Widespread horizontal gene transfer between plants and bacteria.

Plants host a large array of commensal bacteria that interact with the host. The growth of both bacteria and plants is often dependent on nutrients derived from the cognate partners, and the bacteria fine-tune host immunity against pathogens. This ancient interaction is common in all studied land plants and is critical for proper plant health and development. We hypothesized that the spatial vicinity and the long-term relationships between plants and their microbiota may promote cross-kingdom horizontal gene transfer (HGT), a phenomenon that is relatively rare in nature. To test this hypothesis, we analyzed the Arabidopsis thaliana genome and its extensively sequenced microbiome to detect events of horizontal transfer of full-length genes that transferred between plants and bacteria. Interestingly, we detected 75 unique genes that were horizontally transferred between plants and bacteria. Plants and bacteria exchange in both directions genes that are enriched in carbohydrate metabolism functions, and bacteria transferred to plants genes that are enriched in auxin biosynthesis genes. Next, we provided a proof of concept for the functional similarity between a horizontally transferred bacterial gene and its Arabidopsis homologue in planta. The Arabidopsis DET2 gene is essential for biosynthesis of the brassinosteroid phytohormones, and loss of function of the gene leads to dwarfism. We found that expression of the DET2 homologue from Leifsonia bacteria of the Actinobacteria phylum in the Arabidopsis det2 background complements the mutant and leads to normal plant growth. Together, these data suggest that cross-kingdom HGT events shape the metabolic capabilities and interactions between plants and bacteria.

Phosphate deprivation-induced changes in tomato are mediated by an interaction between brassinosteroid signaling and zinc.

Inorganic phosphate (Pi) is a necessary macronutrient for basic biological processes. Plants modulate their root system architecture (RSA) and cellular processes to adapt to Pi deprivation albeit with a growth penalty. Excess application of Pi fertilizer, on the contrary, leads to eutrophication and has a negative environmental impact. We compared RSA, root hair elongation, acid phosphatase activity, metal ion accumulation, and brassinosteroid hormone levels of Solanum lycopersicum (tomato) and Solanum pennellii, which is a wild relative of tomato, under Pi sufficiency and deficiency conditions to understand the molecular mechanism of Pi deprivation response in tomato. We showed that S. pennellii is partially insensitive to phosphate deprivation. Furthermore, it mounts a constitutive response under phosphate sufficiency. We demonstrate that activated brassinosteroid signaling through a tomato BZR1 ortholog gives rise to the same constitutive phosphate deficiency response, which is dependent on zinc overaccumulation. Collectively, these results reveal an additional strategy by which plants can adapt to phosphate starvation.

Optimal BR signalling is required for adequate cell wall orientation in the Arabidopsis root meristem.

Plant brassinosteroid hormones (BRs) regulate growth in part through altering the properties of the cell wall, the extracellular matrix of plant cells. Conversely, feedback signalling from the wall connects the state of cell wall homeostasis to the BR receptor complex and modulates BR activity. Here, we report that both pectin-triggered cell wall signalling and impaired BR signalling result in altered cell wall orientation in the Arabidopsis root meristem. Furthermore, both depletion of endogenous BRs and exogenous supply of BRs triggered these defects. Cell wall signalling-induced alterations in the orientation of newly placed walls appear to occur late during cytokinesis, after initial positioning of the cortical division zone. Tissue-specific perturbations of BR signalling revealed that the cellular malfunction is unrelated to previously described whole organ growth defects. Thus, tissue type separates the pleiotropic effects of cell wall/BR signals and highlights their importance during cell wall placement.

Growth models from a brassinosteroid perspective.

Plant growth relies on interconnected hormonal pathways, their corresponding transcriptional networks and mechanical signals. This work reviews recent brassinosteroid (BR) studies and integrates them with current growth models derived from research in roots. The relevance of spatiotemporal BR signaling in the longitudinal and radial root axes and its multifaceted interaction with auxin, the impact of BR on final cell size determination and its interplay with microtubules and the cell wall are discussed. Also highlighted are emerging variations of canonical BR signaling that could function in developmental-specific context.

Interdependent Nutrient Availability and Steroid Hormone Signals Facilitate Root Growth Plasticity.

Plants acquire essential elements from inherently heterogeneous soils, in which phosphate and iron availabilities vary. Consequently, plants have developed adaptive strategies to cope with low iron or phosphate levels, including alternation between root growth enhancement and attenuation. How this adaptive response is achieved remains unclear. Here, we found that low iron accelerates root growth in Arabidopsis thaliana by activating brassinosteroid signaling, whereas low-phosphate-induced high iron accumulation inhibits it. Altered hormone signaling intensity also modulated iron accumulation in the root elongation and differentiation zones, constituting a feedback response between brassinosteroid and iron. Surprisingly, the early effect of low iron levels on root growth depended on the brassinosteroid receptor but was apparently hormone ligand-independent. The brassinosteroid receptor inhibitor BKI1, the transcription factors BES1/BZR1, and the ferroxidase LPR1 operate at the base of this feedback loop. Hence, shared brassinosteroid and iron regulatory components link nutrient status to root morphology, thereby driving the adaptive response.

Quantitation of Cell Type-Specific Responses to Brassinosteroid by Deep Sequencing of Polysome-Associated Polyadenylated RNA.

Hormonal signaling pathways control almost every aspect of plant physiology and development. Extensive analysis of hormonal signaling output, i.e., gene expression, has therefore been the focus of many studies. These analyses have been primarily conducted on total extracts derived from a mixture of tissues and cell types, consequentially limiting delineation of precise models. In this chapter, methods for tissue-specific functional genomics are overviewed, in which hormonal responses are analyzed at the transcriptional and the translational levels. Deep sequencing of polysome-associated polyadenylated RNA is employed for cell type-specific quantitation of translatome responses to brassinosteroids. Polysomes are purified by the previously established Translating Ribosome Affinity Purification (TRAP) method, in which the expression of a tagged ribosomal protein is targeted to the tissue of interest, allowing tissue-specific immunopurification of the polysome complexes. The methods presented assess establishment and selection of suitable transgenic lines. A protocol for hormonal treatment of the Arabidopsis thaliana root as a case study, TRAP and linear amplification of the purified polysome-associated polyadenylated RNA are described. Finally, a step-by-step presentation is included of the analysis of the RNA deep-sequencing data and Rscript for plotting hierarchically clustered heatmap of the expressed genes.

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications.

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix containing polysaccharides that include cellulose microfibrils that form both crystalline structures and cellulose chains of amorphous organization. The orientation of the cellulose fibers and their concentrations dictate the mechanical properties of the cell. Several methods are used to determine the levels of crystalline cellulose, each bringing both advantages and limitations. Some can distinguish the proportion of crystalline regions within the total cellulose. However, they are limited to whole-organ analyses that are deficient in spatiotemporal information. Others relying on live imaging, are limited by the use of imprecise dyes. Here, we report a sensitive polarized light-based system for specific quantification of relative light retardance, representing crystalline cellulose accumulation in cross sections of Arabidopsis thaliana roots. In this method, the cellular resolution and anatomical data are maintained, enabling direct comparisons between the different tissues composing the growing root. This approach opens a new analytical dimension, shedding light on the link between cell wall composition, cellular behavior and whole-organ growth.

Growth control: brassinosteroid activity gets context.

Brassinosteroid activity controls plant growth and development, often in a seemingly opposing or complex manner. Differential impact of the hormone and its signalling components, acting both as promoters and inhibitors of organ growth, is exemplified by meristem differentiation and cell expansion in above- and below-ground organs. Complex brassinosteroid-based control of stomata count and lateral root development has also been demonstrated. Here, mechanisms underlying these phenotypic outputs are examined. Among these, studies uncovering core brassinosteroid signalling components, which integrate with distinct peptide, hormone, and environmental pathways, are reviewed. Finally, the differential spatiotemporal context of brassinosteroid activity within the organ, as an important determinant of controlled growth, is discussed.

Translatome analyses capture of opposing tissue-specific brassinosteroid signals orchestrating root meristem differentiation.

The mechanisms ensuring balanced growth remain a critical question in developmental biology. In plants, this balance relies on spatiotemporal integration of hormonal signaling pathways, but the understanding of the precise contribution of each hormone is just beginning to take form. Brassinosteroid (BR) hormone is shown here to have opposing effects on root meristem size, depending on its site of action. BR is demonstrated to both delay and promote onset of stem cell daughter differentiation, when acting in the outer tissue of the root meristem, the epidermis, and the innermost tissue, the stele, respectively. To understand the molecular basis of this phenomenon, a comprehensive spatiotemporal translatome mapping of Arabidopsis roots was performed. Analyses of wild type and mutants featuring different distributions of BR revealed autonomous, tissue-specific gene responses to BR, implying its contrasting tissue-dependent impact on growth. BR-induced genes were primarily detected in epidermal cells of the basal meristem zone and were enriched by auxin-related genes. In contrast, repressed BR genes prevailed in the stele of the apical meristem zone. Furthermore, auxin was found to mediate the growth-promoting impact of BR signaling originating in the epidermis, whereas BR signaling in the stele buffered this effect. We propose that context-specific BR activity and responses are oppositely interpreted at the organ level, ensuring coherent growth.

Activity of the brassinosteroid transcription factors BRASSINAZOLE RESISTANT1 and BRASSINOSTEROID INSENSITIVE1-ETHYL METHANESULFONATE-SUPPRESSOR1/BRASSINAZOLE RESISTANT2 blocks developmental reprogramming in response to low phosphate availability.

Plants feature remarkable developmental plasticity, enabling them to respond to and cope with environmental cues, such as limited availability of phosphate, an essential macronutrient for all organisms. Under this condition, Arabidopsis (Arabidopsis thaliana) roots undergo striking morphological changes, including exhaustion of the primary meristem, impaired unidirectional cell expansion, and elevated density of lateral roots, resulting in shallow root architecture. Here, we show that the activity of two homologous brassinosteroid (BR) transcriptional effectors, BRASSINAZOLE RESISTANT1 (BZR1) and BRASSINOSTEROID INSENSITIVE1-ETHYL METHANESULFONATE-SUPPRESSOR1 (BES1)/BZR2, blocks these responses, consequently maintaining normal root development under low phosphate conditions without impacting phosphate homeostasis. We show that phosphate deprivation shifts the intracellular localization of BES1/BZR2 to yield a lower nucleus-to-cytoplasm ratio, whereas replenishing the phosphate supply reverses this ratio within hours. Phosphate deprivation reduces the expression levels of BR biosynthesis genes and the accumulation of the bioactive BR 28-norcastasterone. In agreement, low and high BR levels sensitize and desensitize root response to this adverse condition, respectively. Hence, we propose that the environmentally controlled developmental switch from deep to shallow root architecture involves reductions in BZR1 and BES1/BZR2 levels in the nucleus, which likely play key roles in plant adaptation to phosphate-deficient environments.

Root growth is modulated by differential hormonal sensitivity in neighboring cells.

Coherent plant growth requires spatial integration of hormonal pathways and cell wall remodeling activities. However, the mechanisms governing sensitivity to hormones and how cell wall structure integrates with hormonal effects are poorly understood. We found that coordination between two types of epidermal root cells, hair and nonhair cells, establishes root sensitivity to the plant hormones brassinosteroids (BRs). While expression of the BR receptor BRASSINOSTEROID-INSENSITIVE1 (BRI1) in hair cells promotes cell elongation in all tissues, its high relative expression in nonhair cells is inhibitory. Elevated ethylene and deposition of crystalline cellulose underlie the inhibitory effect of BRI1. We propose that the relative spatial distribution of BRI1, and not its absolute level, fine-tunes growth.

Brassinosteroids in growth control: how, when and where.

The steroid hormones brassinosteroids take on critical roles during various plant growth processes, including control of cell proliferation and cell elongation. In this review, we discuss different strategies that have advanced our understanding of brassinosteroid function. Approaches observing whole-plant responses uncovered regulatory brassinosteroids-dependent modules controlling cell elongation. In these regulatory modules, downstream components of the brassinosteroid signaling pathway directly interact with other hormonal and environmental pathways. In alternative approaches, brassinosteroid activity has been dissected at the tissue and cellular level of above- and below-ground organs. These studies have determined the importance of brassinosteroids in cell cycle progression and in timing of cell differentiation. In addition, they have demonstrated that local reduction of the hormone sets organ boundaries. Finally, these studies uncovered the capacity of the epidermal-derived brassinosteroid signaling to control organ growth. Thus, inter-cellular communication is intimately involved in brassinosteroid-mediated growth control. The current challenge is therefore to decipher the spatiotemporal distribution of brassinosteroid activity and its impact on coherent growth and development.

BRI1 activity in the root meristem involves post-transcriptional regulation of PIN auxin efflux carriers.

Spatiotemporal coordination between multiple hormonal pathways is a key determinant of plant growth. This coordination can be mediated by distribution of the auxin network via the action of PIN auxin efflux carriers. We showed that brassinosteroids (BRs) promote cell proliferation and cell expansion of meristematic cells. Hence, roots with high epidermal expression of the BR receptor BRI1 have enlarged meristem whereas bri1 mutant has a reduced meristem size. Because the extent of mitotic activity and differentiation is tightly linked to auxin gradient we further asked how the BR pathway integrates with current proposed models for PIN regulation.  We showed that the small meristem of BR deficient plants does not involve transcriptional modulation of PIN 1, 3 and 7 genes.  Here, we found that PIN2 and PIN4 are under transcriptional regulation.  However, their accumulation in the epidermis/cortex and columella respectively was also determined by BRs in a post-transcriptional manner.  Thus, BRs impinge on auxin distribution through distinct regulatory modes and the self-organizing auxin system represents at least one mechanism that contributes to BR-mediated growth.

Brassinosteroid perception in the epidermis controls root meristem size.

Multiple small molecule hormones contribute to growth promotion or restriction in plants. Brassinosteroids (BRs), acting specifically in the epidermis, can both drive and restrict shoot growth. However, our knowledge of how BRs affect meristem size is scant. Here, we study the root meristem and show that BRs are required to maintain normal cell cycle activity and cell expansion. These two processes ensure the coherent gradient of cell progression, from the apical to the basal meristem. In addition, BR activity in the meristem is not accompanied by changes in the expression level of the auxin efflux carriers PIN1, PIN3 and PIN7, which are known to control the extent of mitotic activity and differentiation. We further demonstrate that BR signaling in the root epidermis and not in the inner endodermis, quiescent center (QC) cells or stele cell files is sufficient to control root meristem size. Interestingly, expression of the QC and the stele-enriched MADS-BOX gene AGL42 can be modulated by BRI1 activity solely in the epidermis. The signal from the epidermis is probably transmitted by a different component than BES1 and BZR1 transcription factors, as their direct targets, such as DWF4 and BRox2, are regulated in the same cells that express BRI1. Taken together, our study provides novel insights into the role of BRs in controlling meristem size.

New auxin analogs with growth-promoting effects in intact plants reveal a chemical strategy to improve hormone delivery.

Plant growth depends on the integration of environmental cues and phytohormone-signaling pathways. During seedling emergence, elongation of the embryonic stem (hypocotyl) serves as a readout for light and hormone-dependent responses. We screened 10,000 chemicals provided exogenously to light-grown seedlings and identified 100 compounds that promote hypocotyl elongation. Notably, one subset of these chemicals shares structural characteristics with the synthetic auxins, 2,4-dichlorophenoxyacetic acid (2,4-D), and 1-naphthaleneacetic acid (1-NAA); however, traditional auxins (e.g., indole-3-acetic acid [IAA], 2,4-D, 1-NAA) have no effect on hypocotyl elongation. We show that the new compounds act as "proauxins" akin to prodrugs. Our data suggest that these compounds diffuse efficiently to the hypocotyls, where they undergo cleavage at varying rates, releasing functional auxins. To investigate this principle, we applied a masking strategy and designed a pro-2,4-D. Unlike 2,4-D alone, this pro-2,4-D enhanced hypocotyl elongation. We further demonstrated the utility of the proauxins by characterizing auxin responses in light-grown hypocotyls of several auxin receptor mutants. These new compounds thus provide experimental access to a tissue previously inaccessible to exogenous application of auxins. Our studies exemplify the combined power of chemical genetics and biochemical analyses for discovering and refining prohormone analogs with selective activity in specific plant tissues. In addition to the utility of these compounds for addressing questions related to auxin and light-signaling interactions, one can envision using these simple principles to study other plant hormone and small molecule responses in temporally and spatially controlled ways.

Growth coordination and the shoot epidermis.

Cell-cell communication is essential for growth and development of multicellular organisms. In higher plants, the shoot organs are derived from three clonally distinct cell layers present in the meristem. The role of the outermost L1 cell layer and its derived epidermis in coordinating growth of the inner-cell layers has long been debated. This question has been revisited recently using molecular tools to manipulate cell cycle progression or cell expansion, specifically in the epidermis. These studies conclude that cells in the epidermis both promote and restrict growth of the entire shoot by sending growth signals - either physical or chemical - to the inner layers.

The Arabidopsis DESPERADO/AtWBC11 transporter is required for cutin and wax secretion.

The cuticle fulfills multiple roles in the plant life cycle, including protection from environmental stresses and the regulation of organ fusion. It is largely composed of cutin, which consists of C(16-18) fatty acids. While cutin composition and biosynthesis have been studied, the export of cutin monomers out of the epidermis has remained elusive. Here, we show that DESPERADO (AtWBC11) (abbreviated DSO), encoding a plasma membrane-localized ATP-binding cassette transporter, is required for cutin transport to the extracellular matrix. The dso mutant exhibits an array of surface defects suggesting an abnormally functioning cuticle. This was accompanied by dramatic alterations in the levels of cutin monomers. Moreover, electron microscopy revealed unusual lipidic cytoplasmatic inclusions in epidermal cells, disappearance of the cuticle in postgenital fusion areas, and altered morphology of trichomes and pavement cells. We also found that DSO is induced by salt, abscisic acid, and wounding stresses and its loss of function results in plants that are highly susceptible to salt and display reduced root branching. Thus, DSO is not only essential for developmental plasticity but also plays a vital role in stress responses.

The epidermis both drives and restricts plant shoot growth.

The size of an organism is genetically determined, yet how a plant or animal achieves its final size is largely unknown. The shoot of higher plants has a simple conserved body plan based on three major tissue systems: the epidermal (L1), sub-epidermal (L2) and inner ground and vascular (L3) tissues. Which tissue system drives or restricts growth has been a subject of debate for over a century. Here, we use dwarf, brassinosteroid biosynthesis and brassinosteroid response mutants in conjunction with tissue-specific expression of these components as tools to examine the role of the epidermis in shoot growth. We show that expression of the brassinosteroid receptor or a brassinosteroid biosynthetic enzyme in the epidermis, but not in the vasculature, of null mutants is sufficient to rescue their dwarf phenotypes. Brassinosteroid signalling from the epidermis is not sufficient to establish normal vascular organization. Moreover, shoot growth is restricted when brassinosteroids are depleted from the epidermis and brassinosteroids act locally within a leaf. We conclude that the epidermis both promotes and restricts shoot growth by providing a non-autonomous signal to the ground tissues.

Intron retention is a major phenomenon in alternative splicing in Arabidopsis.

Alternative splicing (AS) combines different transcript splice junctions that result in transcripts with shuffled exons, alternative 5' or 3' splicing sites, retained introns and different transcript termini. In this way, multiple mRNA species and proteins can be created from a single gene expanding the potential informational content of eukaryotic genomes. Search algorithms of AS forms in a variety of Arabidopsis databases showed they contained an unusually high fraction of retained introns (above 30%), compared with 10% that was reported for humans. The preponderance of retained introns (65%) were either part of open reading frames, present in the UTR region or present as the last intron in the transcript, indicating that their occurrence would not participate in non-sense-mediated decay. Interestingly, the functional distribution of the transcripts with retained introns is skewed towards stress and external/internal stimuli-related functions. A sampling of the alternative transcripts with retained introns were confirmed by RT-PCR and were shown to co-purify with polyribosomes, indicating their nuclear export. Thus, retained introns are a prominent feature of AS in Arabidopsis and as such may play a regulatory function.