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The plant cell wall plays several crucial roles during plant development with its integrity acting as key signalling component for growth regulation during biotic and abiotic stresses. Cellulose microfibrils, the principal load-bearing components is the major component of the primary cell wall, whose synthesis is mediated by microtubule-associated CELLULOSE SYNTHASE (CESA) COMPLEXES (CSC). Previous studies have shown that CSC interacting proteins COMPANION OF CELLULOSE SYNTHASE (CC) facilitate sustained cellulose synthesis during salt stress by promoting repolymerization of cortical microtubules. However, our understanding of cellulose synthesis during salt stress remains incomplete.
In this study, a pull-down of CC1 protein led to the identification of a novel interactor, termed LEA-like. Phylogenetic analysis revealed that LEA-like belongs to the LATE EMBRYOGENESIS ABUNDANT (LEA) protein family, specifically to the LEA_2 subgroup, showing a close relationship with the CC proteins. Roots of the double mutants lea-like and its closest homolog emb3135 exhibited hypersensitivity when grown on cellulose synthesis inhibitors. Further analysis of higher-order mutants of lea-like, emb3135, and cesa6 demonstrated a genetic interaction between them indicating a significant role in cellulose synthesis.
Live-cell imaging revealed that both LEA-like and EMB3135 migrated with the CSC at the plasma membrane along microtubule tracks in control and oryzalin-treated conditions which destabilize microtubules, suggesting a tight interaction. Investigation of fluorescently labeled lines of different domains of the LEA-like protein revealed that the N-terminal cytosolic domain of LEA-like colocalizes with microtubules, suggesting a physical association between the two.
Considering the established role of LEA proteins in abiotic stress tolerance, we performed phenotypic analysis of the mutant under various stresses. Growth of double mutants of lea-like and emb3135 on NaCl containing media resulted in swelling of root cell indicating a putative role in salt stress tolerance. Supportive of this the quadruple mutant, lacking LEA-like, EMB3135, CC1, and CC2 proteins, exhibited a severe root growth defect on NaCl media compared to control conditions. Live-cell imaging revealed that under salt stress, the LEA-like protein forms aggregates in the plasma membrane.
In conclusion, this study has unveiled two novel interactors of the CSC that act with the CC proteins that regulate plant growth in response to salt stress providing new insights into the intricate regulation of cellulose synthesis, particularly under such conditions.
FORGETTER2 protein phosphatase and phospholipase D modulate heat stress memory in Arabidopsis
(2020)
Plants can mitigate environmental stress conditions through acclimation. In the case of fluctuating stress conditions such as high temperatures, maintaining a stress memory enables a more efficient response upon recurring stress. In a genetic screen forArabidopsis thalianamutants impaired in the memory of heat stress (HS) we have isolated theFORGETTER2(FGT2) gene, which encodes a type 2C protein phosphatase (PP2C) of the D-clade.Fgt2mutants acquire thermotolerance normally; however, they are defective in the memory of HS. FGT2 interacts with phospholipase D alpha 2 (PLD alpha 2), which is involved in the metabolism of membrane phospholipids and is also required for HS memory. In summary, we have uncovered a previously unknown component of HS memory and identified the FGT2 protein phosphatase and PLD alpha 2 as crucial players, suggesting that phosphatidic acid-dependent signaling or membrane composition dynamics underlie HS memory.
For starch metabolism to take place correctly, various enzymes and proteins acting on the starch granule surface are crucial. Recently, two non-catalytic starch-binding proteins, pivotal for normal starch turnover in Arabidopsis leaves, namely, EARLY STARVATION 1 (ESV1) and its homolog LIKE EARLY STARVATION 1 (LESV), have been identified. Both share nearly 38% sequence homology. As ESV1 has been found to influence glucan phosphorylation via two starch-related dikinases, alpha-glucan, water dikinase (GWD) and phosphoglucan, water dikinase (PWD), through modulating the surface glucan structures of the starch granules and thus affecting starch degradation, we assess the impact of its homolog LESV on starch metabolism. Thus, the 65-kDa recombinant protein LESV and the 50-kDa ESV1 were analyzed regarding their influence on the action of GWD and PWD on the surface of the starch granules. We included starches from various sources and additionally assessed the effect of these non-enzymatic proteins on other starch-related enzymes, such as starch synthases (SSI and SSIII), starch phosphorylases (PHS1), isoamylase and beta-amylase. The data obtained indicate that starch phosphorylation, hydrolyses and synthesis were affected by LESV and ESV1. Furthermore, incubation with LESV and ESV1 together exerted an additive effect on starch phosphorylation. In addition, a stable alteration of the glucan structures at the starch granule surface following treatment with LESV and ESV1 was observed. Here, we discuss all the observed changes that point to modifications in the glucan structures at the surface of the native starch granules and present a model to explain the existing processes.
The morphogenesis of sessile plants is mainly driven by directional cell growth and cell division. The organization of their cytoskeleton and the mechanical properties of the cell wall greatly influence morphogenetic events in plants. It is well known that cortical microtubules (CMTs) contribute to directional growth by regulating the deposition of the cellulose microfibrils, as major cell wall fortifying elements. More recent findings demonstrate that mechanical stresses existing in cells and tissues influence microtubule organization. Also, in dividing cells, mechanical stress directions contribute to the orientation of the new cell wall. In comparison to the microtubule cytoskeleton, the role of the actin cytoskeleton in regulating shoot meristem morphogenesis has not been extensively studied.
This thesis focuses on the functional relevance of the actin cytoskeleton during cell and tissue scale morphogenesis in the shoot apical meristem (SAM) of Arabidopsis thaliana. Visualization of transcriptional reporters indicates that ACTIN2 and ACTIN7 are two highly expressed actin genes in the SAM. A link between the actin cytoskeleton and SAM development derives from the observation that the act2-1 act7-1 double mutant has abnormal cell shape and perturbed phyllotactic patterns. Live-cell imaging of the actin cytoskeleton further shows that its organization correlates with cell shape, which indicates a potential role of actin in influencing cellular morphogenesis.
In this thesis, a detailed characterization of the act2-1 act7-1 mutant reveals that perturbation of actin leads to more rectangular cellular geometries with more 90° cell internal angles, and higher incidences of four-way junctions (four cell boundaries intersecting together). This observation deviates from the conventional tricellular junctions found in epidermal cells. Quantitative cellular-level growth data indicates that such differences in the act2-1 act7-1 mutant arise due to the reduced accuracy in the placement of the new cell wall, as well as its mechanical maturation. Changes in cellular morphology observed in the act2-1 act7-1 mutant result in cell packing defects that subsequently compromise the flow of information among cells in the SAM.
The Arabidopsis thaliana NAC transcription factor JUNGBRUNNEN1 (AtJUB1) regulates growth by directly repressing GA3ox1 and DWF4, two key genes involved in gibberellin (GA) and brassinosteroid (BR) biosynthesis, respectively, leading to GA and BR deficiency phenotypes. AtJUB1 also reduces the expression of PIF4, a bHLH transcription factor that positively controls cell elongation, while it stimulates the expression of DELLA genes, which are important repressors of growth. Here, we extend our previous findings by demonstrating that AtJUB1 induces similar GA and BR deficiency phenotypes and changes in gene expression when overexpressed in tomato (Solanum lycopersicum). Importantly, and in accordance with the growth phenotypes observed, AtJUB1 inhibits the expression of growth-supporting genes, namely the tomato orthologs of GA3ox1, DWF4 and PIF4, but activates the expression of DELLA orthologs, by directly binding to their promoters. Overexpression of AtJUB1 in tomato delays fruit ripening, which is accompanied by reduced expression of several ripeningrelated genes, and leads to an increase in the levels of various amino acids (mostly proline, beta-alanine, and phenylalanine), gamma-aminobutyric acid (GABA), and major organic acids including glutamic acid and aspartic acid. The fact that AtJUB1 exerts an inhibitory effect on the GA/BR biosynthesis and PIF4 genes but acts as a direct activator of DELLA genes in both, Arabidopsis and tomato, strongly supports the model that the molecular constituents of the JUNGBRUNNEN1 growth control module are considerably conserved across species.
Leaf senescence is an essential developmental process that involves diverse metabolic changes associated with degradation of macromolecules allowing nutrient recycling and remobilization. In contrast to the significant progress in transcriptomic analysis of leaf senescence, metabolomics analyses have been relatively limited. A broad overview of metabolic changes during leaf senescence including the interactions between various metabolic pathways is required to gain a better understanding of the leaf senescence allowing to link transcriptomics with metabolomics and physiology. In this chapter, we describe how to obtain comprehensive metabolite profiles and how to dissect metabolic shifts during leaf senescence in the model plant Arabidopsis thaliana. Unlike nucleic acid analysis for transcriptomics, a comprehensive metabolite profile can only be achieved by combining a suite of analytic tools. Here, information is provided for measurements of the contents of chlorophyll, soluble proteins, and starch by spectrophotometric methods, ions by ion chromatography, thiols and amino acids by HPLC, primary metabolites by GC/TOF-MS, and secondary metabolites and lipophilic metabolites by LC/ESI-MS. These metabolite profiles provide a rich catalogue of metabolic changes during leaf senescence, which is a helpful database and blueprint to be correlated to future studies such as transcriptome and proteome analyses, forward and reverse genetic studies, or stress-induced senescence studies.
Single molecule RNA fluorescent in situ hybridization (smFISH) enables gene transcription to be assessed at the cellular level. In this point of view article, we describe our recent smFISH research in the model plant Arabidopsis thaliana and discuss how this technique could further knowledge of plant gene transcription in the future.
The process of leaf senescence consists of the final stage of leaf development. It has evolved as a mechanism to degrade macromolecules and micronutrients and remobilize them to other developing parts of the plant; hence it plays a central role for the survival of plants and crop production. During senescence, a range of physiological, morphological, cellular, and molecular events occur, which are generally referred to as the senescence syndrome that includes several hallmarks such as visible yellowing, loss of chlorophyll and water content, increase of ion leakage and cell death, deformation of chloroplast and cell structure, as well as the upregulation of thousands of so-called senescence-associated genes (SAGs) and downregulation of photosynthesis-associated genes (PAGs). This chapter is devoted to methods characterizing the onset and progression of leaf senescence at the morphological, physiological, cellular, and molecular levels. Leaf senescence normally progresses in an age-dependent manner but is also induced prematurely by a variety of environmental stresses in plants. Focused on the hallmarks of the senescence syndrome, a series of protocols is described to asses quantitatively the senescence process caused by developmental cues or environmental perturbations. We first briefly describe the senescence process, the events associated with the senescence syndrome, and the theories and methods to phenotype senescence. Detailed protocols for monitoring senescence in planta and in vitro, using the whole plant and the detached leaf, respectively, are presented. For convenience, most of the protocols use the model plant species Arabidopsis and rice, but they can be easily extended to other plants.
Vacuole integrity maintained by DUF300 proteins is required for brassinosteroid signaling regulation
(2018)
Brassinosteroid (BR) hormone signaling controls multiple processes during plant growth and development and is initiated at the plasma membrane through the receptor kinase BRASSINOSTEROID INSENSITIVE1 (BRI1) together with co-receptors such as BRI1-ASSOCIATED RECEPTOR KINASE1 (BAK1). BRI1 abundance is regulated by endosomal recycling and vacuolar targeting, but the role of vacuole-related proteins in BR receptor dynamics and BR responses remains elusive. Here, we show that the absence of two DUF300 domain-containing tonoplast proteins, LAZARUS1 (LAZ1) and LAZ1 HOMOLOG1 (LAZ1H1), causes vacuole morphology defects, growth inhibition, and constitutive activation of BR signaling. Intriguingly, tonoplast accumulation of BAK1 was substantially increased and appeared causally linked to enhanced BRI1 trafficking and degradation in laz1 laz1h1 plants. Since unrelated vacuole mutants exhibited normal BR responses, our findings indicate that DUF300 proteins play distinct roles in the regulation of BR signaling by maintaining vacuole integrity required to balance subcellular BAK1 pools and BR receptor distribution.
Gene duplication is a major driver for the increase of biological complexity. The divergence of newly duplicated paralogs may allow novel functions to evolve, while maintaining the ancestral one. Alternatively, partitioning the ancestral function among paralogs may allow parts of that role to follow independent evolutionary trajectories. We studied the REDUCED COMPLEXITY (RCO) locus, which contains three paralogs that have evolved through two independent events of gene duplication, and which underlies repeated events of leaf shape evolution within the Brassicaceae. In particular, we took advantage of the presence of three potentially functional paralogs in Capsella to investigate the extent of functional divergence among them. We demonstrate that the RCO copies control growth in different areas of the leaf. Consequently, the copies that are retained active in the different Brassicaceae lineages contribute to define the leaf dissection pattern. Our results further illustrate how successive gene duplication events and subsequent functional divergence can increase trait evolvability by providing independent evolutionary trajectories to specialized functions that have an additive effect on a given trait.