APRF1 Interactome Reveals HSP90 as a New Player in the Complex That Epigenetically Regulates Flowering Time in Arabidopsis thaliana.

WD40 repeat proteins (WDRs) are present in all eukaryotes and include members that are implicated in numerous cellular activities. They act as scaffold proteins and thus as molecular "hubs" for protein-protein interactions, which mediate the assembly of multifunctional complexes that regulate key developmental processes in Arabidopsis thaliana, such as flowering time, hormonal signaling, and stress responses. Despite their importance, many aspects of their putative functions have not been elucidated yet. Here, we show that the late-flowering phenotype of the anthesis promoting factor 1 (aprf1) mutants is temperature-dependent and can be suppressed when plants are grown under mild heat stress conditions. To gain further insight into the mechanism of APRF1 function, we employed a co-immunoprecipitation (Co-IP) approach to identify its interaction partners. We provide the first interactome of APRF1, which includes proteins that are localized in several subcellular compartments and are implicated in diverse cellular functions. The dual nucleocytoplasmic localization of ARRF1, which was validated through the interaction of APRF1 with HEAT SHOCK PROTEIN 1 (HSP90.1) in the nucleus and with HSP90.2 in the cytoplasm, indicates a dynamic and versatile involvement of APRF1 in multiple biological processes. The specific interaction of APRF1 with the chaperon HSP90.1 in the nucleus expands our knowledge regarding the epigenetic regulation of flowering time in A. thaliana and further suggests the existence of a delicate thermoregulated mechanism during anthesis.

BRI1 and BAK1 Canonical Distribution in Plasma Membrane Is HSP90 Dependent.

The activation of BRASSINOSTEROID INSENSITIVE1 (BRI1) and its association with the BRI1 ASSOCIATED RECEPTOR KINASE1 (BAK1) are key steps for the initiation of the BR signaling cascade mediating hypocotyl elongation. Heat shock protein 90 (HSP90) is crucial in the regulation of signaling processes and the activation of hormonal receptors. We report that HSP90 is required for the maintenance of the BRI1 receptor at the plasma membrane (PM) and its association with the BAK1 co-receptor during BL-ligand stimulation. HSP90 mediates BR perception and signal transduction through physical interactions with BRI1 and BAK1, while chaperone depletion resulted in lower levels of BRI1 and BAK1 receptors at the PM and affected the spatial partitioning and organization of BRI1/BAK1 heterocomplexes at the PM. The BRI1/BAK1 interaction relies on the HSP90-dependent activation of the kinase domain of BRI1 which leads to the confinement of the spatial dynamics of the membrane resident BRI1 and the attenuation of the downstream signaling. This is evident by the impaired activation and transcriptional activity of BRI1 EMS SUPPRESSOR 1 (BES1) upon HSP90 depletion. Our findings provide conclusive evidence that further expands the commitment of HSP90 in BR signaling through the HSP90-mediated activation of BRI1 in the control of the BR signaling cascade in plants.

GA-Mediated Disruption of RGA/BZR1 Complex Requires HSP90 to Promote Hypocotyl Elongation.

Circuitries of signaling pathways integrate distinct hormonal and environmental signals, and influence development in plants. While a crosstalk between brassinosteroid (BR) and gibberellin (GA) signaling pathways has recently been established, little is known about other components engaged in the integration of the two pathways. Here, we provide supporting evidence for the role of HSP90 (HEAT SHOCK PROTEIN 90) in regulating the interplay of the GA and BR signaling pathways to control hypocotyl elongation of etiolated seedlings in Arabidopsis. Both pharmacological and genetic depletion of HSP90 alter the expression of GA biosynthesis and catabolism genes. Major components of the GA pathway, like RGA (REPRESSOR of ga1-3) and GAI (GA-INSENSITIVE) DELLA proteins, have been identified as physically interacting with HSP90. Interestingly, GA-promoted DELLA degradation depends on the ATPase activity of HSP90, and inhibition of HSP90 function stabilizes the DELLA/BZR1 (BRASSINAZOLE-RESISTANT 1) complex, modifying the expression of downstream transcriptional targets. Our results collectively reveal that HSP90, through physical interactions with DELLA proteins and BZR1, modulates DELLA abundance and regulates the expression of BZR1-dependent transcriptional targets to promote plant growth.

HSP90 affects root growth in Arabidopsis by regulating the polar distribution of PIN1.

Auxin homeostasis and signaling affect a broad range of developmental processes in plants. The interplay between HSP90 and auxin signaling is channeled through the chaperoning capacity of the HSP90 on the TIR1 auxin receptor. The sophisticated buffering capacity of the HSP90 system through the interaction with diverse signaling protein components drastically shapes genetic circuitries regulating various developmental aspects. However, the elegant networking capacity of HSP90 in the global regulation of auxin response and homeostasis has not been appreciated. Arabidopsis hsp90 mutants were screened for gravity response. Phenotypic analysis of root meristems and cotyledon veins was performed. PIN1 localization in hsp90 mutants was determined. Our results showed that HSP90 affected the asymmetrical distribution of PIN1 in plasma membranes and influenced its expression in prompt cell niches. Depletion of HSP90 distorted polar distribution of auxin, as the acropetal auxin transport was highly affected, leading to impaired root gravitropism and lateral root formation. The essential role of the HSP90 in auxin homeostasis was profoundly evident from early development, as HSP90 depletion affected embryo development and the pattern formation of veins in cotyledons. Our data suggest that the HSP90-mediated distribution of PIN1 modulates auxin distribution and thereby auxin signaling to properly promote plant development.

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.

HSP90 chaperones regulate stomatal differentiation under normal and heat stress conditions.

Stomatal development is tightly connected with the overall plant growth, while changes in environmental conditions, like elevated temperature, affect negatively stomatal formation. Stomatal ontogenesis follows a well-defined series of cell developmental transitions in the cotyledon and leaf epidermis that finally lead to the production of mature stomata. YODA signaling cascade regulates stomatal development mainly through the phosphorylation and inactivation of SPEECHLESS (SPCH) transcription factor, while HSP90 chaperones have a central role in the regulation of YODA cascade. Here, we report that acute heat stress affects negatively stomatal differentiation, leads to high phosphorylation levels of MPK3 and MPK6, and alters the expression of SPCH and MUTE transcription factors. Genetic depletion of HSP90 leads to decreased stomatal differentiation rates. Thus, HSP90 chaperones safeguard the completion of distinct stomatal differentiation steps depending on these two transcription factors under normal and heat stress conditions.

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.

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.

Katanin Effects on Dynamics of Cortical Microtubules and Mitotic Arrays in Arabidopsis thaliana Revealed by Advanced Live-Cell Imaging.

Katanin is the only microtubule severing protein identified in plants so far. Previous studies have documented its role in regulating cortical microtubule organization during cell growth and morphogenesis. Although, some cell division defects are reported in KATANIN mutants, it is not clear whether or how katanin activity may affect microtubule dynamics in interphase cells, as well as the progression of mitosis and cytokinesis and the orientation of cell division plane (CDP). For this reason, we characterized microtubule organization and dynamics in growing and dividing cotyledon cells of Arabidopsis ktn1-2 mutant devoid of KATANIN 1 activity. In interphase epidermal cells of ktn1-2 cortical microtubules exhibited aberrant and largely isotropic organization, reduced bundling and showed excessive branched microtubule formation. End-wise microtubule dynamics were not much affected, although a significantly slower rate of microtubule growth was measured in the ktn1-2 mutant where microtubule severing was completely abolished. KATANIN 1 depletion also brought about significant changes in preprophase microtubule band (PPB) organization and dynamics. In this case, many PPBs exhibited unisided organization and splayed appearance while in most cases they were broader than those of wild type cells. By recording PPB maturation, it was observed that PPBs in the mutant narrowed at a much slower pace compared to those in Col-0. The form of the mitotic spindle and the phragmoplast was not much affected in ktn1-2, however, the dynamics of both processes showed significant differences compared to wild type. In general, both mitosis and cytokinesis were considerably delayed in the mutant. Additionally, the mitotic spindle and the phragmoplast exhibited extensive rotational motions with the equatorial plane of the spindle being essentially uncoupled from the division plane set by the PPB. However, at the onset of its formation the phragmoplast undergoes rotational motion rectifying the expansion of the cell plate to match the original cell division plane. Conclusively, KATANIN 1 contributes to microtubule dynamics during interphase, regulates PPB formation and maturation and is involved in the positioning of the mitotic spindle and the phragmoplast.

KATANIN 1 Is Essential for Embryogenesis and Seed Formation in Arabidopsis.

Cytoskeletal remodeling has a fundamental role, especially during transitional developmental stages when cells rapidly adopt new forms and roles, like gametogenesis, fertilization and concomitant embryogenesis and seed formation. KATANIN 1, a microtubule severing protein, fulfills a major regulatory mechanism of dynamic microtubule turnover in eukaryotes. Herein, we show that three well-established KATANIN 1 mutants, fra2, lue1 and ktn1-2 collectively display lower fertility and seed set in Arabidopsis. These lower fertility and seed set rates of fra2, lue1 and ktn1-2 mutants were correlated to abnormalities in the development of embryo proper and seed. Such phenotypes were rescued by transformation of mutants with functional pKTN1::GFP:KTN1 construct. This study significantly expands the already broad functional repertoire of KATANIN 1 and unravels its new role in embryo and seed development. Thus, KATANIN 1 significantly contributes to the fertility and proper embryo and seed formation in Arabidopsis.

Brassinosteroid nuclear signaling recruits HSP90 activity.

Heat shock protein 90 (HSP90) controls a number of developmental circuits, and serves a sophisticated and highly regulatory function in signaling pathways. Brassinosteroids (BRs) control many aspects of plant development. Genetic, physiological, cytological, gene expression, live cell imaging, and pharmacological approaches provide conclusive evidence for HSP90 involvement in Arabidopsis thalianaBR signaling. Nuclear-localized HSP90s translocate to cytoplasm when their activity is blocked by the HSP90 inhibitor geldanamycin (GDA). GDA treatment promoted the export of BIN2, a regulator of BR signaling, from the nucleus into the cytoplasm, indicating that active HSP90 is required to sustain BIN2 in the nucleus. HSP90 nuclear localization was inhibited by brassinolide (BL). HSP90s interact with BIN2 in the nucleus of untreated cells and in the cytoplasm of BL-treated cells, showing that the site-specific action of HSP90 on BIN2 is controlled by BRs. GDA and BL treatments change the expression of a common set of previously identified BR-responsive genes. This highlights the effect of active HSP90s on the regulation of BR-responsive genes. Our observations reveal that HSP90s have a central role in sustaining BIN2 nuclear function. We propose that BR signaling is mediated by HSP90 activity and via trafficking of BIN2-HSP90 complexes into the cytoplasm.

Complexity of Hsp90 in organelle targeting.

Heat shock protein 90 (Hsp90) is an abundant and highly conserved molecular chaperone. In Arabidopsis, the Hsp90 gene family consists of seven members. Here, we report that the AtHsp90-6 gene gives rise to two mRNA populations, termed AtHsp90-6L and AtHsp90-6S due to alternative initiation of transcription. The AtHsp90-6L and AtHsp90-6S transcription start sites are located 228 nucleotides upstream and 124 nucleotides downstream of the annotated translation start site, respectively. Both transcripts are detected under normal or heat-shock conditions. The inducibility of AtHsp90-6 mRNAs by heat shock implies a potential role of both isoforms in stress management. Stable transformation experiments with fusion constructs between the N-terminal part of each AtHsp90-6 isoform and green fluorescent protein indicated import of both fusion proteins into mitochondria. In planta investigation confirmed that fusion of the AtHsp90-5 N-terminus to green fluorescent protein (GFP) did result in specific chloroplastic localization. The mechanisms of regulation for mitochondria- and plastid-localized chaperone-encoding genes are not well understood. Future work is needed to address the possible roles of harsh environmental conditions and developmental processes on fine-tuning and compartmentalization of the AtHsp90-6L, AtHsp90-6S, and AtHsp90-5 proteins in Arabidopsis.

Hsp90 canalizes developmental perturbation.

Stochastic processes are intrinsic phenomena that perturb developmental processes. However, the canalization process restricts the magnitude of perturbation and hence the magnitude of morphological variation during development. Heat-shock protein 90 (Hsp90) chaperones are a class of proteins stabilizing a network of 'client' proteins that are involved in diverse signal transduction pathways affecting development. Here it is reported that a reduction of Hsp90 gene dose creates canalization perturbations that affect many aspects of Arabidopsis development and results in a plethora of morphological alterations. Hence, Hsp90 restricts stochastic phenomena by minimizing perturbations, thereby canalizing development. It is also shown that morphogenesis is determined by three mutually inter-related parameters: genotype, environment, and time. Hsp90 is involved in the interaction of these three parameters which ultimately affect developmental processes. The amount of phenotypic variation upon the reduction of Hsp90 function could be perceived as adaptive and could have an impact on the evolutionary process.

Tight regulation of expression of two Arabidopsis cytosolic Hsp90 genes during embryo development.

The spatial and temporal distribution of expression of two cytosolic members of the AtHsp90 gene family was assessed during early development. In stressed transgenic plants bearing the AtHsp90-3 promoter, beta-glucuronidase (GUS) activity was strong in meristematic tissues. Expression was also detected in vascular tissues, leaf veins, siliques, and in pollen sacs. The promoter induced gene expression after heat shock in a time-course dependent manner. AtHsp90-1 promoter activity was low throughout the early stages of embryo development but high just before embryo maturation, with expression most prominent in cotyledons. AtHsp90-3 promoter activity was almost constant and restricted to the root and the cotyledon tips of the embryo. This highly specific spatial distribution of GUS activity changed when the tissues were heat-stressed. Both promoters were also active in unstressed mature pollen grains and during pollen germination. The results shown here indicate that different regulatory and developmental mechanisms control and differentiate the expression of the two cytosolic members of the Arabidopsis AtHsp90 gene family under normal conditions. The developmental and restricted pattern of expression of the AtHsp90-1 and -3 gene promoters in unstressed transgenic plants suggest prominent and distinctive roles of these two genes during different developmental processes.