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Membranes of eukaryotic cells contain high lipid-order sterol-rich domains that are thought to mediate temporal and spatial organization of cellular processes. Sterols are crucial for execution of cytokinesis, the last stage of cell division, in diverse eukaryotes. The cell plate of higher-plant cells is the membrane structure that separates daughter cells during somatic cytokinesis. Cell-plate formation in Arabidopsis relies on sterol- and DYNAMIN-RELATED PROTEIN1A (DRP1A)-dependent endocytosis. However, functional relationships between lipid membrane order or lipid packing and endocytic machinery components during eukaryotic cytokinesis have not been elucidated. Using ratiometric live imaging of lipid order-sensitive fluorescent probes, we show that the cell plate of Arabidopsis thaliana represents a dynamic, high lipid-order membrane domain. The cell-plate lipid order was found to be sensitive to pharmacological and genetic alterations of sterol composition. Sterols co-localize with DRP1A at the cell plate, and DRP1A accumulates in detergent-resistant membrane fractions. Modifications of sterol concentration or composition reduce cell-plate membrane order and affect DRP1A localization. Strikingly, DRP1A function itself is essential for high lipid order at the cell plate. Our findings provide evidence that the cell plate represents a high lipid-order domain, and pave the way to explore potential feedback between lipid order and function of dynamin-related proteins during cytokinesis.
Background: The identification of brassinosteroid (BR) deficient and BR insensitive mutants provided conclusive evidence that BR is a potent growth-promoting phytohormone. Arabidopsis mutants are characterized by a compact rosette structure, decreased plant height and reduced root system, delayed development, and reduced fertility. Cell expansion, cell division, and multiple developmental processes depend on BR. The molecular and physiological basis of BR action is diverse. The BR signalling pathway controls the activity of transcription factors, and numerous BR responsive genes have been identified. The analysis of dwarf mutants, however, may to some extent reveal phenotypic changes that are an effect of the altered morphology and physiology. This restriction holds particularly true for the analysis of established organs such as rosette leaves. Results: In this study, the mode of BR action was analysed in established leaves by means of two approaches. First, an inhibitor of BR biosynthesis (brassinazole) was applied to 21-day-old wild-type plants. Secondly, BR complementation of BR deficient plants, namely CPD (constitutive photomorphogenic dwarf)-antisense and cbb1 (cabbage1) mutant plants was stopped after 21 days. BR action in established leaves is associated with stimulated cell expansion, an increase in leaf index, starch accumulation, enhanced CO2 release by the tricarboxylic acid cycle, and increased biomass production. Cell number and protein content were barely affected. Conclusion: Previous analysis of BR promoted growth focused on genomic effects. However, the link between growth and changes in gene expression patterns barely provided clues to the physiological and metabolic basis of growth. Our study analysed comprehensive metabolic data sets of leaves with altered BR levels. The data suggest that BR promoted growth may depend on the increased provision and use of carbohydrates and energy. BR may stimulate both anabolic and catabolic pathways.
Leaf senescence is an active process with a pivotal impact on plant productivity. It results from extensive signalling cross-talk coordinating environmental factors with intrinsic age-related mechanisms. Although many studies have shown that leaf senescence is affected by a range of external parameters, knowledge about the regulatory systems that govern the interplay between developmental programmes and environmental stress is still vague. Salinity is one of the most important environmental stresses that promote leaf senescence and thus affect crop yield. Improving salt tolerance by avoiding or delaying senescence under stress will therefore play an important role in maintaining high agricultural productivity. Experimental evidence suggests that hydrogen peroxide (H2O2) functions as a common signalling molecule in both developmental and salt-induced leaf senescence. In this study, microarray-based gene expression profiling on Arabidopsis thaliana plants subjected to long-term salinity stress to induce leaf senescence was performed, together with co-expression network analysis for H2O2-responsive genes that are mutually up-regulated by salt induced-and developmental leaf senescence. Promoter analysis of tightly co-expressed genes led to the identification of seven cis-regulatory motifs, three of which were known previously, namely CACGTGT and AAGTCAA, which are associated with reactive oxygen species (ROS)-responsive genes, and CCGCGT, described as a stress-responsive regulatory motif, while the others, namely ACGCGGT, AGCMGNC, GMCACGT, and TCSTYGACG were not characterized previously. These motifs are proposed to be novel elements involved in the H2O2-mediated control of gene expression during salinity stress-triggered and developmental senescence, acting through upstream transcription factors that bind to these sites.
Leaf senescence is a developmentally controlled process, which is additionally modulated by a number of adverse environmental conditions. Nitrogen shortage is a well-known trigger of precocious senescence in many plant species including crops, generally limiting biomass and seed yield. However, leaf senescence induced by nitrogen starvation may be reversed when nitrogen is resupplied at the onset of senescence. Here, the transcriptomic, hormonal, and global metabolic rearrangements occurring during nitrogen resupply-induced reversal of senescence in Arabidopsis thaliana were analysed. The changes induced by senescence were essentially in keeping with those previously described; however, these could, by and large, be reversed. The data thus indicate that plants undergoing senescence retain the capacity to sense and respond to the availability of nitrogen nutrition. The combined data are discussed in the context of the reversibility of the senescence programme and the evolutionary benefit afforded thereby. Future prospects for understanding and manipulating this process in both Arabidopsis and crop plants are postulated.
Polyadenylation is a decisive 3’ end processing step during the maturation of pre-mRNAs. The length of the poly(A) tail has an impact on mRNA stability, localization and translatability. Accordingly, many eukaryotic organisms encode several copies of canonical poly(A) polymerases (cPAPs). The disruption of cPAPs in mammals results in lethality. In plants, reduced cPAP activity is non-lethal. Arabidopsis encodes three nuclear cPAPs, PAPS1, PAPS2 and PAPS4, which are constitutively expressed throughout the plant. Recently, the detailed analysis of Arabidopsis paps1 mutants revealed a subset of genes that is preferentially polyadenylated by the cPAP isoform PAPS1 (Vi et al. 2013). Thus, the specialization of cPAPs might allow the regulation of different sets of genes in order to optimally face developmental or environmental challenges.
To gain insights into the cPAP-based gene regulation in plants, the phenotypes of Arabidopsis cPAPs mutants under different conditions are characterized in detail in the following work. An involvement of all three cPAPs in flowering time regulation and stress response regulation is shown. While paps1 knockdown mutants flower early, paps4 and paps2 paps4 knockout mutants exhibit a moderate late-flowering phenotype. PAPS1 promotes the expression of the major flowering inhibitor FLC, supposedly by specific polyadenylation of an FLC activator. PAPS2 and PAPS4 exhibit partially overlapping functions and ensure timely flowering by repressing FLC and at least one other unidentified flowering inhibitor. The latter two cPAPs act in a novel regulatory pathway downstream of the autonomous pathway component FCA and act independently from the polyadenylation factors and flowering time regulators CstF64 and FY. Moreover, PAPS1 and PAPS2/PAPS4 are implicated in different stress response pathways in Arabidopsis. Reduced activity of the poly(A) polymerase PAPS1 results in enhanced resistance to osmotic and oxidative stress. Simultaneously, paps1 mutants are cold-sensitive. In contrast, PAPS2/PAPS4 are not involved in the regulation of osmotic or cold stress, but paps2 paps4 loss-of-function mutants exhibit enhanced sensitivity to oxidative stress provoked in the chloroplast. Thus, both PAPS1 and PAPS2/PAPS4 are required to maintain a balanced redox state in plants. PAPS1 seems to fulfil this function in concert with CPSF30, a polyadenylation factor that regulates alternative polyadenylation and tolerance to oxidative stress.
The individual paps mutant phenotypes and the cPAP-specific genetic interactions support the model of cPAP-dependent polyadenylation of selected mRNAs. The high similarity of the polyadenylation machineries in yeast, mammals and plants suggests that similar regulatory mechanisms might be present in other organism groups. The cPAP-dependent developmental and physiological pathways identified in this work allow the design of targeted experiments to better understand the ecological and molecular context underlying cPAP-specialization.
In plants, SNF1-related kinase (SnRK1) responds to the availability of carbohydrates as well as to environmental stresses by down-regulating ATP consuming biosynthetic processes, while stimulating energy-generating catabolic reactions through gene expression and post-transcriptional regulation. The functional SnRK1 complex is a heterotrimer where the catalytic alpha subunit associates with a regulatory beta subunit and an activating gamma subunit. Several different metabolites as well as the hormone abscisic acid (ABA) have been shown to modulate SnRK1 activity in a cell- and stimulus-type specific manner. It has been proposed that tissue- or stimulus-specific expression of adapter proteins mediating SnRK1 regulation can at least partly explain the differences observed in SnRK1 signaling. By using yeast two-hybrid and in planta bi-molecular fluorescence complementation assays we were able to demonstrate that proteins containing the domain of unknown function (DUF) 581 could interact with both isoforms of the SnRK1 alpha subunit (AKIN10/11) of Arabidopsis. A structure/function analysis suggests that the DUF581 is a generic SnRK1 interaction module and co-expression with DUF581 proteins in plant cells leads to reallocation of the kinase to specific regions within the nucleus. Yeast two-hybrid analyses suggest that SnRK1 and DUF581 proteins share common interaction partners inside the nucleus. The analysis of available microarray data implies that expression of the 19 members of the DUF581 encoding gene family in Arabidopsis is differentially regulated by hormones and environmental cues, indicating specialized functions of individual family members. We hypothesize that DUF581 proteins could act as mediators conferring tissue- and stimulus-type specific differences in SnRK1 regulation.
The coordination of cell polarity within the plane of the tissue layer (planar polarity) is crucial for the development of diverse multicellular organisms. Small Rac/Rho-family GTPases and the actin cytoskeleton contribute to planar polarity formation at sites of polarity establishment in animals and plants. Yet, upstream pathways coordinating planar polarity differ strikingly between kingdoms. In the root of Arabidopsis thaliana, a concentration gradient of the phytohormone auxin coordinates polar recruitment of Rho-of-plant (ROP) to sites of polar epidermal hair initiation. However, little is known about cytoskeletal components and interactions that contribute to this planar polarity or about their relation to the patterning machinery. Here, we show that ACTIN7 (ACT7) represents a main actin isoform required for planar polarity of root hair positioning, interacting with the negative modulator ACTIN-INTERACTING PROTEIN1-2 (AIP1-2). ACT7, AIP1-2 and their genetic interaction are required for coordinated planar polarity of ROP downstream of ethylene signalling. Strikingly, AIP1-2 displays hair cell file-enriched expression, restricted by WEREWOLF (WER)-dependent patterning and modified by ethylene and auxin action. Hence, our findings reveal AIP1-2, expressed under control of the WER-dependent patterning machinery and the ethylene signalling pathway, as a modulator of actin-mediated planar polarity.
The coordination of cell polarity within the plane of the tissue layer (planar polarity) is crucial for the development of diverse multicellular organisms. Small Rac/Rho-family GTPases and the actin cytoskeleton contribute to planar polarity formation at sites of polarity establishment in animals and plants. Yet, upstream pathways coordinating planar polarity differ strikingly between kingdoms. In the root of Arabidopsis thaliana, a concentration gradient of the phytohormone auxin coordinates polar recruitment of Rho-of-plant (ROP) to sites of polar epidermal hair initiation. However, little is known about cytoskeletal components and interactions that contribute to this planar polarity or about their relation to the patterning machinery. Here, we show that ACTIN7 (ACT7) represents a main actin isoform required for planar polarity of root hair positioning, interacting with the negative modulator ACTIN-INTERACTING PROTEIN1-2 (AIP1-2). ACT7, AIP1-2 and their genetic interaction are required for coordinated planar polarity of ROP downstream of ethylene signalling. Strikingly, AIP1-2 displays hair cell file-enriched expression, restricted by WEREWOLF (WER)-dependent patterning and modified by ethylene and auxin action. Hence, our findings reveal AIP1-2, expressed under control of the WER-dependent patterning machinery and the ethylene signalling pathway, as a modulator of actin-mediated planar polarity.
Oxidative stress causes dramatic changes in the expression levels of many genes. The formation of a functional protein through successful mRNA translation is central to a coordinated cellular response. To what extent the response towards reactive oxygen species (ROS) is regulated at the translational level is poorly understood. Here we analysed leaf- and tissue-specific translatomes using a set of transgenic Arabidopsis thaliana lines expressing a FLAG-tagged ribosomal protein to immunopurify polysome-bound mRNAs before and after oxidative stress. We determined transcript levels of 171 ROS-responsive genes upon paraquat treatment, which causes formation of superoxide radicals, at the whole-organ level. Furthermore, the translation of mRNAs was determined for five cell types: mesophyll, bundle sheath, phloem companion, epidermal and guard cells. Mesophyll and bundle sheath cells showed the strongest response to paraquat treatment. Interestingly, several ROS-responsive transcription factors displayed cell type-specific translation patterns, while others were translated in all cell types. In part, cell type-specific translation could be explained by the length of the 5-untranslated region (5-UTR) and the presence of upstream open reading frames (uORFs). Our analysis reveals insights into the translational regulation of ROS-responsive genes, which is important to understanding cell-specific responses and functions during oxidative stress.
The study illustrates the response of different Arabidopsis thaliana leaf cells and tissues to oxidative stress at the translational level, an aspect of reactive oxygen species (ROS) biology that has been little studied in the past. Our data reveal insights into how translational regulation of ROS-responsive genes is fine-tuned at the cellular level, a phenomenon contributing to the integrated physiological response of leaves to stresses involving changes in ROS levels.
Responses to pathogens, including host transcriptional reprogramming, require partially antagonistic signalling pathways dependent on the phytohormones salicylic (SA) and jasmonic (JA) acids. However, upstream factors modulating the interplay of these pathways are not well characterized. Here, we identify the transcription factor ANAC032 from Arabidopsis thaliana as one such regulator in response to the bacterial pathogen Pseudomonas syringae pv. tomato DC3000 (Pst). ANAC032 directly represses MYC2 activation upon Pst attack, resulting in blockage of coronatine-mediated stomatal reopening which restricts entry of bacteria into plant tissue. Furthermore, ANAC032 activates SA signalling by repressing NIMIN1, a key negative regulator of SA-dependent defence. Finally, ANAC032 reduces expression of JA-responsive genes, including PDF1.2A. Thus, ANAC032 enhances resistance to Pst by generating an orchestrated transcriptional output towards key SA- and JA-signalling genes coordinated through direct binding of ANAC032 to the MYC2, NIMIN1 and PDF1.2A promoters.