Highlights in gibberellin research: A tale of the dwarf and the slender.

It has been almost a century since biologically active gibberellin (GA) was isolated. Here, we give a historical overview of the early efforts in establishing the GA biosynthesis and catabolism pathway, characterizing the enzymes for GA metabolism, and elucidating their corresponding genes. We then highlight more recent studies that have identified the GA receptors and early GA signaling components (DELLA repressors and F-box activators), determined the molecular mechanism of DELLA-mediated transcription reprograming, and revealed how DELLAs integrate multiple signaling pathways to regulate plant vegetative and reproductive development in response to internal and external cues. Finally, we discuss the GA transporters and their roles in GA-mediated plant development.

Cytokinin activity - transport and homeostasis at the whole plant, cell, and subcellular levels.

Cytokinins (CKs) are important plant hormones that regulate a variety of biological processes implicated in plant development and stress responses. Here, we summarize the most recent advances in discovering and characterizing the membrane transporters involved in long- and short-distance translocation of CKs and their significance in CK signal activity. We highlight the discovery of PUP7 and PUP21 tonoplast-localized transporters and propose potential mechanisms for CK subcellular homeostasis. Finally, we discuss the importance of subcellular hormone transport in light of the localization of histidine kinase receptors of CKs at the endoplasmic reticulum and plasma membrane.

Gibberellin and abscisic acid transporters facilitate endodermal suberin formation in Arabidopsis.

The plant hormone gibberellin (GA) regulates multiple developmental processes. It accumulates in the root elongating endodermis, but how it moves into this cell file and the significance of this accumulation are unclear. Here we identify three NITRATE TRANSPORTER1/PEPTIDE TRANSPORTER (NPF) transporters required for GA and abscisic acid (ABA) translocation. We demonstrate that NPF2.14 is a subcellular GA/ABA transporter, presumably the first to be identified in plants, facilitating GA and ABA accumulation in the root endodermis to regulate suberization. Further, NPF2.12 and NPF2.13, closely related proteins, are plasma membrane-localized GA and ABA importers that facilitate shoot-to-root GA12 translocation, regulating endodermal hormone accumulation. This work reveals that GA is required for root suberization and that GA and ABA can act non-antagonistically. We demonstrate how the clade of transporters mediates hormone flow with cell-file-specific vacuolar storage at the phloem unloading zone, and slow release of hormone to induce suberin formation in the maturation zone.

Plant Hormone Transport and Localization: Signaling Molecules on the Move.

Plant hormones are a group of small signaling molecules produced by plants at very low concentrations that have the ability to move and function at distal sites. Hormone homeostasis is critical to balance plant growth and development and is regulated at multiple levels, including hormone biosynthesis, catabolism, perception, and transduction. In addition, plants move hormones over short and long distances to regulate various developmental processes and responses to environmental factors. Transporters coordinate these movements, resulting in hormone maxima, gradients, and cellular and subcellular sinks. Here, we summarize the current knowledge of most of the characterized plant hormone transporters with respect to biochemical, physiological, and developmental activities. We further discuss the subcellular localizations of transporters, their substrate specificities, and the need for multiple transporters for the same hormone in the context of plant growth and development.

Multi-Knock-a multi-targeted genome-scale CRISPR toolbox to overcome functional redundancy in plants.

Plant genomes are characterized by large and complex gene families that often result in similar and partially overlapping functions. This genetic redundancy severely hampers current efforts to uncover novel phenotypes, delaying basic genetic research and breeding programmes. Here we describe the development and validation of Multi-Knock, a genome-scale clustered regularly interspaced short palindromic repeat toolbox that overcomes functional redundancy in Arabidopsis by simultaneously targeting multiple gene-family members, thus identifying genetically hidden components. We computationally designed 59,129 optimal single-guide RNAs that each target two to ten genes within a family at once. Furthermore, partitioning the library into ten sublibraries directed towards a different functional group allows flexible and targeted genetic screens. From the 5,635 single-guide RNAs targeting the plant transportome, we generated over 3,500 independent Arabidopsis lines that allowed us to identify and characterize the first known cytokinin tonoplast-localized transporters in plants. With the ability to overcome functional redundancy in plants at the genome-scale level, the developed strategy can be readily deployed by scientists and breeders for basic research and to expedite breeding efforts.

ABCB-mediated shootward auxin transport feeds into the root clock.

Although strongly influenced by environmental conditions, lateral root (LR) positioning along the primary root appears to follow obediently an internal spacing mechanism dictated by auxin oscillations that prepattern the primary root, referred to as the root clock. Surprisingly, none of the hitherto characterized PIN- and ABCB-type auxin transporters seem to be involved in this LR prepatterning mechanism. Here, we characterize ABCB15, 16, 17, 18, and 22 (ABCB15-22) as novel auxin-transporting ABCBs. Knock-down and genome editing of this genetically linked group of ABCBs caused strongly reduced LR densities. These phenotypes were correlated with reduced amplitude, but not reduced frequency of the root clock oscillation. High-resolution auxin transport assays and tissue-specific silencing revealed contributions of ABCB15-22 to shootward auxin transport in the lateral root cap (LRC) and epidermis, thereby explaining the reduced auxin oscillation. Jointly, these data support a model in which LRC-derived auxin contributes to the root clock amplitude.

Hydraulic flux-responsive hormone redistribution determines root branching.

Plant roots exhibit plasticity in their branching patterns to forage efficiently for heterogeneously distributed resources, such as soil water. The xerobranching response represses lateral root formation when roots lose contact with water. Here, we show that xerobranching is regulated by radial movement of the phloem-derived hormone abscisic acid, which disrupts intercellular communication between inner and outer cell layers through plasmodesmata. Closure of these intercellular pores disrupts the inward movement of the hormone signal auxin, blocking lateral root branching. Once root tips regain contact with moisture, the abscisic acid response rapidly attenuates. Our study reveals how roots adapt their branching pattern to heterogeneous soil water conditions by linking changes in hydraulic flux with dynamic hormone redistribution.

ABA homeostasis and long-distance translocation are redundantly regulated by ABCG ABA importers.

The effects of abscisic acid (ABA) on plant growth, development, and response to the environment depend on local ABA concentrations. Here, we show that in Arabidopsis, ABA homeostasis is regulated by two previously unknown ABA transporters. Adenosine triphosphate­binding cassette subfamily G member 17 (ABCG17) and ABCG18 are localized to the plasma membranes of leaf mesophyll and cortex cells to redundantly promote ABA import, leading to conjugated inactive ABA sinks, thus restricting stomatal closure. ABCG17 and ABCG18 double knockdown revealed that the transporters encoded by these genes not only limit stomatal aperture size, conductance, and transpiration while increasing water use efficiency but also control ABA translocation from the shoot to the root to regulate lateral root emergence. Under abiotic stress conditions, ABCG17 and ABCG18 are transcriptionally repressed, promoting active ABA movement and response. The transport mechanism mediated by ABCG17 and ABCG18 allows plants to maintain ABA homeostasis under normal growth conditions.

Transport mechanisms of plant hormones.

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. Here we present an updated snapshot of the known transporters for the hormones abscisic acid, auxin, brassinosteroid, cytokinin, ethylene, gibberellin, jasmonic acid, salicylic acid, and strigolactone. We also describe new findings regarding hormone movement and elaborate on hormone substrate specificity and possible genetic redundancy in hormone transport and distribution. Finally, we discuss subcellular, cell-to-cell, and long-distance hormone movement and local hormone sinks that trigger or prevent hormone-mediated responses.

The GORKY glycoalkaloid transporter is indispensable for preventing tomato bitterness.

Fruit taste is determined by sugars, acids and in some species, bitter chemicals. Attraction of seed-dispersing organisms in nature and breeding for consumer preferences requires reduced fruit bitterness. A key metabolic shift during ripening prevents tomato fruit bitterness by eliminating α-tomatine, a renowned defence-associated Solanum alkaloid. Here, we combined fine mapping with information from 150 resequenced genomes and genotyping a 650-tomato core collection to identify nine bitter-tasting accessions including the 'high tomatine' Peruvian landraces reported in the literature. These 'bitter' accessions contain a deletion in GORKY, a nitrate/peptide family transporter mediating α-tomatine subcellular localization during fruit ripening. GORKY exports α-tomatine and its derivatives from the vacuole to the cytosol and this facilitates the conversion of the entire α-tomatine pool to non-bitter forms, rendering the fruit palatable. Hence, GORKY activity was a notable innovation in the process of tomato fruit domestication and breeding.

Cell kinetics of auxin transport and activity in Arabidopsis root growth and skewing.

Auxin is a key regulator of plant growth and development. Local auxin biosynthesis and intercellular transport generates regional gradients in the root that are instructive for processes such as specification of developmental zones that maintain root growth and tropic responses. Here we present a toolbox to study auxin-mediated root development that features: (i) the ability to control auxin 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 single-cell resolution based on morphokinetic parameters under normal growth conditions and during cell-type-specific induction of auxin biosynthesis. We show directional auxin flow in the root and refine the contributions of key players in this process. In addition, we determine the quantitative kinetics of Arabidopsis root meristem skewing, which depends on local auxin gradients but does not require PIN2 and AUX1 auxin transporter activities. Beyond the mechanistic insights into root development, the tools developed here will enable biologists to study kinetics and morphology of various critical processes at the single cell-level in whole organisms.

Cell-type action specificity of auxin on Arabidopsis root growth.

The plant hormone auxin plays a critical role in root growth and development; however, the contributions or specific roles of cell-type auxin signals in root growth and development are not well understood. Here, we mapped tissue and cell types that are important for auxin-mediated root growth and development by manipulating the local response and synthesis of auxin. Repressing auxin signaling in the epidermis, cortex, endodermis, pericycle or stele strongly inhibited root growth, with the largest effect observed in the endodermis. Enhancing auxin signaling in the epidermis, cortex, endodermis, pericycle or stele also caused reduced root growth, albeit to a lesser extent. Moreover, we established that root growth was inhibited by enhancement of auxin synthesis in specific cell types of the epidermis, cortex and endodermis, whereas increased auxin synthesis in the pericycle and stele had only minor effects on root growth. Our study thus establishes an association between cellular identity and cell type-specific auxin signaling that guides root growth and development.

Characterizing gibberellin flow in planta using photocaged gibberellins.

Gibberellins (GAs) are ubiquitous plant hormones that coordinate central developmental and adaptive growth processes in plants. Accurate movement of GAs throughout the plant from their sources to their destination sites is emerging to be a highly regulated and directed process. We report on the development of novel photocaged gibberellins that, in combination with a genetically encoded GA-response marker, provide a unique platform to study GA movement at high-resolution, in real time and in living, intact plants. By applying this platform to the Arabidopsis thaliana endogenous bioactive gibberellin GA4, we measure kinetic parameters of its flow, such as decay length and velocity, in vivo.

A seed resource for screening functionally redundant genes and isolation of new mutants impaired in CO2 and ABA responses.

The identification of homologous genes with functional overlap in forward genetic screens is severely limited. Here, we report the generation of over 14000 artificial microRNA (amiRNA)-expressing plants that enable screens of the functionally redundant gene space in Arabidopsis. A protocol was developed for isolating robust and reproducible amiRNA mutants. Examples of validation approaches and essential controls are presented for two new amiRNA mutants that exhibit genetically redundant phenotypes and circumvent double mutant lethality. In a forward genetic screen for abscisic acid (ABA)-mediated inhibition of seed germination, amiRNAs that target combinations of known redundant ABA receptor and SnRK2 kinase genes were rapidly isolated, providing a strong proof of principle for this approach. A new ABA-insensitive amiRNA line that targets three avirulence-induced gene 2(-like) genes was isolated . A thermal imaging screen for plants with impaired stomatal opening in response to low CO2 exposure led to the isolation of a new amiRNA targeting two essential proteasomal subunits, PAB1 and PAB2. The seed library of 11000 T2 amiRNA lines (with 3000 lines in progress) generated here provides a new platform for forward genetic screens and has been made available to the Arabidopsis Biological Resource Center (ABRC). Optimized procedures for amiRNA screening and controls are described.

A transportome-scale amiRNA-based screen identifies redundant roles of Arabidopsis ABCB6 and ABCB20 in auxin transport.

Transport of signaling molecules is of major importance for regulating plant growth, development, and responses to the environment. A prime example is the spatial-distribution of auxin, which is regulated via transporters to govern developmental patterning. A critical limitation in our ability to identify transporters by forward genetic screens is their potential functional redundancy. Here, we overcome part of this functional redundancy via a transportome, multi-targeted forward-genetic screen using artificial-microRNAs (amiRNAs). We generate a library of 3000 plant lines expressing 1777 amiRNAs, designed to target closely homologous genes within subclades of transporter families and identify, genotype and quantitatively phenotype, 80 lines showing reproducible shoot growth phenotypes. Within this population, we discover and characterize a strong redundant role for the unstudied ABCB6 and ABCB20 genes in auxin transport and response. The unique multi-targeted lines generated in this study could serve as a genetic resource that is expected to reveal additional transporters.

The KNOXI Transcription Factor SHOOT MERISTEMLESS Regulates Floral Fate in Arabidopsis.

Plants have evolved a unique and conserved developmental program that enables the conversion of leaves into floral organs. Elegant genetic and molecular work has identified key regulators of flower meristem identity. However, further understanding of flower meristem specification has been hampered by redundancy and by pleiotropic effects. The KNOXI transcription factor SHOOT MERISTEMLESS (STM) is a well-characterized regulator of shoot apical meristem maintenance. Arabidopsis thaliana stm loss-of-function mutants arrest shortly after germination; therefore, the knowledge on later roles of STM in later processes, including flower development, is limited. Here, we uncover a role for STM in the specification of flower meristem identity. Silencing STM in the APETALA1 (AP1) expression domain in the ap1-4 mutant background resulted in a leafy-flower phenotype, and an intermediate stm-2 allele enhanced the flower meristem identity phenotype of ap1-4 Transcriptional profiling of STM perturbation suggested that STM activity affects multiple floral fate genes, among them the F-box protein-encoding gene UNUSUAL FLORAL ORGANS (UFO). In agreement with this notion, stm-2 enhanced the ufo-2 floral fate phenotype, and ectopic UFO expression rescued the leafy flowers in genetic backgrounds with compromised AP1 and STM activities. This work suggests a genetic mechanism that underlies the activity of STM in the specification of flower meristem identity.

CRISPys: Optimal sgRNA Design for Editing Multiple Members of a Gene Family Using the CRISPR System.

The development of the CRISPR-Cas9 system in recent years has made eukaryotic genome editing, and specifically gene knockout for reverse genetics, a simple and effective task. The system is directed to a genomic target site by a programmed single-guide RNA (sgRNA) that base-pairs with it, subsequently leading to site-specific modifications. However, many gene families in eukaryotic genomes exhibit partially overlapping functions, and thus, the knockout of one gene might be concealed by the function of the other. In such cases, the reduced specificity of the CRISPR-Cas9 system, which may lead to the modification of genomic sites that are not identical to the sgRNA, can be harnessed for the simultaneous knockout of multiple homologous genes. We introduce CRISPys, an algorithm for the optimal design of sgRNAs that would potentially target multiple members of a given gene family. CRISPys first clusters all the potential targets in the input sequences into a hierarchical tree structure that specifies the similarity among them. Then, sgRNAs are proposed in the internal nodes of the tree by embedding mismatches where needed, such that the efficiency to edit the induced targets is maximized. We suggest several approaches for designing the optimal individual sgRNA and an approach to compute the optimal set of sgRNAs for cases when the experimental platform allows for more than one. The latter may optionally account for the homologous relationships among gene-family members. We further show that CRISPys outperforms simpler alignment-based techniques by in silico examination over all gene families in the Solanum lycopersicum genome.

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 regulating 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 they accumulate remain largely unknown. Here, we summarize recent findings from studies of GA movement and localization, and discuss the importance of GA intermediates in long- and short-distance movement. We further review recently identified Arabidopsis GA transporters and highlight their complex specialization and robust functional redundancy in GA transport activity.

PHB3 Maintains Root Stem Cell Niche Identity through ROS-Responsive AP2/ERF Transcription Factors in Arabidopsis.

The root stem cell niche, which is composed of four mitotically inactive quiescent center (QC) cells and the surrounding actively divided stem cells in Arabidopsis, is critical for growth and root development. Here, we demonstrate that the Arabidopsis prohibitin protein PHB3 is required for the maintenance of root stem cell niche identity by both inhibiting proliferative processes in the QC and stimulating cell division in the proximal meristem (PM). PHB3 coordinates cell division and differentiation in the root apical meristem by restricting the spatial expression of ethylene response factor (ERF) transcription factors 115, 114, and 109. ERF115, ERF114, and ERF109 mediate ROS signaling, in a PLT-independent manner, to control root stem cell niche maintenance and root growth through phytosulfokine (PSK) peptide hormones in Arabidopsis.

Studying microstructure and microstructural changes in plant tissues by advanced diffusion magnetic resonance imaging techniques.

As sessile organisms, plants must respond to the environment by adjusting their growth and development. Most of the plant body is formed post-embryonically by continuous activity of apical and lateral meristems. The development of lateral adventitious roots is a complex process, and therefore the development of methods that can visualize, non-invasively, the plant microstructure and organ initiation that occur during growth and development is of paramount importance. In this study, relaxation-based and advanced diffusion magnetic resonance imaging (MRI) methods including diffusion tensor (DTI), q-space diffusion imaging (QSI), and double-pulsed-field-gradient (d-PFG) MRI, at 14.1 T, were used to characterize the hypocotyl microstructure and the microstructural changes that occurred during the development of lateral adventitious roots in tomato. Better contrast was observed in relaxation-based MRI using higher in-plane resolution but this also resulted in a significant reduction in the signal-to-noise ratio of the T2-weighted MR images. Diffusion MRI revealed that water diffusion is highly anisotropic in the vascular cylinder. QSI and d-PGSE MRI showed that in the vascular cylinder some of the cells have sizes in the range of 6-10 µm. The MR images captured cell reorganization during adventitious root formation in the periphery of the primary vascular bundles, adjacent to the xylem pole that broke through the cortex and epidermis layers. This study demonstrates that MRI and diffusion MRI methods allow the non-invasive study of microstructural features of plants, and enable microstructural changes associated with adventitious root formation to be followed.