Aclonifen targets solanesyl diphosphate synthase, representing a novel mode of action for herbicides.

BACKGROUND: Aclonifen is a unique diphenyl ether herbicide. Despite its structural similarities to known inhibitors of the protoporphyrinogen oxidase (e.g. acifluorfen, bifenox or oxadiazon), which result in leaf necrosis, aclonifen causes a different phenotype that is described as bleaching. This also is reflected by the Herbicide Resistance Action Committee (HRAC) classification that categorizes aclonifen as an inhibitor of pigment biosynthesis with an unknown target. RESULTS: A comprehensive Arabidopsis thaliana RNAseq dataset comprising 49 different inhibitor treatments and covering 40 known target pathways was used to predict the aclonifen mode of action (MoA) by a random forest classifier. The classifier predicts for aclonifen a MoA within the carotenoid biosynthesis pathway similar to the reference compound norflurazon that inhibits the phytoene desaturase. Upon aclonifen treatment, the phytoene desaturation reaction is disturbed, resulting in a characteristic phytoene accumulation in vivo. However, direct enzyme inhibition by the herbicide was excluded for known herbicidal targets such as phytoene desaturase, 4-hydroxyphenylpyruvate dioxygenase and homogentisate solanesyltransferase. Eventually, the solanesyl diphosphate synthase (SPS), providing one of the two homogentisate solanesyltransferase substrate molecules, could be identified as the molecular target of aclonifen. Inhibition was confirmed using biochemical activity assays for the A. thaliana SPSs 1 and 2. Furthermore, a Chlamydomonas reinhardtii homolog was used for co-crystallization of the enzyme-inhibitor complex, showing that one inhibitor molecule binds at the interface between two protein monomers. CONCLUSION: Solanesyl diphosphate synthase was identified as the target of aclonifen, representing a novel mode of action for herbicides. © 2020 Society of Chemical Industry.

Abscisic Acid Receptors and Coreceptors Modulate Plant Water Use Efficiency and Water Productivity.

Improving the water use efficiency (WUE) of crop plants without trade-offs in growth and yield is considered a utopic goal. However, recent studies on model plants show that partial restriction of transpiration can occur without a reduction in CO2 uptake and photosynthesis. In this study, we analyzed the potentials and constraints of improving WUE in Arabidopsis (Arabidopsis thaliana) and in wheat (Triticum aestivum). We show that the analyzed Arabidopsis wild-type plants consume more water than is required for unrestricted growth. WUE was enhanced without a growth penalty by modulating abscisic acid (ABA) responses either by using overexpression of specific ABA receptors or deficiency of ABA coreceptors. Hence, the plants showed higher water productivity compared with the wild-type plants; that is, equal growth with less water. The high WUE trait was resilient to changes in light intensity and water availability, but it was sensitive to the ambient temperature. ABA application to plants generated a partial phenocopy of the water-productivity trait. ABA application, however, was never as effective as genetic modification in enhancing water productivity, probably because ABA indiscriminately targets all ABA receptors. ABA agonists selective for individual ABA receptors might offer an approach to phenocopy the water-productivity trait of the high WUE lines. ABA application to wheat grown under near-field conditions improved WUE without detectable growth trade-offs. Wheat yields are heavily impacted by water deficit, and our identification of this crop as a promising target for WUE improvement may help contribute to greater food security.

Regulation of the heat stress response in Arabidopsis by MPK6-targeted phosphorylation of the heat stress factor HsfA2.

So far little is known on the functional role of phosphorylation in the heat stress response of plants. Here we present evidence that heat stress activates the Arabidopsis mitogen-activated protein kinase MPK6. In vitro and in vivo evidence is provided that MPK6 specifically targets the major heat stress transcription factor HsfA2. Activation of MPK6 results in complex formation with HsfA2. MPK6 phosphorylates HsfA2 on T249 and changes its intracellular localisation. Protein kinase and phosphatase inhibitor studies indicate that HsfA2 protein stability is regulated in a phosphorylation-dependent manner, but this mechanism is independent of MPK6. Overall, our data show that heat stress-induced targeting of HsfA2 by MPK6 participates in the complex regulatory mechanism how plants respond to heat stress.

The plant sHSP superfamily: five new members in Arabidopsis thaliana with unexpected properties.

The small heat shock proteins (sHsps), which are ubiquitous stress proteins proposed to act as chaperones, are encoded by an unusually complex gene family in plants. Plant sHsps are classified into different subfamilies according to amino acid sequence similarity and localization to distinct subcellular compartments. In the whole Arabidopsis thaliana genome, 19 genes were annotated to encode sHsps, of which 14 belong to previously defined plant sHsp families. In this paper, we report studies of the five additional sHsp genes in A. thaliana, which can now be shown to represent evolutionarily distinct sHsp subfamilies also found in other plant species. While two of these five sHsps show expression patterns typical of the other 14 genes, three have unusual tissue specific and developmental profiles and do not respond to heat induction. Analysis of intracellular targeting indicates that one sHsp represents a new class of mitochondrion-targeted sHsps, while the others are cytosolic/nuclear, some of which may cooperate with other sHsps in formation of heat stress granules. Three of the five new proteins were purified and tested for chaperone activity in vitro. Altogether, these studies complete our basic understanding of the sHsp chaperone family in plants.

A cascade of transcription factor DREB2A and heat stress transcription factor HsfA3 regulates the heat stress response of Arabidopsis.

The dehydration-responsive element binding protein (DREB)/C-repeat binding factor (CBF) family are the classical transcriptional regulators involved in plant responses to drought, salt and cold stress. Recently it was demonstrated that DREB2A is induced by heat stress (hs) and is a regulator of the hs response of Arabidopsis. Here we provide molecular insights into the regulation and function of hs transcription factor HsfA3. Among the 21 members of the Arabidopsis Hsf family, HsfA3 is the only Hsf that is transcriptionally induced during hs by DREB2A, and HsfA3 in turn regulates the expression of Hsp-encoding genes. This transcription factor cascade was reconstructed in transient GUS reporter assays in mesophyll protoplasts by showing that DREB2A could activate the HsfA3 promoter, whereas HsfA3 in turn was shown to be a potent activator on the promoters of Hsp genes. Direct binding to the corresponding promoters was demonstrated by electrophoretic mobility shift assays, and the involvement of HsfA3 in the hs response in vivo was shown directly by observation of reduced thermotolerance in HsfA3 mutant lines. Altogether these data demonstrate that HsfA3 is transcriptionally controlled by DREB2A and important for the establishment of thermotolerance.

The diversity of plant heat stress transcription factors.

Compared with other eukaryotes with one to three heat stress transcription factors (Hsf), the plant Hsf family shows a striking multiplicity, with more than 20 members. Despite many conserved features, members of the Hsf family show a strong diversification of expression pattern and function within the family. Research on Arabidopsis Hsfs opened a new era with genome-wide transcriptome profiling in combination with the availability of knockout lines. The output from these analyses provides increasing evidence that individual Hsfs have unique functions as part of different signal transduction pathways operating in response to environmental stress and during development.

Complexity of the heat stress response in plants.

Plants have evolved a variety of responses to elevated temperatures that minimize damage and ensure protection of cellular homeostasis. New information about the structure and function of heat stress proteins and molecular chaperones has become available. At the same time, transcriptome analysis of Arabidopsis has revealed the involvement of factors other than classical heat stress responsive genes in thermotolerance. Recent reports suggest that both plant hormones and reactive oxygen species also contribute to heat stress signaling. Additionally, an increasing number of mutants that have altered thermotolerance have extended our understanding of the complexity of the heat stress response in plants.

A novel transcriptional cascade regulating expression of heat stress proteins during seed development of Arabidopsis.

Within the Arabidopsis thaliana family of 21 heat stress transcription factors (Hsfs), HsfA9 is exclusively expressed in late stages of seed development. Here, we present evidence that developmental expression of HsfA9 is regulated by the seed-specific transcription factor ABSCISIC ACID-INSENSITIVE3 (ABI3). Intriguingly, ABI3 knockout lines lack detectable levels of HsfA9 transcript and protein, and further ectopic expression of ABI3 conferred the ability to accumulate HsfA9 in response to abscisic acid in transgenic plantlets. Consequently, the most abundant heat stress proteins (Hsps) in seeds (Hsp17.4-CI, Hsp17.7-CII, and Hsp101) were not detectable in the ABI3 knockout lines, but their expression could be detected in plants ectopically expressing HsfA9 in vegetative tissues. Furthermore, this seed-specific transcription factor cascade was reconstructed in transient beta-glucuronidase reporter assays in mesophyll protoplasts by showing that ABI3 could activate the HsfA9 promoter, whereas HsfA9 in turn was shown to be a potent activator on the promoters of Hsp genes. Thus, our study establishes a genetic framework in which HsfA9 operates as a specialized Hsf for the developmental expression of Hsp genes during seed maturation.

Arabidopsis immunophilins ROF1 (AtFKBP62) and ROF2 (AtFKBP65) exhibit tissue specificity, are heat-stress induced, and bind HSP90.

The plant co-chaperones FK506-binding proteins (FKBPs) are peptidyl prolyl cis-trans isomerases that function in protein folding, signal transduction and chaperone activity. We report the characterization of the Arabidopsis large FKBPs ROF1 (AtFKBP62) and ROF2 (AtFKBP65) expression and protein accumulation patterns. Transgenic plants expressing ROF1 promoter fused to GUS reporter gene reveal that ROF1 expression is organ specific. High expression was observed in the vascular elements of roots, in hydathodes and trichomes of leaves and in stigma, sepals, and anthers. The tissue specificity and temporal expression of ROF1 and ROF2 show that they are developmentally regulated. Although ROF1 and ROF2 share 85% identity, their expression in response to heat stress is differentially regulated. Both genes are induced in plants exposed to 37 degrees C, but only ROF2 is a bonafide heat-stress protein, undetected when plants are grown at 22 degrees C. ROF1/ROF2 proteins accumulate at 37 degrees C, remain stable for at least 4 h upon recovery at 22 degrees C, whereas, their mRNA level is reduced after 1 h at 22 degrees C. By protein interaction assays, it was demonstrated, that ROF1 is a novel partner of HSP90. The five amino acids identified as essential for recognition and interaction between the mammalian chaperones and HSP90 are conserved in the plant ROF1-HSP90. We suggest that ROF/HSP90 complexes assemble in vivo. We propose that specific complexes formation between an HSP90 and ROF isoforms depends on their spatial and temporal expression. Such complexes might be regulated by environmental conditions such as heat stress or internal cues such as different hormones.

The heat stress transcription factor HsfA2 serves as a regulatory amplifier of a subset of genes in the heat stress response in Arabidopsis.

Within the Arabidopsis family of 21 heat stress transcription factors (Hsfs) HsfA2 is the strongest expressed member under heat stress (hs) conditions. Irrespective of the tissue, HsfA2 accumulates under heat stress similarly to other heat stress proteins (Hsps). A SALK T-DNA insertion line with a complete HsfA2-knockout was analyzed with respect to the changes in the transcriptome under heat stress conditions. Ascorbate peroxidase 2 (APX2) was identified as the most affected transcript in addition to several sHsps, individual members of the Hsp70 and Hsp100 family, as well as many transcripts of genes with yet unknown functions. For functional validation, the transcription activation potential of HsfA2 on GUS reporter constructs containing 1 kb upstream promoter sequences of selected target genes were analyzed using transient reporter assays in mesophyll protoplasts. By deletion analysis the promoter region of the strongest affected target gene APX2 was functionally mapped in detail to verify potential HsfA2 binding sites. By electrophoretic mobility shift assays we identified TATA-Box proximal clusters of heat stress elements (HSE) in the promoters of selected target genes as potential HsfA2 binding sites. The results presented here demonstrate that the expression of HsfA2 in Arabidopsis is strictly heat stress-dependent and this transcription factor represents a regulator of a subset of stress response genes in Arabidopsis.

Heat stress response in plants: a complex game with chaperones and more than twenty heat stress transcription factors.

Compared to the overall multiplicity of more than 20 plant Hsfs, detailed analyses are mainly restricted to tomato and Arabidopsis and to three important representatives of the family (Hsfs A1, A2 and B1). The three Hsfs represent examples of striking functional diversification specialized for the three phases of the heat stress (hs) response (triggering, maintenance and recovery). This is best illustrated for the tomato Hsf system: (i) HsfA1a is the master regulator responsible for hs-induced gene expression including synthesis of HsfA2 and HsfB1. It is indispensible for the development of thermotolerance. (ii) Although functionally equivalent to HsfA1a, HsfA2 is exclusively found after hs induction and represents the dominant Hsf, the "working horse" of the hs response in plants subjected to repeated cycles of hs and recovery in a hot summer period. Tomato HsfA2 is tightly integrated into a network of interacting proteins (HsfA1a, Hsp17-CII, Hsp17-CI) influencing its activity and intracellular distribution. (iii) Because of structural peculiarities, HsfB1 acts as coregulator enhancing the activity of HsfA1a and/or HsfA2. But in addition, it cooperates with yet to be identified other transcription factors in maintaining and/or restoring housekeeping gene expression.

Characterization of C-terminal domains of Arabidopsis heat stress transcription factors (Hsfs) and identification of a new signature combination of plant class A Hsfs with AHA and NES motifs essential for activator function and intracellular localization.

Heat stress transcription factors (Hsfs) are the major regulators of the plant heat stress (hs) response. Sequencing of the Arabidopsis genome revealed the existence of 21 open-reading frames (ORFs) encoding putative Hsfs assigned to classes A-C. Here we present results of a functional genomics approach to the Arabidopsis Hsf family focused on the analysis of their C-terminal domains (CTDs) harboring conserved modules for their function as transcription factors and their intracellular localization. Using reporter assays in tobacco protoplasts and yeast as well as glutathione-S-transferase (GST) pull-down assays, we demonstrate that short peptide motifs enriched with aromatic and large hydrophobic amino acid (aa) residues embedded in an acidic surrounding (AHA motifs) are essential for transcriptional activity of class A Hsfs. In contrast to this, class B and C Hsfs lack AHA motifs and have no activator function on their own. We also provide evidence for the function of a leucine (Leu)-rich region centered around a conserved QMGPhiL motif at the very C-terminus as a nuclear export signal (NES) of class A Hsfs. Sequence comparison indicates that the combination of a C-terminal AHA motif with the consensus sequence FWxxF/L,F/I/L as well as the adjacent NES represents a signature domain for plant class A Hsfs, which allowed to identify more than 60 new Hsfs from the expressed sequence tag (EST) database.

Tomato heat stress transcription factor HsfB1 represents a novel type of general transcription coactivator with a histone-like motif interacting with the plant CREB binding protein ortholog HAC1.

In contrast with the class A heat stress transcription factors (HSFs) of plants, a considerable number of HSFs assigned to classes B and C have no evident function as transcription activators on their own. However, in the following article, we provide evidence that tomato (Lycopersicon peruvianum) HsfB1 represents a novel type of coactivator cooperating with class A HSFs (e.g., with tomato HsfA1). Provided the appropriate promoter architecture, the two HSFs assemble into an enhanceosome-like complex, resulting in strong synergistic activation of reporter gene expression. Moreover, HsfB1 also cooperates in a similar manner with other activators, for example, with the ASF1/2 enhancer binding proteins of the 35S promoter of Cauliflower mosaic virus or with yet unidentified activators controlling housekeeping gene expression. By these effects, HsfB1 may help to maintain and/or restore expression of certain viral or housekeeping genes during ongoing heat stress. The coactivator function of HsfB1 depends on a histone-like motif in its C-terminal domain with an indispensable Lys residue in the center (GRGKMMK). This motif is required for recruitment of the plant CREB binding protein (CBP) ortholog HAC1. HsfA1, HsfB1, and HAC1/CBP form ternary complexes in vitro and in vivo with markedly enhanced efficiency in promoter recognition and transcription activation in plant and mammalian (COS7) cells. Using small interfering RNA-mediated knock down of HAC1 expression in Arabidopsis thaliana mesophyll protoplasts, the crucial role for the coactivator function of HsfB1 was confirmed.