high-resolution-quantification-crystalline-cellulose-accumulation

High Resolution Quantification of Crystalline Cellulose Accumulation in <em>Arabidopsis</em> 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 ...

Technion-Israel Institute of Technology

Alternative splicing is a major contributor to genome complexity, playing a significant role in various cellular functions, including signal transduction, immunity, and development. The spliceosomal machinery is responsible for the processing of nuclear RNA. Several splicing factors associated with this complex are phosphorylated by kinases that possess a conserved LAMMER motif. We demonstrate in BY-2 tobacco cells a novel role for the LAMMER motif in the maintenance of proper subnuclear localization. Furthermore, high expression of the LAMMER kinase in Arabidopsis plants modulated the alternative splicing of specific endogenous genes and resulted in abnormal plant development and a novel transcriptome profile. A prominent feature was the upregulation of genes that play a role in protein turnover, suggesting a moderating function for these gene products in the control of alternative splicing events. Together, these results demonstrate alternative splicing modulation as a result of phosphorylation activity, providing an opportunity to study its global effect on the plasticity of plant development and gene expression at the organism level.

Alternative splicing modulation by a LAMMER kinase impinges on developmental and transcriptome expression.

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.

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

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.

The epidermis both drives and restricts plant shoot growth.

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

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.

Growth coordination and the shoot epidermis.

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.

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

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.

Brassinosteroid perception in the epidermis controls root meristem size.

Plusieurs hormones de petites mol&eacute;cules contribuent &agrave; la promotion de la croissance ou de restriction dans les plantes. Brassinost&eacute;ro&iuml;des (BRs), agissant sp&eacute;cifiquement dans l&#39;&eacute;piderme, peut aussi bien conduire et restreindre la croissance des pousses. Cependant, notre connaissance des cons&eacute;quences BRs sur taille du m&eacute;rist&egrave;me est rares. Ici, nous &eacute;tudions le m&eacute;rist&egrave;me de racine et de montrer que les BRs sont n&eacute;cessaires pour maintenir l&#39;expansion de l&#39;activit&eacute; et la cellule normale du cycle cellulaire. Ces deux processus, assurez-vous que le gradient coh&eacute;rent de la progression de cellules, de l&#39;apicale au m&eacute;rist&egrave;me basal. En outre, l&#39;activit&eacute; BR dans le m&eacute;rist&egrave;me n&#39;est pas accompagn&eacute;e de changements dans le niveau d&#39;expression des transporteurs efflux auxine PIN1, broche 3 et PIN7, qui sont connus pour contr&ocirc;ler l&#39;&eacute;tendue de l&#39;activit&eacute; mitotique et diff&eacute;renciation. De plus, nous d&eacute;montrons que la BR de signalisation dans l&#39;&eacute;piderme racinaire et non dans l&#39;endoderme interne, cellules quiescentes Centre (QC) ou fichiers de cellule de st&egrave;le est suffisant pour contr&ocirc;ler la taille du m&eacute;rist&egrave;me racinaire. Fait int&eacute;ressant, expression de la CQ et le g&egrave;ne de MADS-BOX enrichie en st&egrave;le AGL42 peut &ecirc;tre modul&eacute;e par BRI1 activit&eacute; uniquement dans l&#39;&eacute;piderme. Le signal de l&#39;&eacute;piderme est probablement transmis par un autre composant que BES1 et des facteurs de transcription de BZR1, comme leurs cibles directes, telles que DWF4 et BRox2, sont r&eacute;glement&eacute;es dans les m&ecirc;mes cellules qui expriment BRI1. Pris ensemble, notre &eacute;tude permet de mieux comprendre roman le r&ocirc;le de la BRs en contr&ocirc;lant la taille du m&eacute;rist&egrave;me.

Taille du m&eacute;rist&egrave;me de racine brassinost&eacute;ro&iuml;de perception chez les t&eacute;moins de l&#39;&eacute;piderme.

Pi&ugrave; ormoni di piccola molecola contribuiscono alla promozione della crescita o restrizione nelle piante. Brassinosteroids (BRs), che agisce in modo specifico nell&#39;epidermide, pu&ograve; sia guidare e limitare la crescita di sparare. Tuttavia, la nostra conoscenza di come BRs influenzano la dimensione meristema &egrave; scarsa. Qui, studiamo il meristema della radice e visualizza che BRs sono tenuti a mantenere il normale ciclo cellulare delle cellule e attivit&agrave; di espansione. Questi due processi assicurano il gradiente coerente della progressione delle cellule, da apicale per il meristema basale. Inoltre, attivit&agrave; di BR nel meristema non &egrave; accompagnata da cambiamenti del livello di espressione dei vettori efflusso auxina PIN1, PIN3 e PIN7, che sono conosciuti per controllare la misura dell&#39;attivit&agrave; mitotica e differenziazione. Noi dimostrare ulteriormente che BR segnalando l&#39;epidermide della radice e non la endodermis interna, stele cella file o cellule quiescenti centro (QC) &egrave; sufficiente per controllare la dimensione di radice meristema. &Egrave; interessante notare che, espressione di QC e il gene MADS-BOX arricchita di stele AGL42 pu&ograve; essere modulata da BRI1 attivit&agrave; esclusivamente nell&#39;epidermide. Il segnale dall&#39;epidermide &egrave; probabilmente trasmessa da un componente diverso rispetto a BES1 e BZR1 fattori di trascrizione, come loro obiettivi diretti, quali DWF4 e BRox2, sono disciplinate le stesse cellule che esprimono BRI1. Presi insieme, il nostro studio fornisce nuove intuizioni nel ruolo di BRs nel controllo della dimensione di meristema.

Brassinosteroid percezione nei controlli dell&#39;epidermide radice dimensione meristema.

M&uacute;ltiples hormonas de mol&eacute;cula peque&ntilde;a contribuyen a la promoci&oacute;n del crecimiento o restricci&oacute;n en las plantas. Brasinoesteroides (BRs), espec&iacute;ficamente en la epidermis, pueden conducir y restringir el crecimiento del brote. Sin embargo, nuestro conocimiento de c&oacute;mo BRs afectan el tama&ntilde;o de meristemo es escasa. Aqu&iacute;, estudiamos el meristemo de ra&iacute;z y mostrar que BRs son necesarias para mantener la expansi&oacute;n de actividad y de la c&eacute;lula del ciclo normal de la c&eacute;lula. Estos dos procesos aseguran el gradiente coherente de la progresi&oacute;n de la c&eacute;lula, de la apical al meristemo basal. Adem&aacute;s, actividad de BR en el meristemo no se acompa&ntilde;a de cambios en el nivel de expresi&oacute;n de los portadores de la emanaci&oacute;n de la auxina PIN1, PIN3 y PIN7, que son conocidos para controlar el grado de actividad mit&oacute;tica y diferenciaci&oacute;n. Adem&aacute;s demostramos que es suficiente para controlar el tama&ntilde;o del meristemo de ra&iacute;z BR se&ntilde;alizaci&oacute;n en la epidermis de la ra&iacute;z y no en el interior endodermis, c&eacute;lulas quiescentes centro (QC) o archivos de celular de Estela. Curiosamente, expresi&oacute;n de la QC y el gen de MADS-BOX enriquecida con Estela AGL42 puede ser modulado por BRI1 actividad exclusivamente en la epidermis. La se&ntilde;al de la epidermis se transmite probablemente por un componente diferente que BES1 y BZR1 los factores de transcripci&oacute;n, como sus objetivos directos, como DWF4 y BRox2, est&aacute;n regulados en las mismas c&eacute;lulas que expresan BRI1. Tomados en conjunto, nuestro estudio proporciona nuevas ideas en el papel de BRs en el control de tama&ntilde;o de meristemos.

Tama&ntilde;o del meristemo de la ra&iacute;z de Brasinoesteroides percepci&oacute;n en los controles de la epidermis.

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.

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

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.

Brassinosteroids in growth control: how, when and where.

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.

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

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.

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.

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.

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

Editorial overview: growth and development.

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.

Sigal Savaldi-Goldstein

Faculty of Biology

Technion-Israel Institute of Technology

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

Sigal Savaldi-Goldstein

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Faculty of Biology

Technion-Israel Institute of Technology

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

Faculty of Biology