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.

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.

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.

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.

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.

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.

TEMPRANILLO Reveals the Mesophyll as Crucial for Epidermal Trichome Formation.

Plant trichomes are defensive specialized epidermal cells. In all accepted models, the epidermis is the layer involved in trichome formation, a process controlled by gibberellins (GAs) in Arabidopsis rosette leaves. Indeed, GA activates a genetic cascade in the epidermis for trichome initiation. Here we report that TEMPRANILLO (TEM) genes negatively control trichome initiation not only from the epidermis but also from the leaf layer underneath the epidermis, the mesophyll. Plants over-expressing or reducing TEM specifically in the mesophyll, display lower or higher trichome numbers, respectively. We surprisingly found that fluorescently labeled GA3 accumulates exclusively in the mesophyll of leaves, but not in the epidermis, and that TEM reduces its accumulation and the expression of several newly identified GA transporters. This strongly suggests that TEM plays an essential role, not only in GA biosynthesis, but also in regulating GA distribution in the mesophyll, which in turn directs epidermal trichome formation. Moreover, we show that TEM also acts as a link between GA and cytokinin signaling in the epidermis by negatively regulating downstream genes of both trichome formation pathways. Overall, these results call for a re-evaluation of the present theories of trichome formation as they reveal mesophyll essential during epidermal trichome initiation.

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.

The glucosinolate breakdown product indole-3-carbinol acts as an auxin antagonist in roots of Arabidopsis thaliana.

The glucosinolate breakdown product indole-3-carbinol functions in cruciferous vegetables as a protective agent against foraging insects. While the toxic and deterrent effects of glucosinolate breakdown on herbivores and pathogens have been studied extensively, the secondary responses that are induced in the plant by indole-3-carbinol remain relatively uninvestigated. Here we examined the hypothesis that indole-3-carbinol plays a role in influencing plant growth and development by manipulating auxin signaling. We show that indole-3-carbinol rapidly and reversibly inhibits root elongation in a dose-dependent manner, and that this inhibition is accompanied by a loss of auxin activity in the root meristem. A direct interaction between indole-3-carbinol and the auxin perception machinery was suggested, as application of indole-3-carbinol rescues auxin-induced root phenotypes. In vitro and yeast-based protein interaction studies showed that indole-3-carbinol perturbs the auxin-dependent interaction of Transport Inhibitor Response (TIR1) with auxin/3-indoleacetic acid (Aux/IAAs) proteins, further supporting the possibility that indole-3-carbinol acts as an auxin antagonist. The results indicate that chemicals whose production is induced by herbivory, such as indole-3-carbinol, function not only to repel herbivores, but also as signaling molecules that directly compete with auxin to fine tune plant growth and development.

Auxin response under osmotic stress.

The phytohormone auxin (indole-3-acetic acid, 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, transport and perception. One of the most striking aspects of plant plasticity is the modulation of development in response to changing growth environments. In this review, we explore recent findings correlating auxin response-dependent growth and development with osmotic stresses. Studies of water deficit, dehydration, salt, and other osmotic stresses point towards direct and indirect molecular perturbations in the auxin pathway. Osmotic stress stimuli modulate auxin responses by affecting auxin biosynthesis (YUC, TAA1), transport (PIN), perception (TIR/AFB, Aux/IAA), and inactivation/conjugation (GH3, miR167, IAR3) to coordinate growth and patterning. In turn, stress-modulated auxin gradients drive physiological and developmental mechanisms such as stomata aperture, aquaporin and lateral root positioning. We conclude by arguing that auxin-mediated growth inhibition under abiotic stress conditions is one of the developmental and physiological strategies to acclimate to the changing environment.

Stronger sink demand for metabolites supports dominance of the apical bud in etiolated growth.

The potato tuber is a swollen underground stem that can sprout under dark conditions. Sprouting initiates in the tuber apical bud (AP), while lateral buds (LTs) are repressed by apical dominance (AD). Under conditions of lost AD, removal of tuber LTs showed that they partially inhibit AP growth only at the AD stage. Detached buds were inhibited by exogenous application of naphthaleneacetic acid (NAA), whereas 6-benzyladenine (6-BA) and gibberellic acid (GA3) induced bud burst and elongation, respectively. NAA, applied after 6-BA or GA3, nullified the latters' growth-stimulating effect in both the AP and LTs. GA3 applied to the fifth-position LT was transported mainly to the tuber's AP. GA3 treatment also resulted in increased indole-3-acetic acid (IAA) concentration and cis-zeatin O-glucoside in the AP. In a tuber tissue strip that included two or three buds connected by the peripheral vascular system, treatment of a LT with GA3 affected only the AP side of the strip, suggesting that the AP is the strongest sink for GA3, which induces its etiolated elongation. Dipping etiolated sprouts in labeled GA3 showed specific accumulation of the signal in the AP. Transcriptome analysis of GA3's effect showed that genes related to the cell cycle, cell proliferation, and hormone transport are up-regulated in the AP as compared to the LT. Sink demand for metabolites is suggested to support AD in etiolated stem growth by inducing differential gene expression in the AP.

Gibberellins accumulate in the elongating endodermal cells of Arabidopsis root.

Plant hormones are small-molecule signaling compounds that are collectively involved in all aspects of plant growth and development. Unlike animals, plants actively regulate the spatial distribution of several of their hormones. For example, auxin transport results in the formation of auxin maxima that have a key role in developmental patterning. However, the spatial distribution of the other plant hormones, including gibberellic acid (GA), is largely unknown. To address this, we generated two bioactive fluorescent GA compounds and studied their distribution in Arabidopsis thaliana roots. The labeled GAs specifically accumulated in the endodermal cells of the root elongation zone. Pharmacological studies, along with examination of mutants affected in endodermal specification, indicate that GA accumulation is an active and highly regulated process. Our results strongly suggest the presence of an active GA transport mechanism that would represent an additional level of GA regulation.

BSKs are partially redundant positive regulators of brassinosteroid signaling in Arabidopsis.

Arabidopsis thaliana brassinosteroid signaling kinases (BSKs) constitute a receptor-like cytoplasmic kinase sub-family (RLCK-XII) with 12 members. Previous analysis demonstrated a positive role for BSK1 and BSK3 in the initial steps of brassinosteroid (BR) signal transduction. To investigate the function of BSKs in plant growth and BR signaling, we characterized T-DNA insertion lines for eight BSK genes (BSK1-BSK8) and multiple mutant combinations. Simultaneous elimination of three BSK genes caused alterations in growth and the BR response, and the most severe phenotypes were observed in the bsk3,4,7,8 quadruple and bsk3,4,6,7,8 pentuple mutants, which displayed reduced rosette size, leaf curling and enhanced leaf inclination. In addition, upon treatment with 24-epibrassinolide, these mutants showed reduced hypocotyl elongation, enhanced root growth and alteration in the expression of BR-responsive genes. Some mutant combinations also showed antagonistic interactions. In support of a redundant function in BR signaling, multiple BSKs interacted in vivo with the BR receptor BRI1, and served as its phosphorylation substrates in vitro. The BIN2 and BIL2 GSK3-like kinases, which are negative regulators of BR signaling, interacted in vivo with BSKs and phosphorylated them in vitro, probably at different sites to BRI1. This study demonstrates redundant biological functions for BSKs, and suggests the existence of a regulatory link between BSKs and GSK3-like kinases.

A map of cell type-specific auxin responses.

In plants, changes in local auxin concentrations can trigger a range of developmental processes as distinct tissues respond differently to the same auxin stimulus. However, little is known about how auxin is interpreted by individual cell types. We performed a transcriptomic analysis of responses to auxin within four distinct tissues of the Arabidopsis thaliana root and demonstrate that different cell types show competence for discrete responses. The majority of auxin-responsive genes displayed a spatial bias in their induction or repression. The novel data set was used to examine how auxin influences tissue-specific transcriptional regulation of cell-identity markers. Additionally, the data were used in combination with spatial expression maps of the root to plot a transcriptomic auxin-response gradient across the apical and basal meristem. The readout revealed a strong correlation for thousands of genes between the relative response to auxin and expression along the longitudinal axis of the root. This data set and comparative analysis provide a transcriptome-level spatial breakdown of the response to auxin within an organ where this hormone mediates many aspects of development.

ENTIRE and GOBLET promote leaflet development in tomato by modulating auxin response.

Compound leaves produce leaflets in a highly controlled yet flexible pattern. Here, we investigate the interaction between auxin, the putative auxin response inhibitor ENTIRE (E, SlIAA9) and the CUC transcription factor GOBLET (GOB) in compound-leaf development in tomato (Solanum lycopersicum). Auxin maxima, monitored by the auxin response sensor DR5, marked and preceded leaflet and lobe initiation. The DR5 signal increased, but maxima were partially retained in response to the external or internal elevation of auxin levels. E directly interacted with the auxin receptors SlTIR1 and SlAFB6. Furthermore, E was stabilized by a mutation in domain II of the protein and by the inhibition of auxin or proteasome activity, implying that E is subjected to auxin-mediated degradation. In e mutants the DR5 signal expanded to include the complete leaf margin, and leaf-specific overexpression of a stabilized form of E inhibited the DR5 signal and lamina expansion. Genetic manipulation of GOB activity altered the distribution of the DR5 signal, and the inhibition of auxin transport or activity suppressed the GOB overexpression phenotype, suggesting that auxin mediates GOB-regulated leaf patterning. Whereas leaves of single e or gob mutants developed only primary leaflets, the downregulation of both E and GOB resulted in the complete abolishment of leaflet initiation, and in a strong DR5 signal throughout the leaf margin. These results suggest that E and GOB modulate auxin response and leaflet morphogenesis via partly redundant pathways, and that proper leaflet initiation and separation requires distinct boundaries between regions of lamina growth and adjacent regions in which growth is inhibited.

Cytokinin regulates compound leaf development in tomato.

Leaf shape diversity relies on transient morphogenetic activity in leaf margins. However, how this morphogenetic capacity is maintained is still poorly understood. Here, we uncover a role for the hormone cytokinin (CK) in the regulation of morphogenetic activity of compound leaves in tomato (Solanum lycopersicum). Manipulation of CK levels led to alterations in leaf complexity and revealed a unique potential for prolonged growth and morphogenesis in tomato leaves. We further demonstrate that the effect of CK on leaf complexity depends on proper localization of auxin signaling. Genetic analysis showed that reduction of CK levels suppresses the effect of Knotted1 like homeobox (KNOXI) proteins on leaf shape and that CK can substitute for KNOXI activity at the leaf margin, suggesting that CK mediates the activity of KNOXI proteins in the regulation of leaf shape. These results imply that CK regulates flexible leaf patterning by dynamic interaction with additional hormones and transcription factors.

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.

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.

Gibberellin partly mediates LANCEOLATE activity in tomato.

Elaboration of a compound leaf shape depends on extended morphogenetic activity in developing leaves. In tomato (Solanum lycopersicum), the CIN-TCP transcription factor LANCEOLATE (LA) promotes leaf differentiation. LA is negatively regulated by miR319 during the early stages of leaf development, and decreased sensitivity of LA mRNA to miR319 recognition in the semi-dominant mutant La leads to prematurely increased LA expression, precocious leaf differentiation and a simpler and smaller leaf. Increased levels or responses of the plant hormone gibberellin (GA) in tomato leaves also led to a simplified leaf form. Here, we show that LA activity is mediated in part by GA. Expression of the SlGA20 oxidase1 (SlGA20ox1) gene, which encodes an enzyme in the GA biosynthesis pathway, is increased in gain-of-function La mutants and reduced in plants that over-express miR319. Conversely, the transcript levels of the GA deactivation gene SlGA2 oxidase4 (SlGA2ox4) are increased in plants over-expressing miR319. The miR319 over-expression phenotype is suppressed by exogenous GA application and by a mutation in the PROCERA (PRO) gene, which encodes an inhibitor of the GA response. SlGA2ox4 is expressed in initiating leaflets during early leaf development. Its expression expands as a result of miR319 over-expression, and its over-expression leads to increased leaf complexity. These results suggest that LA activity is partly mediated by positive regulation of the GA response, probably by regulation of GA levels.