Functional and regulatory diversity of homeobox-leucine zipper transcription factors BnaHB6 under dehydration and salt stress in Brassica napus L.

The plant-specific homeodomain-leucine zipper I subfamily is involved in the regulation of various biological processes, particularly growth, development and stress response. In the present study, we characterized four BnaHB6 homologues from Brassica napus. All BnaHB6 proteins have transcriptional activation activity. Structural and functional data indicate the complex role of BnaHB6 genes in regulating biological processes, with some functions conserved and others diverged. Transcriptional analyzes revealed that they are induced in a similar manner in different tissues but show different expression patterns in response to stress and circadian rhythm. Only the BnaA09HB6 and BnaC08HB6 genes are expressed under dehydration and salt stress, and in darkness. The partial transcriptional overlap of BnaHB6s with the evolutionarily related genes BnaHB5 and BnaHB16 was also observed. Transgenic Arabidopsis thaliana plants expressing a single proBnaHB6::GUS partially confirmed the expression results. Bioinformatic analysis allowed the identification of TF-binding sites in the BnaHB6 promoters that may control their expression under stress and circadian rhythm. ChIP-qPCR analysis revealed that BnaA09HB6 and BnaC08HB6 bind directly to the promoters of the target genes BnaABF4 and BnaDREB2A. Comparison of their expression patterns in the WT plants and the bnac08hb6 mutant showed that BnaC08HB6 positively regulates the expression of the BnaABF4 and BnaDREB2A genes under dehydration and salt stress. We conclude that four BnaHB6 homologues have distinct functions in response to stress despite high sequence similarity, possibly indicating different binding preferences with BnaABF4 and BnaDREB2A. We hypothesize that BnaC08HB6 and BnaA09HB6 function in a complex regulatory network under stress.

Organelles and phytohormones: a network of interactions in plant stress responses.

Phytohormones are major signaling components that contribute to nearly all aspects of plant life. They constitute an interconnected communication network to fine-tune growth and development in response to the ever-changing environment. To this end, they have to coordinate with other signaling components, such as reactive oxygen species and calcium signals. On the one hand, the two endosymbiotic organelles, plastids and mitochondria, control various aspects of phytohormone signaling and harbor important steps of hormone precursor biosynthesis. On the other hand, phytohormones have feedback actions on organellar functions. In addition, organelles and phytohormones often act in parallel in a coordinated matter to regulate cellular functions. Therefore, linking organelle functions with increasing knowledge of phytohormone biosynthesis, perception, and signaling will reveal new aspects of plant stress tolerance. In this review, we highlight recent work on organelle-phytohormone interactions focusing on the major stress-related hormones abscisic acid, jasmonates, salicylic acid, and ethylene.

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.

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.

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.

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.

Arabidopsis protein phosphatase 2C ABI1 interacts with type I ACC synthases and is involved in the regulation of ozone-induced ethylene biosynthesis.

Ethylene plays a crucial role in various biological processes and therefore its biosynthesis is strictly regulated by multiple mechanisms. Posttranslational regulation, which is pivotal in controlling ethylene biosynthesis, impacts 1-aminocyclopropane 1-carboxylate synthase (ACS) protein stability via the complex interplay of specific factors. Here, we show that the Arabidopsis thaliana protein phosphatase type 2C, ABI1, a negative regulator of abscisic acid signaling, is involved in the regulation of ethylene biosynthesis under oxidative stress conditions. We found that ABI1 interacts with ACS6 and dephosphorylates its C-terminal fragment, a target of the stress-responsive mitogen-activated protein kinase, MPK6. In addition, ABI1 controls MPK6 activity directly and by this means also affects the ACS6 phosphorylation level. Consistently with this, ozone-induced ethylene production was significantly higher in an ABI1 knockout strain (abi1td) than in wild-type plants. Importantly, an increase in stress-induced ethylene production in the abi1td mutant was compensated by a higher ascorbate redox state and elevated antioxidant activities. Overall, the results of this study provide evidence that ABI1 restricts ethylene synthesis by affecting the activity of ACS6. The ABI1 contribution to stress phenotype underpins its role in the interplay between the abscisic acid (ABA) and ethylene signaling pathways.