High-resolution genome mapping and functional dissection of chlorogenic acid production in Lonicera maackii.

Amur honeysuckle (Lonicera maackii) is a widely used medicinal plant of the Caprifoliaceae family that produces chlorogenic acid. Research on this plant mainly focuses on its ornamental value and medicinal compounds, but a reference genome sequence and molecular resources for accelerated breeding are currently lacking. Herein, nanopore sequencing and high-throughput chromosome conformation capture (Hi-C) allowed a chromosome-level genome assembly of L. maackii (2n = 18). A global view of the gene regulatory network involved in the biosynthesis of chlorogenic acid and the dynamics of fruit coloration in L. maackii was established through metabolite profiling and transcriptome analyses. Moreover, we identified the genes encoding hydroxycinnamoyl-CoA quinate transferase (LmHQT) and hydroxycinnamoyl-CoA shikimic/quinate transferase (LmHCT), which localized to the cytosol and nucleus. Heterologous overexpression of these genes in Nicotiana benthamiana leaves resulted in elevated chlorogenic acid contents. Importantly, HPLC analyses revealed that LmHCT and LmHQTs recombinant proteins modulate the accumulation of chlorogenic acid (CGA) using quinic acid and caffeoyl CoA as substrates, highlighting the importance of LmHQT and LmHCT in CGA biosynthesis. These results confirmed that LmHQTs and LmHCT catalyze the biosynthesis of CGA in vitro. The genomic data presented in this study will offer a valuable resource for the elucidation of CGA biosynthesis and facilitating selective molecular breeding.

Spatial proteomics of vesicular trafficking: coupling mass spectrometry and imaging approaches in membrane biology.

In plants, membrane compartmentalization requires vesicle trafficking for communication among distinct organelles. Membrane proteins involved in vesicle trafficking are highly dynamic and can respond rapidly to changes in the environment and to cellular signals. Capturing their localization and dynamics is thus essential for understanding the mechanisms underlying vesicular trafficking pathways. Quantitative mass spectrometry and imaging approaches allow a system-wide dissection of the vesicular proteome, the characterization of ligand-receptor pairs and the determination of secretory, endocytic, recycling and vacuolar trafficking pathways. In this review, we highlight major proteomics and imaging methods employed to determine the location, distribution and abundance of proteins within given trafficking routes. We focus in particular on methodologies for the elucidation of vesicle protein dynamics and interactions and their connections to downstream signalling outputs. Finally, we assess their biological applications in exploring different cellular and subcellular processes.

Imaging plant cells and organs with light-sheet and super-resolution microscopy.

The documentation of plant growth and development requires integrative and scalable approaches to investigate and spatiotemporally resolve various dynamic processes at different levels of plant body organization. The present update deals with vigorous developments in mesoscopy, microscopy and nanoscopy methods that have been translated to imaging of plant subcellular compartments, cells, tissues and organs over the past 3 years with the aim to report recent applications and reasonable expectations from current light-sheet fluorescence microscopy (LSFM) and super-resolution microscopy (SRM) modalities. Moreover, the shortcomings and limitations of existing LSFM and SRM are discussed, particularly for their ability to accommodate plant samples and regarding their documentation potential considering spherical aberrations or temporal restrictions prohibiting the dynamic recording of fast cellular processes at the three dimensions. For a more comprehensive description, advances in living or fixed sample preparation methods are also included, supported by an overview of developments in labeling strategies successfully applied in plants. These strategies are practically documented by current applications employing model plant Arabidopsis thaliana (L.) Heynh., but also robust crop species such as Medicago sativa L. and Hordeum vulgare L. Over the past few years, the trend towards designing of integrative microscopic modalities has become apparent and it is expected that in the near future LSFM and SRM will be bridged to achieve broader multiscale plant imaging with a single platform.

Knockout of MITOGEN-ACTIVATED PROTEIN KINASE 3 causes barley root resistance against Fusarium graminearum.

The roles of mitogen-activated protein kinases (MAPKs) in plant-fungal pathogenic interactions are poorly understood in crops. Here, microscopic, phenotypic, proteomic, and biochemical analyses revealed that roots of independent transcription activator-like effector nuclease (TALEN)-based knockout lines of barley (Hordeum vulgare L.) MAPK 3 (HvMPK3 KO) were resistant against Fusarium graminearum infection. When co-cultured with roots of the HvMPK3 KO lines, F. graminearum hyphae were excluded to the extracellular space, the growth pattern of extracellular hyphae was considerably deregulated, mycelia development was less efficient, and number of appressoria-like structures and their penetration potential were substantially reduced. Intracellular penetration of hyphae was preceded by the massive production of reactive oxygen species (ROS) in attacked cells of the wild-type (WT), but ROS production was mitigated in the HvMPK3 KO lines. Suppression of ROS production in these lines coincided with elevated abundance of catalase (CAT) and ascorbate peroxidase (APX). Moreover, differential proteomic analysis revealed downregulation of several defense-related proteins in WT, and the upregulation of pathogenesis-related protein 1 (PR-1) and cysteine proteases in HvMPK3 KO lines. Proteins involved in suberin formation, such as peroxidases, lipid transfer proteins (LTPs), and the GDSL esterase/lipase (containing "GDSL" aminosequence motif) were differentially regulated in HvMPK3 KO lines after F. graminearum inoculation. Consistent with proteomic analysis, microscopic observations showed enhanced suberin accumulation in roots of HvMPK3 KO lines, most likely contributing to the arrested infection by F. graminearum. These results suggest that TALEN-based knockout of HvMPK3 leads to barley root resistance against Fusarium root rot.

Phosphorylation-Mediated Dynamics of Nitrate Transceptor NRT1.1 Regulate Auxin Flux and Nitrate Signaling in Lateral Root Growth.

The dual-affinity nitrate transceptor NITRATE TRANSPORTER1.1 (NRT1.1) has two modes of transport and signaling, governed by Thr-101 (T101) phosphorylation. NRT1.1 regulates lateral root (LR) development by modulating nitrate-dependent basipetal auxin export and nitrate-mediated signal transduction. Here, using the Arabidopsis (Arabidopsis thaliana) NRT1.1T101D phosphomimetic and NRT1.1T101A nonphosphorylatable mutants, we found that the phosphorylation state of NRT1.1 plays a key role in NRT1.1 function during LR development. Single-particle tracking revealed that phosphorylation affected NRT1.1 spatiotemporal dynamics. The phosphomimetic NRT1.1T101D form showed fast lateral mobility and membrane partitioning that facilitated auxin flux under low-nitrate conditions. By contrast, nonphosphorylatable NRT1.1T101A showed low lateral mobility and oligomerized at the plasma membrane (PM), where it induced endocytosis via the clathrin-mediated endocytosis and microdomain-mediated endocytosis pathways under high-nitrate conditions. These behaviors promoted LR development by suppressing NRT1.1-controlled auxin transport on the PM and stimulating Ca2+-ARABIDOPSIS NITRATE REGULATED1 signaling from the endosome.

Dynamics and organization of cortical microtubules as revealed by superresolution structured illumination microscopy.

Plants employ acentrosomal mechanisms to organize cortical microtubule arrays essential for cell growth and differentiation. Using structured illumination microscopy (SIM) adopted for the optimal documentation of Arabidopsis (Arabidopsis thaliana) hypocotyl epidermal cells, dynamic cortical microtubules labeled with green fluorescent protein fused to the microtubule-binding domain of the mammalian microtubule-associated protein MAP4 and with green fluorescent protein-fused to the alpha tubulin6 were comparatively recorded in wild-type Arabidopsis plants and in the mitogen-activated protein kinase mutant mpk4 possessing the former microtubule marker. The mpk4 mutant exhibits extensive microtubule bundling, due to increased abundance and reduced phosphorylation of the microtubule-associated protein MAP65-1, thus providing a very useful genetic tool to record intrabundle microtubule dynamics at the subdiffraction level. SIM imaging revealed nano-sized defects in microtubule bundling, spatially resolved microtubule branching and release, and finally allowed the quantification of individual microtubules within cortical bundles. Time-lapse SIM imaging allowed the visualization of subdiffraction, short-lived excursions of the microtubule plus end, and dynamic instability behavior of both ends during free, intrabundle, or microtubule-templated microtubule growth and shrinkage. Finally, short, rigid, and nondynamic microtubule bundles in the mpk4 mutant were observed to glide along the parent microtubule in a tip-wise manner. In conclusion, this study demonstrates the potential of SIM for superresolution time-lapse imaging of plant cells, showing unprecedented details accompanying microtubule dynamic organization.

Proteomic and biochemical analyses show a functional network of proteins involved in antioxidant defense of the Arabidopsis anp2anp3 double mutant.

Disentanglement of functional complexity associated with plant mitogen-activated protein kinase (MAPK) signaling has benefited from transcriptomic, proteomic, phosphoproteomic, and genetic studies. Published transcriptomic analysis of a double homozygous recessive anp2anp3 mutant of two MAPK kinase kinase (MAPKKK) genes called Arabidopsis thaliana Homologues of Nucleus- and Phragmoplast-localized Kinase 2 (ANP2) and 3 (ANP3) showed the upregulation of stress-related genes. In this study, a comparative proteomic analysis of anp2anp3 mutant against its respective Wassilevskaja ecotype (Ws) wild type background is provided. Such differential proteomic analysis revealed overabundance of core enzymes such as FeSOD1, MnSOD, DHAR1, and FeSOD1-associated regulatory protein CPN20, which are involved in the detoxification of reactive oxygen species in the anp2anp3 mutant. The proteomic results were validated at the level of single protein abundance by Western blot analyses and by quantitative biochemical determination of antioxidant enzymatic activities. Finally, the functional network of proteins involved in antioxidant defense in the anp2anp3 mutant was physiologically linked with the increased resistance of mutant seedlings against paraquat treatment.

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

BACKGROUND: Small 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. RESULTS: The 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. CONCLUSIONS: RabA1d 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.

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.

Salt-induced subcellular kinase relocation and seedling susceptibility caused by overexpression of Medicago SIMKK in Arabidopsis.

Dual-specificity mitogen-activated protein kinases kinases (MAPKKs) are the immediate upstream activators of MAPKs. They simultaneously phosphorylate the TXY motif within the activation loop of MAPKs, allowing them to interact with and regulate multiple substrates. Often, the activation of MAPKs triggers their nuclear translocation. However, the spatiotemporal dynamics and the physiological consequences of the activation of MAPKs, particularly in plants, are still poorly understood. Here, we studied the activation and localization of the Medicago sativa stress-induced MAPKK (SIMKK)-SIMK module after salt stress. In the inactive state, SIMKK and SIMK co-localized in the cytoplasm and in the nucleus. Upon salt stress, however, a substantial part of the nuclear pool of both SIMKK and SIMK relocated to cytoplasmic compartments. The course of nucleocytoplasmic shuttling of SIMK correlated temporally with the dual phosphorylation of the pTEpY motif. SIMKK function was further studied in Arabidopsis plants overexpressing SIMKK-yellow fluorescent protein (YFP) fusions. SIMKK-YFP plants showed enhanced activation of Arabidopsis MPK3 and MPK6 kinases upon salt treatment and exhibited high sensitivity against salt stress at the seedling stage, although they were salt insensitive during seed germination. Proteomic analysis of SIMKK-YFP overexpressors indicated the differential regulation of proteins directly or indirectly involved in salt stress responses. These proteins included catalase, peroxiredoxin, glutathione S-transferase, nucleoside diphosphate kinase 1, endoplasmic reticulum luminal-binding protein 2, and finally plasma membrane aquaporins. In conclusion, Arabidopsis seedlings overexpressing SIMKK-YFP exhibited higher salt sensitivity consistent with their proteome composition and with the presumptive MPK3/MPK6 hijacking of the salt response pathway.

Arabidopsis homologs of nucleus- and phragmoplast-localized kinase 2 and 3 and mitogen-activated protein kinase 4 are essential for microtubule organization.

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.

Effect of Glyceryl Monoolein Addition on the Foaming Properties and Stability of Whipped Oleogels.

Medium Chain Triglyceride (MCT) oil was successfully combined with Glyceryl Monostearate (GMS) and Glyceryl Monoolein (GMO) to form oleogels that were subsequently whipped to form stable oleofoams. The co-crystallization of GMS and GMO at a ratio of 20:1, 20:2.5, and 20:5 within MCT oil was studied through Differential Scanning Calorimetry (DSC), X-ray Diffraction analysis (XRD), rheological analysis, Fluorescence Recovery after Photobleaching (FRAP), Fourier Transform Infrared Spectroscopy (FTIR), and polarized microscopy. The addition of 5% GMO resulted in the production of more stable oleogels in terms of crystal structure and higher peak melting point, rendering this formulation suitable for pharmaceutical applications that are intended to be used internally and those that require stability at temperatures close to 40 °C. All formulations were whipped to form oleofoams that were evaluated for their storage stability for prolonged period at different temperatures. The results show that oleofoams containing 5% MGO retained their foam characteristics even after 3 months of storage under different temperature conditions.

Arabidopsis MPK6 is involved in cell division plane control during early root development, and localizes to the pre-prophase band, phragmoplast, trans-Golgi network and plasma membrane.

The proper spatial and temporal expression and localization of mitogen-activated protein kinases (MAPKs) is essential for developmental and cellular signalling in all eukaryotes. Here, we analysed expression, subcellular localization and function of MPK6 in roots of Arabidopsis thaliana using wild-type plants and three mpk6 knock-out mutant lines. The MPK6 promoter showed two expression maxima in the most apical part of the root meristem and in the root transition zone. This expression pattern was highly consistent with 'no root' and 'short root' phenotypes, as well as with ectopic cell divisions and aberrant cell division planes, resulting in disordered cell files in the roots of these mpk6 knock-out mutants. In dividing root cells, MPK6 was localized on the subcellular level to distinct fine spots in the pre-prophase band and phragmoplast, representing the two most important cytoskeletal structures controlling the cell division plane. By combining subcellular fractionation and microscopic in situ and in vivo co-localization methods, MPK6 was localized to the plasma membrane (PM) and the trans-Golgi network (TGN). In summary, these data suggest that MPK6 localizing to mitotic microtubules, secretory TGN vesicles and the PM is involved in cell division plane control and root development in Arabidopsis.

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.

GR24, A Synthetic Strigolactone Analog, and Light Affect the Organization of Cortical Microtubules in Arabidopsis Hypocotyl Cells.

Strigolactones are plant hormones regulating cytoskeleton-mediated developmental events in roots, such as lateral root formation and elongation of root hairs and hypocotyls. The latter process was addressed herein by the exogenous application of a synthetic strigolactone, GR24, and an inhibitor of strigolactone biosynthesis, TIS108, on hypocotyls of wild-type Arabidopsis and a strigolactone signaling mutant max2-1 (more axillary growth 2-1). Owing to the interdependence between light and strigolactone signaling, the present work was extended to seedlings grown under a standard light/dark regime, or under continuous darkness. Given the essential role of the cortical microtubules in cell elongation, their organization and dynamics were characterized under the conditions of altered strigolactone signaling using fluorescence microscopy methods with different spatiotemporal capacities, such as confocal laser scanning microscopy (CLSM) and structured illumination microscopy (SIM). It was found that GR24-dependent inhibition of hypocotyl elongation correlated with changes in cortical microtubule organization and dynamics, observed in living wild-type and max2-1 seedlings stably expressing genetically encoded fluorescent molecular markers for microtubules. Quantitative assessment of microscopic datasets revealed that chemical and/or genetic manipulation of strigolactone signaling affected microtubule remodeling, especially under light conditions. The application of GR24 in dark conditions partially alleviated cytoskeletal rearrangement, suggesting a new mechanistic connection between cytoskeletal behavior and the light-dependence of strigolactone signaling.

TALEN-Based HvMPK3 Knock-Out Attenuates Proteome and Root Hair Phenotypic Responses to flg22 in Barley.

Mitogen activated protein kinases (MAPKs) integrate elicitor perception with both early and late responses associated with plant defense and innate immunity. Much of the existing knowledge on the role of plant MAPKs in defense mechanisms against microbes stems from extensive research in the model plant Arabidopsis thaliana. In the present study, we investigated the involvement of barley (Hordeum vulgare) MPK3 in response to flagellin peptide flg22, a well-known bacterial elicitor. Using differential proteomic analysis we show that TALEN-induced MPK3 knock-out lines of barley (HvMPK3 KO) exhibit constitutive downregulation of defense related proteins such as PR proteins belonging to thaumatin family and chitinases. Further analyses showed that the same protein families were less prone to flg22 elicitation in HvMPK3 KO plants compared to wild types. These results were supported and validated by chitinase activity analyses and immunoblotting for HSP70. In addition, differential proteomes correlated with root hair phenotypes and suggested tolerance of HvMPK3 KO lines to flg22. In conclusion, our study points to the specific role of HvMPK3 in molecular and root hair phenotypic responses of barley to flg22.

Spatiotemporal Pattern of Ectopic Cell Divisions Contribute to Mis-Shaped Phenotype of Primary and Lateral Roots of katanin1 Mutant.

Pattern formation, cell proliferation, and directional cell growth, are driving factors of plant organ shape, size, and overall vegetative development. The establishment of vegetative morphogenesis strongly depends on spatiotemporal control and synchronization of formative and proliferative cell division patterns. In this context, the progression of cell division and the regulation of cell division plane orientation are defined by molecular mechanisms converging to the proper positioning and temporal reorganization of microtubule arrays such as the preprophase microtubule band, the mitotic spindle and the cytokinetic phragmoplast. By focusing on the tractable example of primary root development and lateral root emergence in Arabidopsis thaliana, genetic studies have highlighted the importance of mechanisms underlying microtubule reorganization in the establishment of the root system. In this regard, severe alterations of root growth, and development found in extensively studied katanin1 mutants of A. thaliana (fra2, lue1, and ktn1-2), were previously attributed to defective rearrangements of cortical microtubules and aberrant cell division plane reorientation. How KATANIN1-mediated microtubule severing contributes to tissue patterning and organ morphogenesis, ultimately leading to anisotropy in microtubule organization is a trending topic under vigorous investigation. Here we addressed this issue during root development, using advanced light-sheet fluorescence microscopy (LSFM) and long-term imaging of ktn1-2 mutant expressing the GFP-TUA6 microtubule marker. This method allowed spatial and temporal monitoring of cell division patterns in growing roots. Analysis of acquired multidimensional data sets revealed the occurrence of ectopic cell divisions in various tissues including the calyptrogen and the protoxylem of the main root, as well as in lateral root primordia. Notably the ktn1-2 mutant exhibited excessive longitudinal cell divisions (parallel to the root axis) at ectopic positions. This suggested that changes in the cell division pattern and the occurrence of ectopic cell divisions contributed significantly to pleiotropic root phenotypes of ktn1-2 mutant. LSFM provided evidence that KATANIN1 is required for the spatiotemporal control of cell divisions and establishment of tissue patterns in living A. thaliana roots.

Complementary Superresolution Visualization of Composite Plant Microtubule Organization and Dynamics.

Microtubule bundling is an essential mechanism underlying the biased organization of interphase and mitotic microtubular systems of eukaryotes in ordered arrays. Microtubule bundle formation can be exemplified in plants, where the formation of parallel microtubule systems in the cell cortex or the spindle midzone is largely owing to the microtubule crosslinking activity of a family of microtubule associated proteins, designated as MAP65s. Among the nine members of this family in Arabidopsis thaliana, MAP65-1 and MAP65-2 are ubiquitous and functionally redundant. Crosslinked microtubules can form high-order arrays, which are difficult to track using widefield or confocal laser scanning microscopy approaches. Here, we followed spatiotemporal patterns of MAP65-2 localization in hypocotyl cells of Arabidopsis stably expressing fluorescent protein fusions of MAP65-2 and tubulin. To circumvent imaging difficulties arising from the density of cortical microtubule bundles, we use different superresolution approaches including Airyscan confocal laser scanning microscopy (ACLSM), structured illumination microscopy (SIM), total internal reflection SIM (TIRF-SIM), and photoactivation localization microscopy (PALM). We provide insights into spatiotemporal relations between microtubules and MAP65-2 crossbridges by combining SIM and ACLSM. We obtain further details on MAP65-2 distribution by single molecule localization microscopy (SMLM) imaging of either mEos3.2-MAP65-2 stochastic photoconversion, or eGFP-MAP65-2 stochastic emission fluctuations under specific illumination conditions. Time-dependent dynamics of MAP65-2 were tracked at variable time resolution using SIM, TIRF-SIM, and ACLSM and post-acquisition kymograph analysis. ACLSM imaging further allowed to track end-wise dynamics of microtubules labeled with TUA6-GFP and to correlate them with concomitant fluctuations of MAP65-2 tagged with tagRFP. All different microscopy modules examined herein are accompanied by restrictions in either the spatial resolution achieved, or in the frame rates of image acquisition. PALM imaging is compromised by speed of acquisition. This limitation was partially compensated by exploiting emission fluctuations of eGFP which allowed much higher photon counts at substantially smaller time series compared to mEos3.2. SIM, TIRF-SIM, and ACLSM were the methods of choice to follow the dynamics of MAP65-2 in bundles of different complexity. Conclusively, the combination of different superresolution methods allowed for inferences on the distribution and dynamics of MAP65-2 within microtubule bundles of living A. thaliana cells.

YODA-HSP90 Module Regulates Phosphorylation-Dependent Inactivation of SPEECHLESS to Control Stomatal Development under Acute Heat Stress in Arabidopsis.

Stomatal ontogenesis, patterning, and function are hallmarks of environmental plant adaptation, especially to conditions limiting plant growth, such as elevated temperatures and reduced water availability. The specification and distribution of a stomatal cell lineage and its terminal differentiation into guard cells require a master regulatory protein phosphorylation cascade involving the YODA mitogen-activated protein kinase kinase kinase. YODA signaling results in the activation of MITOGEN-ACTIVATED PROTEIN KINASEs (MPK3 and MPK6), which regulate transcription factors, including SPEECHLESS (SPCH). Here, we report that acute heat stress affects the phosphorylation and deactivation of SPCH and modulates stomatal density. By using complementary molecular, genetic, biochemical, and cell biology approaches, we provide solid evidence that HEAT SHOCK PROTEINS 90 (HSP90s) play a crucial role in transducing heat-stress response through the YODA cascade. Genetic studies revealed that YODA and HSP90.1 are epistatic, and they likely function linearly in the same developmental pathway regulating stomata formation. HSP90s interact with YODA, affect its cellular polarization, and modulate the phosphorylation of downstream targets, such as MPK6 and SPCH, under both normal and heat-stress conditions. Thus, HSP90-mediated specification and differentiation of the stomatal cell lineage couples stomatal development to environmental cues, providing an adaptive heat stress response mechanism in plants.