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

Leishmania donovani are the causative agents of kala-azar in humans. They undergo a developmental program following changes in the environment, resulting in the reversible transformation between the extracellular promastigote form in the sand fly vector and the obligatory intracellular amastigote form in phagolysosomes of macrophages. A host-free differentiation system for L. donovani was used to investigate the initial process of promastigote to amastigote differentiation. Within an hour after exposing promastigotes to differentiation signal (concomitant exposure to 37 degrees C and pH 5.5), they expressed the amastigote-specific A2 protein family. At 5 h they started to transform to amastigote-shaped cells, a process that was completed 7 h later. This morphological transformation occurred synchronously, while cells arrested at G1. By sequential exposure to elevated temperature (for 24 h) and then acidic pH, we found that heat was responsible for the growth arrest and acidic pH to its release and subsequent route to differentiation into amastigotes. Lastly, ethanol and Azetidine 2 carboxylic acid (a synthetic proline analog) that induced heat shock response in promastigotes were capable of replacing heat in the differentiation signal.

Differentiation of Leishmania donovani in host-free system: analysis of signal perception and response.

We describe the first functional and molecular characterization of an amino acid permease (LdAAP3) from the human parasitic protozoan Leishmania donovani, the causative agent of visceral leishmaniasis in humans. This permease contains 480 amino acids with 11 predicted trans-membrane domains. Expressing LdAAP3 in Saccharomyces cerevisiae mutants revealed that LdAAP3 codes for a high-affinity arginine transporter (Km 1.9 microM). LdAAP3 is highly specific for arginine as its transport was not inhibited by other amino acids or arginine-related compounds. Using green fluorescence protein (GFP) fused to the N-terminus of LdAAP3, this transporter was localized to the surface membrane of promastigotes. The GFP-LdAAP3 chimera mediated a threefold increase in arginine transport in promastigotes, indicating that it is active and confirmed that LdAAP3 codes for an arginine transporter in parasite cells as well. LdAAP3 is novel as it shares a high level of homology with amino acid permeases from other trypanosomatidae but almost none with permeases from other phyla. The results of this work suggest that LdAAP3 might play a role in host-parasite interaction.

A novel high-affinity arginine transporter from the human parasitic protozoan Leishmania donovani.

Adjustment of catalytic activity in response to diverse ambient temperatures is fundamental to life on Earth. A crucial example of this is photosynthesis, where solar energy is converted into electrochemical potential that drives oxygen and biomass generation at temperatures ranging from those of frigid Antarctica to those of scalding hot springs. The energy conversion proceeds by concerted mobilization of electrons and protons on photoexcitation of reaction centre protein complexes. Following physicochemical paradigms, the rates of imperative steps in this process were predicted to increase exponentially with rising temperatures, resulting in different yields of solar energy conversion at the distinct growth temperatures of photosynthetic mesophiles and extremophiles. In contrast, here we show a meticulous adjustment of energy conversion rate, resulting in similar yields from mesophiles and thermophiles. The key molecular players in the temperature adjustment process consist of a cluster of hitherto unrecognized protein cavities and an adjacent packing motif that jointly impart local flexibility crucial to the reaction centre proteins. Mutations within the packing motif of mesophiles that increase the bulkiness of the amino-acid side chains, and thus reduce the size of the cavities, promote thermophilic behaviour. This novel biomechanical mechanism accounts for the slowing of the catalytic reaction above physiological temperatures in contradiction to the classical Arrhenius paradigm. The mechanism provides new guidelines for manipulating the acclimatization of enzymes to the ambient temperatures of diverse habitats. More generally, it reveals novel protein elements that are of potential significance for modulating structure-activity relationships in membrane and globular proteins alike.

Protein flexibility acclimatizes photosynthetic energy conversion to the ambient temperature.

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.

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.

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.

Neta Holland

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

Neta Holland

.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