combining-clearing-fluorescence-microscopy-for-visualising-changes

Combining Clearing and Fluorescence Microscopy for Visualising Changes in Gene Expression and Physiological Responses to <em>Plasmodiophora brassicae</em>

Infection of Brassica crops by the soilborne protist Plasmodiophora brassicae leads to gall formation on the underground organs. The formation of galls ...

University of Tübingen

Secondary growth occurs in dicotyledons and gymnosperms, and results in an increased girth of plant organs. It is driven primarily by the vascular cambium, which produces thousands of cells throughout the life of several plant species. For instance, even in the small herbaceous model plant Arabidopsis, manual quantification of this massive process is impractical. Here, we provide a comprehensive overview of current methods used to measure radial growth. We discuss the issues and problematics related to its quantification. We highlight recent advances and tools developed for automated cellular phenotyping and its future applications.

Novel tools for quantifying secondary growth.

Secondary growth, the increase in girth of plant organs, is primarily driven by the vascular and cork cambium. In perennial dicotyledons and gymnosperms, it represents a major source of biomass accumulation in the form of wood. However, the molecular framework underlying secondary growth is largely based on studies in the annual herbaceous plant Arabidopsis thaliana. In this review, we will focus on a selection of major regulators of stem secondary growth, which have recently been shown to play a role in woody species. In particular, we will focus on thermospermine and its bivalent role in controlling xylem differentiation and cell proliferation and we will highlight the contributions of the different LRR-Receptor-Like Kinase signaling hubs.

Secondary development in the stem: when Arabidopsis and trees are closer than it seems.

Quantification of vascular morphodynamics during secondary growth has been hampered by the scale of the process. Even in the tiny model plant Arabidopsis thaliana, the xylem can include more than 2000 cells in a single cross section, rendering manual counting impractical. Moreover, due to its deep location, xylem is an inaccessible tissue, limiting live imaging. A novel method to visualize and measure secondary growth progression has been proposed: "the Quantitative Histology" approach. This method is based on a detailed anatomical atlas, and image segmentation coupled with machine learning to automatically extract cell shapes and identify cell type. Here we present a new version of this approach, with a user-friendly interface implemented in the open source software LithoGraphX.

Vascular Morphodynamics During Secondary Growth.

Plants are the primary producers of biomass on earth. As an almost stereotypic feature, higher plants generate continuously growing bodies mediated by the activity of different groups of stem cells, the meristems. Shoot and root thickening is one of the fundamental growth processes determining form and function of these bodies. Mediated by a group of cylindrical meristems located below organ surfaces, vascular and protective tissues are continuously generated in a highly plastic manner, a competence essential for the survival in an ever changing environment. Acknowledging the fundamental role of this process, which is overall designated as secondary growth, we discuss in this review our current knowledge about the evolution and molecular regulation of the vascular cambium. The cambium is the meristem responsible for the formation of wood and bast, the two types of vascular tissues important for long-distance transport of water and assimilates, respectively. Although regulatory patterns are only beginning to emerge, we show that cambium activity represents a highly rewarding model for studying cell fate decisions, tissue patterning and differentiation, which has experienced an outstanding phylogenetic diversification.

Secondary growth as a determinant of plant shape and form.

During secondary growth in most eudicots and gymnosperms, the periderm replaces the epidermis as the frontier tissue protecting the vasculature from biotic and abiotic stresses. Despite its importance, the mechanisms underlying periderm establishment and formation are largely unknown. The herbaceous Arabidopsis thaliana undergoes secondary growth, including periderm formation in the root and hypocotyl. Thus, we focused on these two organs to establish a framework to study periderm development in a model organism. We identified a set of characteristic developmental stages describing periderm growth from the first cell division in the pericycle to the shedding of the cortex and epidermis. We highlight that two independent mechanisms are involved in the loosening of the outer tissues as the endodermis undergoes programmed cell death, whereas the epidermis and the cortex are abscised. Moreover, the phellem of Arabidopsis, as in trees, is suberized, lignified and peels off. In addition, putative regulators from oak and potato are also expressed in the Arabidopsis periderm. Collectively, the periderm of Arabidopsis shares many characteristics/features of woody and tuberous periderms, rendering Arabidopsis thaliana an attractive model for cork biology.

A molecular framework to study periderm formation in Arabidopsis.

Tree bark is a highly specialized array of tissues that plays important roles in plant protection and development. Bark tissues develop from two lateral meristems; the phellogen (cork cambium) produces the outermost stem-environment barrier called the periderm, while the vascular cambium contributes with phloem tissues. Although bark is diverse in terms of tissues, functions and species, it remains understudied at higher resolution. We dissected the stem of silver birch (Betula pendula) into eight major tissue types, and characterized these by a combined transcriptomics and metabolomics approach. We further analyzed the varying bark types within the Betulaceae family. The two meristems had a distinct contribution to the stem transcriptomic landscape. Furthermore, inter- and intraspecies analyses illustrated the unique molecular profile of the phellem. We identified multiple tissue-specific metabolic pathways, such as the mevalonate/betulin biosynthesis pathway, that displayed differential evolution within the Betulaceae. A detailed analysis of suberin and betulin biosynthesis pathways identified a set of underlying regulators and highlighted the important role of local, small-scale gene duplication events in the evolution of metabolic pathways. This work reveals the transcriptome and metabolic diversity among bark tissues and provides insights to its development and evolution, as well as its biotechnological applications.

Tissue-specific study across the stem reveals the chemistry and transcriptome dynamics of birch bark.

The periderm acts as the first line of defence for a plant, protecting wood and phloem from abiotic and biotic stresses. During secondary growth, through the increase in girth of plant organs, the periderm replaces the epidermis as the outermost tissue. The phellogen, a bifacial post-embryonic meristem, forms the phelloderm inwards (toward the vasculature) and the suberized phellem outwards (toward the environment). These three tissues are collectively referred to as the periderm. Here, we summarize recent findings on the molecular mechanisms of periderm development by describing periderm formation in connection to the fate of the surrounding tissues, by discussing common regulatory hubs between the vascular cambium and the phellogen, and by highlighting transcription factors (TFs) controlling phellem differentiation.

The development of the periderm: the final frontier between a plant and its environment.

Vascular cambium, a lateral plant meristem, is a central producer of woody biomass. Although a few transcription factors have been shown to regulate cambial activity, the phenotypes of the corresponding loss-of-function mutants are relatively modest, highlighting our limited understanding of the underlying transcriptional regulation. Here, we use cambium cell-specific transcript profiling followed by a combination of transcription factor network and genetic analyses to identify 62 new transcription factor genotypes displaying an array of cambial phenotypes. This approach culminated in virtual loss of cambial activity when both WUSCHEL-RELATED HOMEOBOX 4 (WOX4) and KNOTTED-like from Arabidopsis thaliana 1 (KNAT1; also known as BREVIPEDICELLUS) were mutated, thereby unlocking the genetic redundancy in the regulation of cambium development. We also identified transcription factors with dual functions in cambial cell proliferation and xylem differentiation, including WOX4, SHORT VEGETATIVE PHASE (SVP) and PETAL LOSS (PTL). Using the transcription factor network information, we combined overexpression of the cambial activator WOX4 and removal of the putative inhibitor PTL to engineer Arabidopsis for enhanced radial growth. This line also showed ectopic cambial activity, thus further highlighting the central roles of WOX4 and PTL in cambium development.

Transcriptional regulatory framework for vascular cambium development in Arabidopsis roots.

During post-embryonic development, the pericycle specifies the stem cells that give rise to both lateral roots (LRs) and the periderm, a suberized barrier that protects the plant against biotic and abiotic stresses. Comparable auxin-mediated signaling hubs regulate meristem establishment in many developmental contexts; however, it is unknown how specific outputs are achieved. Using the Arabidopsis root as a model, we show that while LR formation is the main auxin-induced program after de-etiolation, plants with age become competent to form a periderm in response to auxin. The establishment of the vascular cambium acts as the developmental switch required to trigger auxin-mediated periderm initiation. Moreover, distinct auxin signaling components and targets control LR versus periderm formation. Among the periderm-specific-promoting transcription factors, WUSCHEL-RELATED HOMEOBOX 4 (WOX4) and KNAT1/BREVIPEDICELLUS (BP) stand out as their specific overexpression in the periderm results in an increased number of periderm layers, a trait of agronomical importance in breeding programs targeting stress tolerance. These findings reveal that specificity in pericycle stem cell fate is achieved by the integration of developmental cues into distinct regulatory modules.

Pluripotent Pericycle Cells Trigger Different Growth Outputs by Integrating Developmental Cues into Distinct Regulatory Modules.

Plants deposit hydrophobic polymers, such as lignin or suberin, in their root cell walls to protect inner tissues and facilitate selective uptake of solutes. Insights into how individual root tissues contribute to polymer formation are important for elucidation of ultrastructure, function, and development of these protective barriers. Although the pathways responsible for production of the barrier constituents are established, our models lack spatiotemporal resolution-especially in roots-thus, the source of monomeric barrier components is not clear. This is mainly due to our restricted ability to manipulate synthesis of the broadly important phenylpropanoid pathway, as mutants in this pathway display lethal or pleiotropic phenotypes. Here, we overcome this challenge by exploiting highly controlled in vivo repression systems. We provide strong evidence that autonomous production of phenylpropanoids is essential for establishment of the endodermal Casparian strip as well as adherence of the suberin matrix to the cell wall of endodermis and cork. Our work highlights that, in roots, the phenylpropanoid pathway is under tight spatiotemporal control and serves distinct roles in barrier formation across tissues and developmental zones. This becomes evident in the late endodermis, where repression of phenylpropanoid production leads to active removal of suberin in pre-suberized cells, indicating that endodermal suberin depositions might embody a steady state between continuous synthesis and degradation.

Tissue-Autonomous Phenylpropanoid Production Is Essential for Establishment of Root Barriers.

During secondary growth, the thickening of plant organs, wood (xylem) and bast (phloem) is continuously produced by the vascular cambium. In Arabidopsis hypocotyl and root, we can distinguish two phases of secondary growth based on cell morphology and production rate. The first phase, in which xylem and phloem are equally produced, precedes the xylem expansion phase in which xylem formation is enhanced and xylem fibers differentiate. It is known that gibberellins (GA) trigger this developmental transition via degradation of DELLA proteins and that the cambium master regulator BREVIPEDICELLUS/KNAT1 (BP/KNAT1) and receptor like kinases ERECTA and ERL1 regulate this process downstream of GA. However, our understanding of the regulatory network underlying GA-mediated secondary growth is still limited. Here, we demonstrate that DELLA-mediated xylem expansion in Arabidopsis hypocotyl is mainly achieved through DELLA family members RGA and GAI, which promote cambium senescence. We further show that AUXIN RESPONSE FACTOR 6 (ARF6) and ARF8, which physically interact with DELLAs, specifically repress phloem proliferation and induce cambium senescence during the xylem expansion phase. Moreover, the inactivation of BP in arf6 arf8 background revealed an essential role for ARF6 and ARF8 in cambium establishment and maintenance. Overall, our results shed light on a pivotal hormone cross-talk between GA and auxin in the context of plant secondary growth.

Auxin and gibberellin signaling cross-talk promotes hypocotyl xylem expansion and cambium homeostasis.

Drought stress impacts the quality and yield of Pisum sativum. Here, we show how short periods of limited water availability during the vegetative stage of pea alters phloem sap content and how these changes are connected to strategies used by plants to cope with water deficit. We have investigated the metabolic content of phloem sap exudates and explored how this reflects P. sativum physiological and developmental responses to drought. Our data show that drought is accompanied by phloem-mediated redirection of the components that are necessary for cellular respiration and the proper maintenance of carbon/nitrogen balance during stress. The metabolic content of phloem sap reveals a shift from anabolic to catabolic processes as well as the developmental plasticity of P. sativum plants subjected to drought. Our study underlines the importance of phloem-mediated transport for plant adaptation to unfavourable environmental conditions. We also show that phloem exudate analysis can be used as a useful proxy to study stress responses in plants. We propose that the decrease in oleic acid content within phloem sap could be considered as a potential marker of early signalling events mediating drought response.

Phloem exudate metabolic content reflects the response to water-deficit stress in pea plants (Pisum sativum L.).

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 , ABA homeostasis is regulated by two previously unknown ABA transporters. Adenosine triphosphate–binding 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.  and  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,  and  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.

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

The periderm acts as armor protecting the plant's inner tissues from biotic and abiotic stress. It forms during the radial thickening of plant organs such as stems and roots and replaces the function of primary protective tissues such as the epidermis and the endodermis. A wound periderm also forms to heal and protect injured tissues. The periderm comprises a meristematic tissue called the phellogen, or cork cambium, and its derivatives: the lignosuberized phellem and the phelloderm. Research on the periderm has mainly focused on the chemical composition of the phellem due to its relevance as a raw material for industrial processes. Today, there is increasing interest in the regulatory network underlying periderm development as a novel breeding trait to improve plant resilience and to sequester CO. Here, we discuss our current understanding of periderm formation, focusing on aspects of periderm evolution, mechanisms of periderm ontogenesis, regulatory networks underlying phellogen initiation and cork differentiation, and future challenges of periderm research.

Laura Ragni

ZMBP-Center for Plant Molecular Biology

University of Tübingen

Combining Clearing and Fluorescence Microscopy for Visualising Changes in Gene Expression and Physiological Responses to Plasmodiophora brassicae

Laura Ragni

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}ZMBP-Center for Plant Molecular Biology

University of Tübingen

Combining Clearing and Fluorescence Microscopy for Visualising Changes in Gene Expression and Physiological Responses to Plasmodiophora brassicae

ZMBP-Center for Plant Molecular Biology