HOP stabilizes the HSFA1a and plays a main role in the onset of thermomorphogenesis.

Living organisms have the capacity to respond to environmental stimuli, including warm conditions. Upon sensing mild temperature, plants launch a transcriptional response that promotes morphological changes, globally known as thermomorphogenesis. This response is orchestrated by different hormonal networks and by the activity of different transcription factors, including the heat shock factor A1 (HSFA1) family. Members of this family interact with heat shock protein 70 (HSP70) and heat shock protein 90 (HSP90); however, the effect of this binding on the regulation of HSFA1 activity or of the role of cochaperones, such as the HSP70-HSP90 organizing protein (HOP) on HSFA1 regulation, remains unknown. Here, we show that AtHOPs are involved in the folding and stabilization of the HSFA1a and are required for the onset of the transcriptional response associated to thermomorphogenesis. Our results demonstrate that the three members of the AtHOP family bind in vivo to the HSFA1a and that the expression of multiple HSFA1a-responsive-responsive genes is altered in the hop1 hop2 hop3 mutant under warm temperature. Interestingly, HSFA1a is accumulated at lower levels in the hop1 hop2 hop3 mutant, while control levels are recovered in the presence of the proteasome inhibitor MG132 or the synthetic chaperone tauroursodeoxycholic acid (TUDCA). This uncovers the HSFA1a as a client of HOP complexes in plants and reveals the participation of HOPs in HSFA1a stability.

HOPping up with the homeostasis engine in plant development and stress.

Proteins need to acquire their native structure in order to become fully functional. In specific cases, active conformation is obtained spontaneously; nevertheless, many proteins need the assistance of chaperones and co-chaperones to be properly folded. These proteins help to maintain protein homeostasis under control conditions and under different stresses. HOP (HSP70-HSP90 Organizing Protein) is a highly conserved family of co-chaperones that assist HSP70 and HSP90 in the folding of specific proteins. In the last few years, exciting findings in mammals and yeast have revealed novel functions of HOP and re-defined the role of HOP in protein folding. In this article, we give an overview of the most important aspects of HOP regulation and function in other eukaryotes and analyze if these aspects are conserved in plants. In addition, we put special emphasis on the HOP clients described in plants and on the role of HOP in plant development and stress response.

HOP co-chaperones contribute to GA signaling by promoting the accumulation of the F-box protein SNE in Arabidopsis.

Gibberellins (GAs) play important roles in multiple developmental processes and in plant response to the environment. Within the GA pathway, a central regulatory step relies on GA-dependent degradation of the DELLA transcriptional regulators. Nevertheless, the relevance of the stability of other key proteins in this pathway, such as SLY1 and SNE (the F-box proteins involved in DELLA degradation), remains unknown. Here, we take advantage of mutants in the HSP70-HSP90 organizing protein (HOP) co-chaperones and reveal that these proteins contribute to the accumulation of SNE in Arabidopsis. Indeed, HOP proteins, along with HSP90 and HSP70, interact in vivo with SNE, and SNE accumulation is significantly reduced in the hop mutants. Concomitantly, greater accumulation of the DELLA protein RGA is observed in these plants. In agreement with these molecular phenotypes, hop mutants show a hypersensitive response to the GA inhibitor paclobutrazol and display a partial response to the ectopic addition of GA when GA-regulated processes are assayed. These mutants also display different phenotypes associated with alterations in the GA pathway, such as reduced germination rate, delayed bolting, and reduced hypocotyl elongation in response to warm temperatures. Remarkably, ectopic overexpression of SNE reverts the delay in germination and the thermally dependent hypocotyl elongation defect of the hop1 hop2 hop3 mutant, revealing that SNE accumulation is the key aspect of the hop mutant phenotypes. Together, these data reveal a pivotal role for HOP in SNE accumulation and GA signaling.

The co-chaperone HOP participates in TIR1 stabilisation and in auxin response in plants.

HOP (HSP70-HSP90 organising protein) is a conserved family of co-chaperones well known in mammals for its role in the folding of signalling proteins associated with development. In plants, HOP proteins have been involved in the response to multiple stresses, but their role in plant development remains elusive. Herein, we describe that the members of the HOP family participate in different aspects of plant development as well as in the response to warm temperatures through the regulation of auxin signalling. Arabidopsis hop1 hop2 hop3 triple mutant shows different auxin-related phenotypes and a reduced auxin sensitivity. HOP interacts with TIR1 auxin coreceptor in vivo. Furthermore, TIR1 accumulation and auxin transcriptional response are reduced in the hop1 hop2 hop3 triple mutant, suggesting that HOP's function in auxin signalling is related, at least, to TIR1 interaction and stabilisation. Interestingly, HOP proteins form part of the same complexes as SGT1b (a different HSP90 co-chaperone) and these co-chaperones synergistically cooperate in auxin signalling. This study provides relevant data about the role of HOP in auxin regulation in plants and uncovers that both co-chaperones, SGT1b and HOP, cooperate in the stabilisation of common targets involved in plant development.

The co-chaperone HOP3 participates in jasmonic acid signaling by regulating CORONATINE-INSENSITIVE 1 activity.

HOPs (HSP70-HSP90 organizing proteins) are a highly conserved family of HSP70 and HSP90 co-chaperones whose role in assisting the folding of various hormonal receptors has been extensively studied in mammals. In plants, HOPs are mainly associated with stress response, but their potential involvement in hormonal networks remains completely unexplored. In this article we describe that a member of the HOP family, HOP3, is involved in the jasmonic acid (JA) pathway and is linked to plant defense responses not only to pathogens, but also to a generalist herbivore. The JA pathway regulates responses to Botrytis cinerea infection and to Tetranychus urticae feeding; our data demonstrate that the Arabidopsis (Arabidopsis thaliana) hop3-1 mutant shows an increased susceptibility to both. The hop3-1 mutant exhibits reduced sensitivity to JA derivatives in root growth assays and downregulation of different JA-responsive genes in response to methyl jasmonate, further revealing the relevance of HOP3 in the JA pathway. Interestingly, yeast two-hybrid assays and in planta co-immunoprecipitation assays found that HOP3 interacts with COI1, suggesting that COI1 is a target of HOP3. Consistent with this observation, COI1 activity is reduced in the hop3-1 mutant. All these data strongly suggest that, specifically among HOPs, HOP3 plays a relevant role in the JA pathway by regulating COI1 activity in response to JA and, consequently, participating in defense signaling to biotic stresses.

Peculiarities of the regulation of translation initiation in plants.

Protein synthesis is a fundamental process for life and, as such, plays a crucial role in the adaptation to energy, developmentaland environmental conditions. For these reasons, and despite the general conservation of the eukaryotic translational machinery, it is not surprising that organisms with different lifestyles have evolved distinct mechanisms of regulation to adapt translation initiation to their intrinsic growth and development. Plants have clear peculiarities compared with other eukaryotes that have also extended to translation control. This review describes the plant-specific mechanisms for regulation of translation initiation, with a focus on those that modulate the eIF4F complexes, central translational regulatory hubs in all eukaryotes, and highlights the latest discoveries on the signaling pathways that regulate their constituents and activity.

High overexpression of CERES, a plant regulator of translation, induces different phenotypical defence responses during TuMV infection.

Mutations in the eukaryotic translation initiation factors eIF4E and eIF(iso)4E confer potyvirus resistance in a range of plant hosts. This supports the notion that, in addition to their role in translation of cellular mRNAs, eIF4E isoforms are also essential for the potyvirus cycle. CERES is a plant eIF4E- and eIF(iso)4E-binding protein that, through its binding to the eIF4Es, modulates translation initiation; however, its possible role in potyvirus resistance is unknown. In this article, we analyse if the ectopic expression of AtCERES is able to interfere with turnip mosaic virus replication in plants. Our results demonstrate that, during infection, the ectopic expression of CERES in Nicotiana benthamiana promotes the development of a mosaic phenotype when it is accumulated to moderate levels, but induces veinal necrosis when it is accumulated to higher levels. This necrotic process resembles a hypersensitive response (HR)-like response that occurs with different HR hallmarks. Remarkably, Arabidopsis plants inoculated with a virus clone that promotes high expression of CERES do not show signs of infection. These final phenotypical outcomes are independent of the capacity of CERES to bind to eIF4E. All these data suggest that CERES, most likely due to its leucine-rich repeat nature, could act as a resistance protein, able to promote a range of different defence responses when it is highly overexpressed from viral constructs.

HOP, a Co-chaperone Involved in Response to Stress in Plants.

Protein folding is an essential step for protein functionality. In eukaryotes this process is carried out by multiple chaperones that act in a cooperative manner to maintain the proteome homeostasis. Some of these chaperones are assisted during protein folding by different co-chaperones. One of these co-chaperones is HOP, the HSP70-HSP90 organizing protein. This assistant protein, due to its importance, has been deeply analyzed in other eukaryotes, but its function has only recently started to be envisaged in plants. In this kingdom, the role of HOP has been associated to plant response to different cellular, biotic and abiotic stresses. In this article, we analyze the current knowledge about HOP in eukaryotes, paying a special attention to the recently described roles of HOP in plants. In addition, we discuss the recent breakthroughs in the field and the possible new avenues for the study of plant HOP proteins in the future.

eIF2α Phosphorylation by GCN2 Is Induced in the Presence of Chitin and Plays an Important Role in Plant Defense against B. cinerea Infection.

Translation plays an important role in plant adaptation to different abiotic and biotic stresses; however, the mechanisms involved in translational regulation during each specific response and their effect in translation are poorly understood in plants. In this work, we show that GCN2 promotes eIF2α phosphorylation upon contact with Botrytis cinerea spores, and that this phosphorylation is required for the proper establishment of plant defense against the fungus. In fact, independent gcn2 mutants display an enhanced susceptibility to B. cinerea infection, which is highlighted by an increased cell death and reduced expression of ethylene- and jasmonic-related genes in the gcn2 mutants. eIF2α phosphorylation is not only triggered in the presence of the fungus, but interestingly, is also achieved in the sole presence of the microbe-associated molecular pattern (MAMP) chitin. Moreover, analysis of de novo protein synthesis by 35SMet-35SCys incorporation indicates that chitin treatment promotes a global inhibition of translation. Taken together, these results suggest that eIF2α phosphorylation by GCN2 is promoted in the presence of chitin and plays an important role in plant defense against B. cinerea infection.

A novel eIF4E-interacting protein that forms non-canonical translation initiation complexes.

Translation is a fundamental step in gene expression that regulates multiple developmental and stress responses. One key step of translation initiation is the association between eIF4E and eIF4G. This process is regulated in different eukaryotes by proteins that bind to eIF4E; however, evidence of eIF4E-interacting proteins able to regulate translation is missing in plants. Here, we report the discovery of CERES, a plant eIF4E-interacting protein. CERES contains an LRR domain and a canonical eIF4E-binding site. Although the CERES-eIF4E complex does not include eIF4G, CERES forms part of cap-binding complexes, interacts with eIF4A, PABP and eIF3, and co-sediments with translation initiation complexes in vivo. Moreover, CERES promotes translation in vitro and general translation in vivo, while it modulates the translation of specific mRNAs related to light and carbohydrate response. These data suggest that CERES is a non-canonical translation initiation factor that modulates translation in plants.

The polyadenylation factor FIP1 is important for plant development and root responses to abiotic stresses.

Root development and its response to environmental changes is crucial for whole plant adaptation. These responses include changes in transcript levels. Here, we show that the alternative polyadenylation (APA) of mRNA is important for root development and responses. Mutations in FIP1, a component of polyadenylation machinery, affects plant development, cell division and elongation, and response to different abiotic stresses. Salt treatment increases the amount of poly(A) site usage within the coding region and 5' untranslated regions (5'-UTRs), and the lack of FIP1 activity reduces the poly(A) site usage within these non-canonical sites. Gene ontology analyses of transcripts displaying APA in response to salt show an enrichment in ABA signaling, and in the response to stresses such as salt or cadmium (Cd), among others. Root growth assays show that fip1-2 is more tolerant to salt but is hypersensitive to ABA or Cd. Our data indicate that FIP1-mediated alternative polyadenylation is important for plant development and stress responses.

Coimmunoprecipitation of Interacting Proteins in Plants.

Protein-protein interactions discovered by yeast two-hybrid systems must be confirmed in vivo in a homologous system. In the case of plants, one of the easiest and fastest methods to validate protein interactions in vivo is the transient expression of the proteins in Nicotiana benthamiana leaves followed by coimmunoprecipitation. This method consists of the following steps: growth of the appropriate Agrobacterium tumefaciens cultures, preparation of the infiltration mixtures, infiltration into N. benthamiana leaves, protein extraction and immunoprecipitation. The utilization of epitope tags to immunoprecipitate and detect the proteins of interest is very useful in this procedure. In this chapter we describe a standard protocol to coimmunoprecipitate proteins expressed in N. benthamiana leaves.

HOP family plays a major role in long-term acquired thermotolerance in Arabidopsis.

HSP70-HSP90 organizing protein (HOP) is a family of cytosolic cochaperones whose molecular role in thermotolerance is quite unknown in eukaryotes and unexplored in plants. In this article, we describe that the three members of the AtHOP family display a different induction pattern under heat, being HOP3 highly regulated during the challenge and the attenuation period. Despite HOP3 is the most heat-regulated member, the analysis of the hop1 hop2 hop3 triple mutant demonstrates that the three HOP proteins act redundantly to promote long-term acquired thermotolerance in Arabidopsis. HOPs interact strongly with HSP90 and part of the bulk of HOPs shuttles from the cytoplasm to the nuclei and to cytoplasmic foci during the challenge. RNAseq analyses demonstrate that, although the expression of the Hsf targets is not generally affected, the transcriptional response to heat is drastically altered during the acclimation period in the hop1 hop2 hop3 triple mutant. This mutant also displays an unusual high accumulation of insoluble and ubiquitinated proteins under heat, which highlights the additional role of HOP in protein quality control. These data reveal that HOP family is involved in different aspects of the response to heat, affecting the plant capacity to acclimate to high temperatures for long periods.

Regulation of Translation by TOR, eIF4E and eIF2α in Plants: Current Knowledge, Challenges and Future Perspectives.

An important step in eukaryotic gene expression is the synthesis of proteins from mRNA, a process classically divided into three stages, initiation, elongation, and termination. Translation is a precisely regulated and conserved process in eukaryotes. The presence of plant-specific translation initiation factors and the lack of well-known translational regulatory pathways in this kingdom nonetheless indicate how a globally conserved process can diversify among organisms. The control of protein translation is a central aspect of plant development and adaptation to environmental stress, but the mechanisms are still poorly understood. Here we discuss current knowledge of the principal mechanisms that regulate translation initiation in plants, with special attention to the singularities of this eukaryotic kingdom. In addition, we highlight the major recent breakthroughs in the field and the main challenges to address in the coming years.

HOP3 a new regulator of the ER stress response in Arabidopsis with possible implications in plant development and response to biotic and abiotic stresses.

HOPs (heat shock protein 70 (HSP70)-heat shock protein 90 (HSP90) organizing proteins) are a highly conserved family of cytosolic cochaperones. In a recent study we showed that HOP3, a member of the HOP family in Arabidopsis, plays an essential role during endoplasmic reticulum (ER) stress in plants. Interestingly, we also demonstrated that AtHOP3 interacts with binding immunoglobulin protein (BiP), a major ER-resident chaperone. All these data suggest that HOP3 could assist BiP in protein folding in the ER. These findings open the exciting possibility that HOP3, through its role in the alleviation of ER stress, could play an important function during different developmental processes and in response to different biotic and abiotic stresses.

HOP3, a member of the HOP family in Arabidopsis, interacts with BiP and plays a major role in the ER stress response.

HSP70-HSP90 organizing protein (HOP) is a well-studied family of cytosolic cochaperones. However, the possible role of HOP during the endoplasmic reticulum (ER) stress response and the identity of its interactors within the ER were not previously addressed in any eukaryote. We have demonstrated that Arabidopsis HOP3, whose function was not studied before, interacts in vivo with cytosolic HSP90 and HSP70, and, unexpectedly, with binding immunoglobulin protein (BiP), a HSP70 ER-resident protein. Although BiP lacks the domain described in other eukaryotes for HOP-HSP70 binding, it interacts with HOP3 through a non-canonical association to its nucleotide binding domain. Consistent with this interaction with BiP, HOP3 is partially localized at the ER. Moreover, HOP3 is induced both at transcript and protein levels by unfolded protein response (UPR) inducer agents by a mechanism dependent on inositol-requiring enzyme 1 (IRE1). Importantly, hop3 loss-of-function mutants show a reduction in pollen germination and a hypersensitive phenotype in the presence of ER stress inducer agents, a phenotype that is reverted by the addition of the chemical chaperone tauroursodeoxycholic acid (TUDCA). All these data demonstrate, for the first time in any eukaryote, a main role of HOP as an important regulator of the ER stress response, a process intimately linked in plants to important specific developmental programs and to environmental stress sensing and response.

Dissecting the proteome dynamics of the early heat stress response leading to plant survival or death in Arabidopsis.

In many plant species, an exposure to a sublethal temperature triggers an adaptative response called acclimation. This response involves an extensive molecular reprogramming that allows the plant to further survive to an otherwise lethal increase of temperature. A related response is also launched under an abrupt and lethal heat stress that, in this case, is unable to successfully promote thermotolerance and therefore ends up in plant death. Although these molecular programmes are expected to have common players, the overlapping degree and the specific regulators of each process are currently unknown. We have carried out a high-throughput comparative proteomics analysis during acclimation and during the early stages of the plant response to a severe heat stress that lead Arabidopsis seedlings either to survival or death. This analysis dissects these responses, unravels the common players and identifies the specific proteins associated with these different fates. Thermotolerance assays of mutants in genes with an uncharacterized role in heat stress demonstrate the relevance of this study to uncover both positive and negative heat regulators and pinpoint a pivotal role of JR1 and BAG6 in heat tolerance.

Analysis of genome-wide changes in the translatome of Arabidopsis seedlings subjected to heat stress.

Heat stress is one of the most prominent and deleterious environmental threats affecting plant growth and development. Upon high temperatures, plants launch specialized gene expression programs that promote stress protection and survival. These programs involve global and specific changes at the transcriptional and translational levels. However, the coordination of these processes and their specific role in the establishment of the heat stress response is not fully elucidated. We have carried out a genome-wide analysis to monitor the changes in the translation efficiency of individual mRNAs of Arabidopsis thaliana seedlings after the exposure to a heat shock stress. Our results demonstrate that translation exerts a wide but dual regulation of gene expression. For the majority of mRNAs, translation is severely repressed, causing a decreased of 50% in the association of the bulk of mRNAs to polysomes. However, some relevant mRNAs involved in different aspects of homeostasis maintenance follow a differential pattern of translation. Sequence analyses of the differentially translated mRNAs unravels that some features, such as the 5'UTR G+C content and the cDNA length, may take part in the discrimination mechanisms for mRNA polysome loading. Among the differentially translated genes, master regulators of the stress response stand out, highlighting the main role of translation in the early establishment of the physiological response of plants to elevated temperatures.