Repressive ZINC FINGER OF ARABIDOPSIS THALIANA proteins promote programmed cell death in the Arabidopsis columella root cap.

Developmental programmed cell death (dPCD) controls a plethora of functions in plant growth and reproduction. In the root cap of Arabidopsis (Arabidopsis thaliana), dPCD functions to control organ size in balance with the continuous stem cell activity in the root meristem. Key regulators of root cap dPCD including SOMBRERO/ANAC033 (SMB) belong to the NAC family of transcription factors. Here, we identify the C2H2 zinc finger protein ZINC FINGER OF ARABIDOPSIS THALIANA 14 ZAT14 as part of the gene regulatory network of root cap dPCD acting downstream of SMB. Similar to SMB, ZAT14-inducible misexpression leads to extensive ectopic cell death. Both the canonical EAR motif and a conserved L-box motif of ZAT14 act as transcriptional repression motifs and are required to trigger cell death. While a single zat14 mutant does not show a cell death-related phenotype, a quintuple mutant knocking out 5 related ZAT paralogs shows a delayed onset of dPCD execution in the columella and the adjacent lateral root cap. While ZAT14 is co-expressed with established dPCD-associated genes, it does not activate their expression. Our results suggest that ZAT14 acts as a transcriptional repressor controlling a so far uncharacterized subsection of the dPCD gene regulatory network active in specific root cap tissues.

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.

HEAT SHOCK PROTEIN 90 proteins and YODA regulate main body axis formation during early embryogenesis.

The YODA (YDA) kinase pathway is intimately associated with the control of Arabidopsis (Arabidopsis thaliana) embryo development, but little is known regarding its regulators. Using genetic analysis, HEAT SHOCK PROTEIN 90 (HSP90) proteins emerge as potent regulators of YDA in the process of embryo development and patterning. This study is focused on the characterization and quantification of early embryonal traits of single and double hsp90 and yda mutants. HSP90s genetic interactions with YDA affected the downstream signaling pathway to control the development of both basal and apical cell lineage of embryo. Our results demonstrate that the spatiotemporal expression of WUSCHEL-RELATED HOMEOBOX 8 (WOX8) and WOX2 is changed when function of HSP90s or YDA is impaired, suggesting their essential role in the cell fate determination and possible link to auxin signaling during early embryo development. Hence, HSP90s together with YDA signaling cascade affect transcriptional networks shaping the early embryo development.

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.

Multifaceted roles of HEAT SHOCK PROTEIN 90 molecular chaperones in plant development.

HEAT SHOCK PROTEINS 90 (HSP90s) are molecular chaperones that mediate correct folding and stability of many client proteins. These chaperones act as master molecular hubs involved in multiple aspects of cellular and developmental signalling in diverse organisms. Moreover, environmental and genetic perturbations affect both HSP90s and their clients, leading to alterations of molecular networks determining respectively plant phenotypes and genotypes and contributing to a broad phenotypic plasticity. Although HSP90 interaction networks affecting the genetic basis of phenotypic variation and diversity have been thoroughly studied in animals, such studies are just starting to emerge in plants. Here, we summarize current knowledge and discuss HSP90 network functions in plant development and cellular homeostasis.

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.

Phosphorylation of Plant Microtubule-Associated Proteins During Cell Division.

Progression of mitosis and cytokinesis depends on the reorganization of cytoskeleton, with microtubules driving the segregation of chromosomes and their partitioning to two daughter cells. In dividing plant cells, microtubules undergo global reorganization throughout mitosis and cytokinesis, and with the aid of various microtubule-associated proteins (MAPs), they form unique systems such as the preprophase band (PPB), the acentrosomal mitotic spindle, and the phragmoplast. Such proteins include nucleators of de novo microtubule formation, plus end binding proteins involved in the regulation of microtubule dynamics, crosslinking proteins underlying microtubule bundle formation and members of the kinesin superfamily with microtubule-dependent motor activities. The coordinated function of such proteins not only drives the continuous remodeling of microtubules during mitosis and cytokinesis but also assists the positioning of the PPB, the mitotic spindle, and the phragmoplast, affecting tissue patterning by controlling cell division plane (CDP) orientation. The affinity and the function of such proteins is variably regulated by reversible phosphorylation of serine and threonine residues within the microtubule binding domain through a number of protein kinases and phosphatases which are differentially involved throughout cell division. The purpose of the present review is to provide an overview of the function of protein kinases and protein phosphatases involved in cell division regulation and to identify cytoskeletal substrates relevant to the progression of mitosis and cytokinesis and the regulation of CDP orientation.