Arabidopsis HECT and RING-type E3 Ligases Promote MAPKKK18 Degradation to Regulate Abscisic Acid Signaling.

Mitogen-activated protein kinase (MAPK) cascades are conserved signaling pathways that transduce extracellular signals into diverse cellular responses. Arabidopsis MAPKKK18 is a component of the MAPKKK17/18-MKK3-MPK1/2/7/14 cascades, which play critical roles in abscisic acid (ABA) signaling, drought tolerance and senescence. A very important aspect of MAP kinase signaling is both its activation and its termination, which must be tightly controlled to achieve appropriate biological responses. Recently, the ubiquitin-proteasome system (UPS) has received increasing attention as a key mechanism for maintaining the homeostasis of MAPK cascade components and other ABA signaling effectors. Previous studies have shown that the stability of MAPKKK18 is regulated by the UPS via the ABA core pathway. Here, using multiple proteomic approaches, we found that MAPKKK17/18 turnover is tightly controlled by three E3 ligases, UPL1, UPL4 and KEG. We also identified lysines 154 and 237 as critical for MAPKKK18 stability. Taken together, this study sheds new light on the mechanism that controls MAPKKK17/18 activity and function.

The Bro1-like domain-containing protein, AtBro1, modulates growth and abiotic stress responses in Arabidopsis.

Abscisic acid (ABA) affects plant physiology by altering gene expression, enabling plants to adapt to a wide range of environments. Plants have evolved protective mechanisms to allow seed germination in harsh conditions. Here, we explore a subset of these mechanisms involving the AtBro1 gene, which encodes one of a small family of poorly characterised Bro1-like domain-containing proteins, in Arabidopsis thaliana plants subjected to multiple abiotic stresses. AtBro1 transcripts were upregulated by salt, ABA and mannitol stress, while AtBro1-overexpression lines demonstrated robust tolerance to drought and salt stress. Furthermore, we found that ABA elicits stress-resistance responses in loss-of-function bro1-1 mutant plants and AtBro1 regulates drought resistance in Arabidopsis. When the AtBro1 promoter was fused to the ß-glucuronidase (GUS) gene and introduced into plants, GUS was expressed mainly in rosette leaves and floral clusters, especially in anthers. Using a construct expressing an AtBro1-GFP fusion protein, AtBro1 was found to be localized in the plasma membrane in Arabidopsis protoplasts. A broad RNA-sequencing analysis revealed specific quantitative differences in the early transcriptional responses to ABA treatment between wild-type and loss-of-function bro1-1 mutant plants, suggesting that ABA stimulates stress-resistance responses via AtBro1. Additionally, transcripts levels of MOP9.5, MRD1, HEI10, and MIOX4 were altered in bro1-1 plants exposed to different stress conditions. Collectively, our results show that AtBro1 plays a significant role in the regulation of the plant transcriptional response to ABA and the induction of resistance responses to abiotic stress.

The effects of nature-inspired amino acid substitutions on structural and biochemical properties of the E. coli L-asparaginase EcAIII.

The Escherichia coli enzyme EcAIII catalyzes the hydrolysis of L-Asn to L-Asp and ammonia. Using a nature-inspired mutagenesis approach, we designed and produced five new EcAIII variants (M200I, M200L, M200K, M200T, M200W). The modified proteins were characterized by spectroscopic and crystallographic methods. All new variants were enzymatically active, confirming that the applied mutagenesis procedure has been successful. The determined crystal structures revealed new conformational states of the EcAIII molecule carrying the M200W mutation and allowed a high-resolution observation of an acyl-enzyme intermediate with the M200L mutant. In addition, we performed structure prediction, substrate docking, and molecular dynamics simulations for 25 selected bacterial orthologs of EcAIII, to gain insights into how mutations at the M200 residue affect the active site and substrate binding mode. This comprehensive strategy, including both experimental and computational methods, can be used to guide further enzyme engineering and can be applied to the study of other proteins of medicinal or biotechnological importance.

Identification of Novel miRNAs and Their Target Genes in the Response to Abscisic Acid in Arabidopsis.

miRNAs are involved in various biological processes, including adaptive responses to abiotic stress. To understand the role of miRNAs in the response to ABA, ABA-responsive miRNAs were identified by small RNA sequencing in wild-type Arabidopsis, as well as in abi1td, mkkk17, and mkkk18 mutants. We identified 10 novel miRNAs in WT after ABA treatment, while in abi1td, mkkk17, and mkkk18 mutants, three, seven, and nine known miRNAs, respectively, were differentially expressed after ABA treatment. One novel miRNA (miRn-8) was differentially expressed in the mkkk17 mutant. Potential target genes of the miRNA panel were identified using psRNATarget. Sequencing results were validated by quantitative RT-PCR of several known and novel miRNAs in all genotypes. Of the predicted targets of novel miRNAs, seven target genes of six novel miRNAs were further validated by 5' RLM-RACE. Gene ontology analyses showed the potential target genes of ABA-responsive known and novel miRNAs to be involved in diverse cellular processes in plants, including development and stomatal movement. These outcomes suggest that a number of the identified miRNAs have crucial roles in plant responses to environmental stress, as well as in plant development, and might have common regulatory roles in the core ABA signaling pathway.

Identification of Novel Inhibitors of a Plant Group A Protein Phosphatase Type 2C Using a Combined In Silico and Biochemical Approach.

Type 2C protein phosphatases (PP2Cs) of group A play a significant role in the regulation of various processes in plants including growth, development, ion transport, and stress acclimation. In this study, we selected potential PP2C group A inhibitors using a structure-based virtual screening method followed by biochemical and in vitro validation. Over twenty million chemical compounds from the ZINC database were used for docking studies. The precision of the calculations was increased by an induced-fit docking protocol and the molecular mechanics/generalized Born surface area (MM/GBSA) method, which yielded approximate values for the binding energy of the protein-ligand complex. After clustering and ranking their activity, the top-ranking compounds were tested against PP2C group A members in vitro and their in vivo activity was also explored. Phosphatase activity assays identified two compounds with significant inhibitory activity against ABI1 protein ranging from around 57 to 91% at a concentration of 100 µM. Importantly, this in vitro activity correlated well with in vivo inhibition of seed germination, as expected for PP2C inhibitors. The results should promote the design of novel inhibitors with improved potency against ABI1-like and other PP2Cs that might be used in agriculture for the protection of crops against stress.

Protein Phosphatases Type 2C Group A Interact with and Regulate the Stability of ACC Synthase 7 in Arabidopsis.

Ethylene is an important plant hormone that controls growth, development, aging and stress responses. The rate-limiting enzymes in ethylene biosynthesis, the 1-aminocyclopropane-1-carboxylate synthases (ACSs), are strictly regulated at many levels, including posttranslational control of protein half-life. Reversible phosphorylation/dephosphorylation events play a pivotal role as signals for ubiquitin-dependent degradation. We showed previously that ABI1, a group A protein phosphatase type 2C (PP2C) and a key negative regulator of abscisic acid signaling regulates type I ACS stability. Here we provide evidence that ABI1 also contributes to the regulation of ethylene biosynthesis via ACS7, a type III ACS without known regulatory domains. Using various approaches, we show that ACS7 interacts with ABI1, ABI2 and HAB1. We use molecular modeling to predict the amino acid residues involved in ABI1/ACS7 complex formation and confirm these predictions by mcBiFC-FRET-FLIM analysis. Using a cell-free degradation assay, we show that proteasomal degradation of ACS7 is delayed in protein extracts prepared from PP2C type A knockout plants, compared to a wild-type extract. This study therefore shows that ACS7 undergoes complex regulation governed by ABI1, ABI2 and HAB1. Furthermore, this suggests that ACS7, together with PP2Cs, plays an essential role in maintaining appropriate levels of ethylene in Arabidopsis.

Allies or Enemies: The Role of Reactive Oxygen Species in Developmental Processes of Black Cottonwood (Populus trichocarpa).

In contrast to aboveground organs (stems and leaves), developmental events and their regulation in underground organs, such as pioneer and fine roots, are quite poorly understood. The objective of the current study was to achieve a better understanding of the physiological and molecular role of reactive oxygen species (ROS) and ROS-related enzymes in the process of stem and pioneer root development in black cottonwood (Populus trichocarpa), as well as in the senescence of leaves and fine roots. Results of a transcriptomic analysis revealed that primary/secondary growth and senescence are accompanied by substantial changes in the expression of genes related to oxidative stress metabolism. We observed that some mechanisms common for above- and under-ground organs, e.g., the expression of superoxide dismutase (SOD) genes and SOD activity, declined during stems' and pioneer roots' development. Moreover, the localization of hydrogen peroxide (H2O2) and superoxide (O2•-) in the primary and secondary xylem of stems and pioneer roots confirms their involvement in xylem cell wall lignification and the induction of programmed cell death (PCD). H2O2 and O2•- in senescing fine roots were present in the same locations as demonstrated previously for ATG8 (AuTophaGy-related) proteins, implying their participation in cell degradation during senescence, while O2•- in older leaves was also localized similarly to ATG8 in chloroplasts, suggesting their role in chlorophagy. ROS and ROS-related enzymes play an integral role in the lignification of xylem cell walls in Populus trichocarpa, as well as the induction of PCD during xylogenesis and senescence.

Abscisic Acid and Jasmonate Metabolisms Are Jointly Regulated During Senescence in Roots and Leaves of Populus trichocarpa.

Plant senescence is a highly regulated process that allows nutrients to be mobilized from dying tissues to other organs. Despite that senescence has been extensively studied in leaves, the senescence of ephemeral organs located underground is still poorly understood, especially in the context of phytohormone engagement. The present study focused on filling this knowledge gap by examining the roles of abscisic acid (ABA) and jasmonate in the regulation of senescence of fine, absorptive roots and leaves of Populus trichocarpa. Immunohistochemical (IHC), chromatographic, and molecular methods were utilized to achieve this objective. A transcriptomic analysis identified significant changes in gene expression that were associated with the metabolism and signal transduction of phytohormones, especially ABA and jasmonate. The increased level of these phytohormones during senescence was detected in both organs and was confirmed by IHC. Based on the obtained data, we suggest that phytohormonal regulation of senescence in roots and leaves is organ-specific. We have shown that the regulation of ABA and JA metabolism is tightly regulated during senescence processes in both leaves and roots. The results were discussed with respect to the role of ABA in cold tolerance and the role of JA in resistance to pathogens.

Seasonal senescence of leaves and roots of Populus trichocarpa-is the scenario the same or different?

The remobilization and resorption of plant nutrients is considered as a crucial aspect of the seasonal senescence of plant organs. In leaves, the mechanisms responsible for the relocation of valuable compounds are well understood while the related processes in roots are still being debated. Some research indicates that remobilization in roots occurs, while other studies have not found evidence of this process. Considering that the total biomass of fine roots is equal to or greater than that of leaves, clarifying the conflicting reports and ambiguities may provide critical information on the circulation of chemical elements in forest ecosystems. This study provides new information concerning the basis for remobilization processes in roots by combining physiological data with gene expression and protein levels. We suggest that, as in leaves, molecular mechanisms involved in nitrogen (N) resorption are also activated in senescent roots. An analysis of N concentration indicated that N levels decreased during the senescence of both organs. The decrease was associated with an increase in the expression of a glutamine synthetase (GS) gene and a concomitant elevation in the amount of GS-one of the most important enzymes in N metabolism. In addition, significant accumulation of carbohydrates was observed in fine roots, which may represent an adaptation to unfavorable weather conditions that would allow remobilization to occur rather than a rapid death in response to ground frost or cold. Our results provide new insights into the senescence of plant organs and clarify contentious topics related to the remobilization process in fine roots.

Xylem Cell Wall Formation in Pioneer Roots and Stems of Populus trichocarpa (Torr. & Gray).

Regulation of gene expression, as determined by the genetics of the tree species, is a major factor in determining wood quality. Therefore, the identification of genes that play a role in xylogenesis is extremely important for understanding the mechanisms shaping the plant phenotype. Efforts to develop new varieties characterized by higher yield and better wood quality will greatly benefit from recognizing and understanding the complex transcriptional network underlying wood development. The present study provides a detailed comparative description of the changes that occur in genes transcription and the biosynthesis of cell-wall-related compounds during xylogenesis in Populus trichocarpa pioneer roots and stems. Even though results of microarray analysis indicated that only approximately 10% of the differentially expressed genes were common to both organs, many fundamental mechanisms were similar; e.g. the pattern of expression of genes involved in the biosynthesis of cell wall proteins, polysaccharides, and lignins. Gas chromatography time-of-flight mass spectrometry (GC-TOF-MS) shows that the composition of monosaccharides was also very similar, with an increasing amount of xylose building secondary cell wall hemicellulose and pectins, especially in the stems. While hemicellulose degradation was typical for stems, possibly due to the intensive level of cell wall lignification. Notably, the main component of lignins in roots were guiacyl units, while syringyl units were dominant in stems, where fibers are especially needed for support. Our study is the first comprehensive analysis, at the structural and molecular level, of xylogenesis in under- and aboveground tree parts, and clearly reveals the great complexity of molecular mechanisms underlying cell wall formation and modification during xylogenesis in different plant organs.

Mitogen-Activated Protein Kinase Cascades in Plant Hormone Signaling.

Mitogen-activated protein kinase (MAPK) modules play key roles in the transduction of environmental and developmental signals through phosphorylation of downstream signaling targets, including other kinases, enzymes, cytoskeletal proteins or transcription factors, in all eukaryotic cells. A typical MAPK cascade consists of at least three sequentially acting serine/threonine kinases, a MAP kinase kinase kinase (MAPKKK), a MAP kinase kinase (MAPKK) and finally, the MAP kinase (MAPK) itself, with each phosphorylating, and hence activating, the next kinase in the cascade. Recent advances in our understanding of hormone signaling pathways have led to the discovery of new regulatory systems. In particular, this research has revealed the emerging role of crosstalk between the protein components of various signaling pathways and the involvement of this crosstalk in multiple cellular processes. Here we provide an overview of current models and mechanisms of hormone signaling with a special emphasis on the role of MAPKs in cell signaling networks. One-sentence summary: In this review we highlight the mechanisms of crosstalk between MAPK cascades and plant hormone signaling pathways and summarize recent findings on MAPK regulation and function in various cellular processes.

A Role for Barley Calcium-Dependent Protein Kinase CPK2a in the Response to Drought.

Increasing the drought tolerance of crops is one of the most challenging goals in plant breeding. To improve crop productivity during periods of water deficit, it is essential to understand the complex regulatory pathways that adapt plant metabolism to environmental conditions. Among various plant hormones and second messengers, calcium ions are known to be involved in drought stress perception and signaling. Plants have developed specific calcium-dependent protein kinases that convert calcium signals into phosphorylation events. In this study we attempted to elucidate the role of a calcium-dependent protein kinase in the drought stress response of barley (Hordeum vulgare L.), one of the most economically important crops worldwide. The ongoing barley genome project has provided useful information about genes potentially involved in the drought stress response, but information on the role of calcium-dependent kinases is still limited. We found that the gene encoding the calcium-dependent protein kinase HvCPK2a was significantly upregulated in response to drought. To better understand the role of HvCPK2a in drought stress signaling, we generated transgenic Arabidopsis plants that overexpressed the corresponding coding sequence. Overexpressing lines displayed drought sensitivity, reduced nitrogen balance index (NBI), an increase in total chlorophyll content and decreased relative water content. In addition, in vitro kinase assay experiments combined with mass spectrometry allowed HvCPK2a autophosphorylation sites to be identified. Our results suggest that HvCPK2a is a dual-specificity calcium-dependent protein kinase that functions as a negative regulator of the drought stress response in barley.

Phosphatase ABI1 and okadaic acid-sensitive phosphoprotein phosphatases inhibit salt stress-activated SnRK2.4 kinase.

BACKGROUND: SNF1-related protein kinases 2 (SnRK2s) are key regulators of the plant response to osmotic stress. They are transiently activated in response to drought and salinity. Based on a phylogenetic analysis SnRK2s are divided into three groups. The classification correlates with their response to abscisic acid (ABA); group 1 consists SnRK2s non-activated in response to ABA, group 2, kinases non-activated or weakly activated (depending on the plant species) by ABA treatment, and group 3, ABA-activated kinases. The activity of all SnRK2s is regulated by phosphorylation. It is well established that clade A phosphoprotein phosphatases 2C (PP2Cs) are negative regulators of ABA-activated SnRK2s, whereas regulators of SnRK2s from group 1 remain unidentified. RESULTS: Here, we show that ABI1, a PP2C clade A phosphatase, interacts with SnRK2.4, member of group 1 of the SnRK2 family, dephosphorylates Ser158, whose phosphorylation is needed for the kinase activity, and inhibits the kinase, both in vitro and in vivo. Our data indicate that ABI1 and the kinase regulate primary root growth in response to salinity; the phenotype of ABI1 knockout mutant (abi1td) exposed to salt stress is opposite to that of the snrk2.4 mutant. Moreover, we show that the activity of SnRK2s from group 1 is additionally regulated by okadaic acid-sensitive phosphatase(s) from the phosphoprotein phosphatase (PPP) family. CONCLUSIONS: Phosphatase ABI1 and okadaic acid-sensitive phosphatases of the PPP family are negative regulators of salt stress-activated SnRK2.4. The results show that ABI1 inhibits not only the ABA-activated SnRK2s but also at least one ABA-non-activated SnRK2, suggesting that the phosphatase is involved in the cross talk between ABA-dependent and ABA-independent stress signaling pathways in plants.

Regulation of Arabidopsis MAPKKK18 by ABI1 and SnRK2, components of the ABA signaling pathway.

The plant hormone abscisic acid (ABA), a key regulator in many crucial developmental and physiological processes, recruits diverse components into precisely regulated signaling network. We recently discovered that MAPKKK18, an ABA-activated kinase, is regulated by the protein phosphatase type 2C (PP2C) ABI1 and the kinase SnRK2.6, both components of the ABA core signaling pathway. ABI1 acts to inhibit MAPKKK18 kinase activity, but also affects MAPKKK18 protein turnover via the ubiquitin-proteasome pathway. SnRK2.6 kinase also seems to be important for the regulation of MAPKKK18 function. In this review we summarize the mechanisms that are exclusively involved in MAPKKK18 kinase regulation and that ensure specificity in its activation.

Arabidopsis ABA-Activated Kinase MAPKKK18 is Regulated by Protein Phosphatase 2C ABI1 and the Ubiquitin-Proteasome Pathway.

Phosphorylation and dephosphorylation events play an important role in the transmission of the ABA signal. Although SnRK2 [sucrose non-fermenting1-related kinase2] protein kinases and group A protein phosphatase type 2C (PP2C)-type phosphatases constitute the core ABA pathway, mitogen-activated protein kinase (MAPK) pathways are also involved in plant response to ABA. However, little is known about the interplay between MAPKs and PP2Cs or SnRK2 in the regulation of ABA pathways. In this study, an effort was made to elucidate the role of MAP kinase kinase kinase18 (MKKK18) in relation to ABA signaling and response. The MKKK18 knockout lines showed more vigorous root growth, decreased abaxial stomatal index and increased stomatal aperture under normal growth conditions, compared with the control wild-type Columbia line. In addition to transcriptional regulation of the MKKK18 promoter by ABA, we demonstrated using in vitro and in vivo kinase assays that the kinase activity of MKKK18 was regulated by ABA. Analysis of the cellular localization of MKKK18 showed that the active kinase was targeted specifically to the nucleus. Notably, we identified abscisic acid insensitive 1 (ABI1) PP2C as a MKKK18-interacting protein, and demonstrated that ABI1 inhibited its activity. Using a cell-free degradation assay, we also established that MKKK18 was unstable and was degraded by the proteasome pathway. The rate of MKKK18 degradation was delayed in the ABI1 knockout line. Overall, we provide evidence that ABI1 regulates the activity and promotes proteasomal degradation of MKKK18.

Involvement of genes encoding ABI1 protein phosphatases in the response of Brassica napus L. to drought stress.

In this report we characterized the Arabidopsis ABI1 gene orthologue and Brassica napus gene paralogues encoding protein phosphatase 2C (PP2C, group A), which is known to be a negative regulator of the ABA signaling pathway. Six homologous B. napus sequences were identified and characterized as putative PP2C group A members. To gain insight into the conservation of ABI1 function in Brassicaceae, and understand better its regulatory effects in the drought stress response, we generated transgenic B. napus plants overexpressing A. thaliana ABI1. Transgenic plants subjected to drought showed a decrease in relative water content, photosynthetic pigments content and expression level of RAB18- and RD19A-drought-responsive marker genes relative to WT plants. We present the characterization of the drought response of B. napus with the participation of ABI1-like paralogues. The expression pattern of two evolutionarily distant paralogues, BnaA01.ABI1.a and BnaC07.ABI1.b in B. napus and their promoter activity in A. thaliana showed differences in the induction of the paralogues under dehydration stress. Comparative sequence analysis of both BnaABI1 promoters showed variation in positions of cis-acting elements that are especially important for ABA- and stress-inducible expression. Together, these data reveal that subfunctionalization following gene duplication may be important in the maintenance and functional divergence of the BnaABI1 paralogues. Our results provide a framework for a better understanding of (1) the role of ABI1 as a hub protein regulator of the drought response, and (2) the differential involvement of the duplicated BnaABI1 genes in the response of B. napus to dehydration-related stresses.

Targeting proteins for proteasomal degradation-a new function of Arabidopsis ABI1 protein phosphatase 2C.

The ubiquitin/26S proteasome system (UPS) has been implicated in the regulation of many physiological processes including hormone signaling. The plant hormone abscisic acid (ABA) employs the UPS to control its own synthesis and signaling and to regulate stress response and tolerance. Among the known effectors of ABA signaling, the ABI1 (abscisic acid-insensitive 1) protein phosphatase, which belongs to group A of the type 2C protein phosphatases, is recognized as a key component of the pathway. Molecular and genetic evidence implicates this protein phosphatase in numerous plant responses. This mini-review discusses recent progress in understanding the role of ABI1 in ABA signaling, with particular emphasis on recent data that link ABI1 to protein degradation via the UPS.

AtEAF1 is a potential platform protein for Arabidopsis NuA4 acetyltransferase complex.

BACKGROUND: Histone acetyltransferase complex NuA4 and histone variant exchanging complex SWR1 are two chromatin modifying complexes which act cooperatively in yeast and share some intriguing structural similarities. Protein subunits of NuA4 and SWR1-C are highly conserved across eukaryotes, but form different multiprotein arrangements. For example, the human TIP60-p400 complex consists of homologues of both yeast NuA4 and SWR1-C subunits, combining subunits necessary for histone acetylation and histone variant exchange. It is currently not known what protein complexes are formed by the plant homologues of NuA4 and SWR1-C subunits. RESULTS: We report on the identification and molecular characterization of AtEAF1, a new subunit of Arabidopsis NuA4 complex which shows many similarities to the platform protein of the yeast NuA4 complex. AtEAF1 copurifies with Arabidopsis homologues of NuA4 and SWR1-C subunits ARP4 and SWC4 and interacts physically with AtYAF9A and AtYAF9B, homologues of the YAF9 subunit. Plants carrying a T-DNA insertion in one of the genes encoding AtEAF1 showed decreased FLC expression and early flowering, similarly to Atyaf9 mutants. Chromatin immunoprecipitation analyses of the single mutant Ateaf1b-2 and artificial miRNA knock-down Ateaf1 lines showed decreased levels of H4K5 acetylation in the promoter regions of major flowering regulator genes, further supporting the role of AtEAF1 as a subunit of the plant NuA4 complex. CONCLUSIONS: Growing evidence suggests that the molecular functions of the NuA4 and SWR1 complexes are conserved in plants and contribute significantly to plant development and physiology. Our work provides evidence for the existence of a yeast-like EAF1 platform protein in A. thaliana, filling an important gap in the knowledge about the subunit organization of the plant NuA4 complex.