transport, for plant development.

EXO70C2 Is a Key Regulatory Factor for Optimal Tip Growth of Pollen.

The exocyst, a eukaryotic tethering complex, coregulates targeted exocytosis as an effector of small GTPases in polarized cell growth. In land plants, several exocyst subunits are encoded by double or triple paralogs, culminating in tens of EXO70 paralogs. Out of 23 Arabidopsis thaliana EXO70 isoforms, we analyzed seven isoforms expressed in pollen. Genetic and microscopic analyses of single mutants in EXO70A2, EXO70C1, EXO70C2, EXO70F1, EXO70H3, EXO70H5, and EXO70H6 genes revealed that only a loss-of-function EXO70C2 allele resulted in a significant male-specific transmission defect (segregation 40%:51%:9%) due to aberrant pollen tube growth. Mutant pollen tubes grown in vitro exhibited an enhanced growth rate and a decreased thickness of the tip cell wall, causing tip bursts. However, exo70C2 pollen tubes could frequently recover and restart their speedy elongation, resulting in a repetitive stop-and-go growth dynamics. A pollen-specific depletion of the closest paralog, EXO70C1, using artificial microRNA in the exo70C2 mutant background, resulted in a complete pollen-specific transmission defect, suggesting redundant functions of EXO70C1 and EXO70C2. Both EXO70C1 and EXO70C2, GFP tagged and expressed under the control of their native promoters, localized in the cytoplasm of pollen grains, pollen tubes, and also root trichoblast cells. The expression of EXO70C2-GFP complemented the aberrant growth of exo70C2 pollen tubes. The absent EXO70C2 interactions with core exocyst subunits in the yeast two-hybrid assay, cytoplasmic localization, and genetic effect suggest an unconventional EXO70 function possibly as a regulator of exocytosis outside the exocyst complex. In conclusion, EXO70C2 is a novel factor contributing to the regulation of optimal tip growth of Arabidopsis pollen tubes.

Analysis of Exocyst Subunit EXO70 Family Reveals Distinct Membrane Polar Domains in Tobacco Pollen Tubes.

The vesicle-tethering complex exocyst is one of the crucial cell polarity regulators. The EXO70 subunit is required for the targeting of the complex and is represented by many isoforms in angiosperm plant cells. This diversity could be partly responsible for the establishment and maintenance of membrane domains with different composition. To address this hypothesis, we employed the growing pollen tube, a well-established cell polarity model system, and performed large-scale expression, localization, and functional analysis of tobacco (Nicotiana tabacum) EXO70 isoforms. Various isoforms localized to different regions of the pollen tube plasma membrane, apical vesicle-rich inverted cone region, nucleus, and cytoplasm. The overexpression of major pollen-expressed EXO70 isoforms resulted in growth arrest and characteristic phenotypic deviations of tip swelling and apical invaginations. NtEXO70A1a and NtEXO70B1 occupied two distinct and mutually exclusive plasma membrane domains. Both isoforms partly colocalized with the exocyst subunit NtSEC3a at the plasma membrane, possibly forming different exocyst complex subpopulations. NtEXO70A1a localized to the small area previously characterized as the site of exocytosis in the tobacco pollen tube, while NtEXO70B1 surprisingly colocalized with the zone of clathrin-mediated endocytosis. Both NtEXO70A1a and NtEXO70B1 colocalized to different degrees with markers for the anionic signaling phospholipids phosphatidylinositol 4,5-bisphosphate and phosphatidic acid. In contrast, members of the EXO70 C class, which are specifically expressed in tip-growing cells, exhibited exocytosis-related functional effects in pollen tubes despite the absence of apparent plasma membrane localization. Taken together, our data support the existence of multiple membrane-trafficking domains regulated by different EXO70-containing exocyst complexes within a single cell.

Microtubule-dependent targeting of the exocyst complex is necessary for xylem development in Arabidopsis.

Cortical microtubules (MTs) play a major role in the patterning of secondary cell wall (SCW) thickenings in tracheary elements (TEs) by determining the sites of SCW deposition. The EXO70A1 subunit of the exocyst secretory vesicle tethering complex was implicated to be important for TE development via the MT interaction. We investigated the subcellular localization of several exocyst subunits in the xylem of Arabidopsis thaliana and analyzed the functional significance of exocyst-mediated trafficking in TE development. Live cell imaging of fluorescently tagged exocyst subunits in TE using confocal microscopy and protein-protein interaction assays were performed to describe the role of the exocyst and its partners in TE development. In TEs, exocyst subunits were localized to the sites of SCW deposition in an MT-dependent manner. We propose that the mechanism of exocyst targeting to MTs involves the direct interaction of exocyst subunits with the COG2 protein. We demonstrated the importance of a functional exocyst subunit EXO84b for normal TE development and showed that the deposition of SCW constituents is partially compromised, possibly as a result of the mislocalization of secondary cellulose synthase in exocyst mutants. We conclude that the exocyst complex is an important factor bridging the pattern defined by cortical MTs with localized secretion of the SCW in developing TEs.

Exocyst SEC3 and Phosphoinositides Define Sites of Exocytosis in Pollen Tube Initiation and Growth.

Polarized exocytosis is critical for pollen tube growth, but its localization and function are still under debate. The exocyst vesicle-tethering complex functions in polarized exocytosis. Here, we show that a sec3a exocyst subunit null mutant cannot be transmitted through the male gametophyte due to a defect in pollen tube growth. The green fluorescent protein (GFP)-SEC3a fusion protein is functional and accumulates at or proximal to the pollen tube tip plasma membrane. Partial complementation of sec3a resulted in the development of pollen with multiple tips, indicating that SEC3 is required to determine the site of pollen germination pore formation. Time-lapse imaging demonstrated that SEC3a and SEC8 were highly dynamic and that SEC3a localization on the apical plasma membrane predicts the direction of growth. At the tip, polar SEC3a domains coincided with cell wall deposition. Labeling of GFP-SEC3a-expressing pollen with the endocytic marker FM4-64 revealed the presence of subdomains on the apical membrane characterized by extensive exocytosis. In steady-state growing tobacco (Nicotiana tabacum) pollen tubes, SEC3a displayed amino-terminal Pleckstrin homology-like domain (SEC3a-N)-dependent subapical membrane localization. In agreement, SEC3a-N interacted with phosphoinositides in vitro and colocalized with a phosphatidylinositol 4,5-bisphosphate (PIP2) marker in pollen tubes. Correspondingly, molecular dynamics simulations indicated that SEC3a-N associates with the membrane by interacting with PIP2 However, the interaction with PIP2 is not required for polar localization and the function of SEC3a in Arabidopsis (Arabidopsis thaliana). Taken together, our findings indicate that SEC3a is a critical determinant of polar exocytosis during tip growth and suggest differential regulation of the exocytotic machinery depending on pollen tube growth modes.

Tethering Complexes in the Arabidopsis Endomembrane System.

Targeting of endomembrane transport containers is of the utmost importance for proper land plant growth and development. Given the immobility of plant cells, localized membrane vesicle secretion and recycling are amongst the main processes guiding proper cell, tissue and whole plant morphogenesis. Cell wall biogenesis and modification are dependent on vectorial membrane traffic, not only during normal development, but also in stress responses and in plant defense against pathogens and/or symbiosis. It is surprising how little we know about these processes in plants, from small GTPase regulation to the tethering complexes that act as their effectors. Tethering factors are single proteins or protein complexes mediating first contact between the target membrane and arriving membrane vesicles. In this review we focus on the tethering complexes of the best-studied plant model-Arabidopsis thaliana. Genome-based predictions indicate the presence of all major tethering complexes in plants that are known from a hypothetical last eukaryotic common ancestor (LECA). The evolutionary multiplication of paralogs of plant tethering complex subunits has produced the massively expanded EXO70 family, indicating a subfunctionalization of the terminal exocytosis machinery in land plants. Interpretation of loss of function (LOF) mutant phenotypes has to consider that related, yet clearly functionally-specific complexes often share some common core subunits. It is therefore impossible to conclude with clarity which version of the complex is responsible for the phenotypic deviations observed. Experimental interest in the analysis of plant tethering complexes is growing and we hope to contribute with this review by attracting even more attention to this fascinating field of plant cell biology.

Dissecting a hidden gene duplication: the Arabidopsis thaliana SEC10 locus.

Repetitive sequences present a challenge for genome sequence assembly, and highly similar segmental duplications may disappear from assembled genome sequences. Having found a surprising lack of observable phenotypic deviations and non-Mendelian segregation in Arabidopsis thaliana mutants in SEC10, a gene encoding a core subunit of the exocyst tethering complex, we examined whether this could be explained by a hidden gene duplication. Re-sequencing and manual assembly of the Arabidopsis thaliana SEC10 (At5g12370) locus revealed that this locus, comprising a single gene in the reference genome assembly, indeed contains two paralogous genes in tandem, SEC10a and SEC10b, and that a sequence segment of 7 kb in length is missing from the reference genome sequence. Differences between the two paralogs are concentrated in non-coding regions, while the predicted protein sequences exhibit 99% identity, differing only by substitution of five amino acid residues and an indel of four residues. Both SEC10 genes are expressed, although varying transcript levels suggest differential regulation. Homozygous T-DNA insertion mutants in either paralog exhibit a wild-type phenotype, consistent with proposed extensive functional redundancy of the two genes. By these observations we demonstrate that recently duplicated genes may remain hidden even in well-characterized genomes, such as that of A. thaliana. Moreover, we show that the use of the existing A. thaliana reference genome sequence as a guide for sequence assembly of new Arabidopsis accessions or related species has at least in some cases led to error propagation.