George Komis

Arabidopsis Homologs of Nucleus- and Phragmoplast-Localized Kinase 2 and 3 and Mitogen-Activated Protein Kinase 4 Are Essential for Microtubule Organization

Arabidopsis mutants defective in MAPK signaling were found to have aberrant microtubule organization and cell growth. This study shows that two mitogen-activated protein kinase kinase kinase isoforms, mitogen-activated protein kinase 4 and microtubule-associated protein 65, play a role in the organization of cortical microtubules. A double homozygous recessive mutant in the Arabidopsis thaliana homologs of nucleus- and phragmoplast-localized kinase 2 (ANP2) and 3 (ANP3) genes and a homozygous recessive mutant in the mitogen-activated protein kinase 4 (MPK4) gene of Arabidopsis exhibit deficiencies in the overall microtubule (MT) organization, which result in abnormal cell growth patterns, such as branching of root hairs and swelling of diffusely growing epidermal cells. Genetic, pharmacological, molecular, cytological, and biochemical analyses show that the major underlying mechanism for these phenotypes is excessive MT stabilization manifested in both mutants as heavy MT bundling, disorientation, and drug stability. The above defects in MAPK signaling result in the adverse regulation of members of the microtubule-associated protein (MAP65) protein family, including strongly diminished phosphorylation of MAP65-1. These data suggest that ANP2/ANP3, MPK4, and the microtubule-associated protein MAP65-1, a putative target of MPK4 signaling, are all essential for the proper organization of cortical microtubules in Arabidopsis epidermal cells.

Crosstalk between secondary messengers, hormones and MAPK modules during abiotic stress signalling in plants

The crosstalk between second messengers, hormones and mitogen-activated protein kinases (MAPKs) in plant signalling systems facilitates adaptation and survival in the face of diverse environmental stresses. This review focuses on the transduction of second messenger and hormone signals by MAPK modules in plant abiotic stress responses. We discuss how this crosstalk regulates gene expression (e.g. by controlling transcription factor activity) and other cellular and physiological responses to enable adaptation and/or resistance to abiotic stresses.

Mitogen‐activated protein kinase 4 is involved in the regulation of mitotic and cytokinetic microtubule transitions in Arabidopsis thaliana

• A mitogen-activated protein kinase kinase kinase (MAPKKK) double mutant, Arabidopsis homologue of nucleus and phragmoplast associated kinase (anp) anp2anp3, and the mitogen-activated protein kinase (MAPK) 4 mutant mpk4 of Arabidopsis thaliana show prominent cytokinetic defects. This prompted the analysis of mitotic and cytokinetic progression as a function of MAPK signalling. Mutants were compared with wild types untreated or treated with the specific MAPKK inhibitor PD98059. • This study included phenotype analysis, expression analysis of the MPK4 promoter, immunofluorescent localization of MPK4, tubulin and MAP65-1, and time-lapse microscopic visualization of the mitotic microtubule (MT) transitions in control, mutant and inhibitor-treated cells. • Mutant and inhibitor-treated cells showed defects in mitosis and cytokinesis, including aberrant spindle and phragmoplast formation and drastically delayed or abortive mitosis and cytokinesis. As a result, bi- and multinucleate cells were formed, ultimately disturbing the vegetative tissue patterning. MPK4 was localized to all stages of the expanding phragmoplast, in a pattern similar to that of its putative substrate MAP65-1. • In this study, MPK4 is shown to be involved in the regulation of mitosis/cytokinesis through modulation of the cell division plane and cytokinetic progression.

Involvement of YODA and mitogen activated protein kinase 6 in Arabidopsis post‐embryogenic root development through auxin up‐regulation and cell division plane orientation

The role of YODA MITOGEN ACTIVATED PROTEIN KINASE KINASE KINASE 4 (MAPKKK4) upstream of MITOGEN ACTIVATED PROTEIN KINASE 6 (MPK6) was studied during post-embryonic root development of Arabidopsis thaliana. Loss- and gain-of-function mutants of YODA (yda1 and ΔNyda1) were characterized in terms of root patterning, endogenous auxin content and global proteomes. We surveyed morphological and cellular phenotypes of yda1 and ΔNyda1 mutants suggesting possible involvement of auxin. Endogenous indole-3-acetic acid (IAA) levels were up-regulated in both mutants. Proteomic analysis revealed up-regulation of auxin biosynthetic enzymes tryptophan synthase and nitrilases in these mutants. The expression, abundance and phosphorylation of MPK3, MPK6 and MICROTUBULE ASSOCIATED PROTEIN 65-1 (MAP65-1) were characterized by quantitative polymerase chain reaction (PCR) and western blot analyses and interactions between MAP65-1, microtubules and MPK6 were resolved by quantitative co-localization studies and co-immunoprecipitations. yda1 and ΔNyda1 mutants showed disoriented cell divisions in primary and lateral roots, abortive cytokinesis, and differential subcellular localization of MPK6 and MAP65-1. They also showed deregulated expression of TANGLED1 (TAN1), PHRAGMOPLAST ORIENTING KINESIN 1 (POK1), and GAMMA TUBULIN COMPLEX PROTEIN 4 (GCP4). The findings that MPK6 localized to preprophase bands (PPBs) and phragmoplasts while the mpk6-4 mutant transformed with MPK6AEF (alanine (A)-glutamic acid (E)-phenylanine (F)) showed a root phenotype similar to that of yda1 demonstrated that MPK6 is an important player downstream of YODA. These data indicate that YODA and MPK6 are involved in post-embryonic root development through an auxin-dependent mechanism regulating cell division and mitotic microtubule (PPB and phragmoplast) organization.

Emerging topics in the cell biology of mitogen-activated protein kinases

Signaling through mitogen-activated protein kinase (MAPK) cascades is organized in complex interconnected subcellular networks. Upon MAPK activation, signals are transferred to targets in different subcellular compartments able to regulate various cellular processes. Therefore, subcellular dissection of individual MAPK modules is vital to understand how a single MAPK can simultaneously mediate many tasks and how a single stimulus can direct different MAPK modules to separated tasks. In this opinion article, we present a subcellular localization prediction of all members of Arabidopsis thaliana MAPK modules validated wherever possible with experimental data. Furthermore, we propose, that at least in part, the complexity of plant MAPK signaling can be explained by unique strategies of subcellular targeting, which will be worth investigating in the near future.

Superresolution live imaging of plant cells using structured illumination microscopy

Although superresolution (SR) approaches have been routinely used for fixed or living material from other organisms, the use of time-lapse structured illumination microscopy (SIM) imaging in plant cells still remains under-developed. Here we describe a validated method for time-lapse SIM that focuses on cortical microtubules of different plant cell types. By using one of the existing commercially available SIM platforms, we provide a user-friendly and easy-to-follow protocol that may be widely applied to the imaging of plant cells. This protocol includes steps describing calibration of the microscope and channel alignment, generation of an experimental point spread function (PSF), preparation of appropriate observation chambers with available plant material, image acquisition, reconstruction and validation. This protocol can be carried out within two to three working days.

Transient plant transformation mediated by Agrobacterium tumefaciens: Principles, methods and applications.

Agrobacterium tumefaciens is widely used as a versatile tool for development of stably transformed model plants and crops. However, the development of Agrobacterium based transient plant transformation methods attracted substantial attention in recent years. Transient transformation methods offer several applications advancing stable transformations such as rapid and scalable recombinant protein production and in planta functional genomics studies. Herein, we highlight Agrobacterium and plant genetics factors affecting transfer of T-DNA from Agrobacterium into the plant cell nucleus and subsequent transient transgene expression. We also review recent methods concerning Agrobacterium mediated transient transformation of model plants and crops and outline key physical, physiological and genetic factors leading to their successful establishment. Of interest are especially Agrobacterium based reverse genetics studies in economically important crops relying on use of RNA interference (RNAi) or virus-induced gene silencing (VIGS) technology. The applications of Agrobacterium based transient plant transformation technology in biotech industry are presented in thorough detail. These involve production of recombinant proteins (plantibodies, vaccines and therapeutics) and effectoromics-assisted breeding of late blight resistance in potato. In addition, we also discuss biotechnological potential of recombinant GFP technology and present own examples of successful Agrobacterium mediated transient plant transformations.

Super-resolution Microscopy in Plant Cell Imaging

Although the development of super-resolution microscopy methods dates back to 1994, relevant applications in plant cell imaging only started to emerge in 2010. Since then, the principal super-resolution methods, including structured-illumination microscopy (SIM), photoactivation localization microscopy (PALM), stochastic optical reconstruction microscopy (STORM), and stimulated emission depletion microscopy (STED), have been implemented in plant cell research. However, progress has been limited due to the challenging properties of plant material. Here we summarize the basic principles of existing super-resolution methods and provide examples of applications in plant science. The limitations imposed by the nature of plant material are reviewed and the potential for future applications in plant cell imaging is highlighted.

Trans-Golgi network localized small GTPase RabA1d is involved in cell plate formation and oscillatory root hair growth

BackgroundSmall Rab GTPases are important regulators of vesicular trafficking in plants. AtRabA1d, a member of the RabA1 subfamily of small GTPases, was previously found in the vesicle-rich apical dome of growing root hairs suggesting a role during tip growth; however, its specific intracellular localization and role in plants has not been well described.ResultsThe transient expression of 35S::GFP:RabA1d construct in Allium porrum and Nicotiana benthamiana revealed vesicular structures, which were further corroborated in stable transformed Arabidopsis thaliana plants. GFP-RabA1d colocalized with the trans-Golgi network marker mCherry-VTI12 and with early FM4-64-labeled endosomal compartments. Late endosomes and endoplasmic reticulum labeled with FYVE-DsRed and ER-DsRed, respectively, were devoid of GFP-RabA1d. The accumulation of GFP-RabA1d in the core of brefeldin A (BFA)-induced-compartments and the quantitative upregulation of RabA1d protein levels after BFA treatment confirmed the association of RabA1d with early endosomes/TGN and its role in vesicle trafficking. Light-sheet microscopy revealed involvement of RabA1d in root development. In root cells, GFP-RabA1d followed cell plate expansion consistently with cytokinesis-related vesicular trafficking and membrane recycling. GFP-RabA1d accumulated in disc-like structures of nascent cell plates, which progressively evolved to marginal ring-like structures of the growing cell plates. During root hair growth and development, GFP-RabA1d was enriched at root hair bulges and at the apical dome of vigorously elongating root hairs. Importantly, GFP-RabA1d signal intensity exhibited an oscillatory behavior in-phase with tip growth. Progressively, this tip localization dissapeared in mature root hairs suggesting a link between tip localization of RabA1d and root hair elongation. Our results support a RabA1d role in events that require vigorous membrane trafficking.ConclusionsRabA1d is located in early endosomes/TGN and is involved in vesicle trafficking. RabA1d participates in both cell plate formation and root hair oscillatory tip growth. The specific GFP-RabA1d subcellular localization confirms a correlation between its specific spatio-temporal accumulation and local vesicle trafficking requirements during cell plate and root hair formation.

Preparation of plants for developmental and cellular imaging by light-sheet microscopy

Long-term fluorescence live-cell imaging experiments have long been limited by the effects of excitation-induced phototoxicity. The advent of light-sheet microscopy now allows users to overcome this limitation by restricting excitation to a narrow illumination plane. In addition, light-sheet imaging allows for high-speed image acquisition with uniform illumination of samples composed of multiple cell layers. The majority of studies conducted thus far have used custom-built platforms with specialized hardware and software, along with specific sample handling approaches. The first versatile commercially available light-sheet microscope, Lightsheet Z.1, offers a number of innovative solutions, but it requires specific strategies for sample handling during long-term imaging experiments. There are currently no standard procedures describing the preparation of plant specimens for imaging with the Lightsheet Z.1. Here we describe a detailed protocol to prepare plant specimens for light-sheet microscopy, in which Arabidopsis seeds or seedlings are placed in solid medium within glass capillaries or fluorinated ethylene propylene tubes. Preparation of plant material for imaging may be completed within one working day.