Signaling through mitogen-activated protein kinases (MPKs) cascades is a complex and fundamental process in eukaryotes, requiring MPK-activating kinases (MKKs) and MKK-activating kinases (MKKKs). ...However, to date only a limited number of MKK-MPK interactions and MPK phosphorylation substrates have been revealed. We determined which Arabidopsis thaliana MKKs preferentially activate 10 different MPKs in vivo and used the activated MPKs to probe high-density protein microarrays to determine their phosphorylation targets. Our analyses revealed known and novel signaling modules encompassing 570 MPK phosphorylation substrates; these substrates were enriched in transcription factors involved in the regulation of development, defense, and stress responses. Selected MPK substrates were validated by in planta reconstitution experiments. A subset of activated and wild-type MKKs induced cell death, indicating a possible role for these MKKs in the regulation of cell death. Interestingly, MKK7- and MKK9-induced death requires Sgt1, a known regulator of cell death induced during plant innate immunity. Our predicted MKK-MPK phosphorylation network constitutes a valuable resource to understand the function and specificity of MPK signaling systems.
Plant protein kinases form redundant signaling pathways to perceive microbial pathogens and activate immunity. Bacterial pathogens repress cellular immune responses by secreting effectors, some of ...which bind and inhibit multiple host kinases. To understand how broadly bacterial effectors may bind protein kinases and the function of these kinase interactors, we first tested kinase-effector (K-E) interactions using the Pseudomonas syringae pv. tomato-tomato pathosystem. We tested interactions between five individual effectors (HopAI1, AvrPto, HopA1, HopM1, and HopAF1) and 279 tomato kinases in tomato cells. Over half of the tested kinases interacted with at least one effector, and 48% of these kinases interacted with more than three effectors, suggesting a role in the defense. Next, we characterized the role of select multi-effector-interacting kinases and revealed their roles in basal resistance, effector-triggered immunity (ETI), or programmed cell death (PCD). The immune function of several of these kinases was only detectable in the presence of effectors, suggesting that these kinases are critical when particular cell functions are perturbed or that their role is typically masked. To visualize the kinase networks underlying the cellular responses, we derived signal-specific networks. A comparison of the networks revealed a limited overlap between ETI and basal immunity networks. In addition, the basal immune network complexity increased when exposed to some of the effectors. The networks were used to successfully predict the role of a new set of kinases in basal immunity. Our work indicates the complexity of the larger kinase-based defense network and demonstrates how virulence- and avirulence-associated bacterial effectors alter sectors of the defense network.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Nonhost resistance (NHR) is the most prevalent form of plant immunity. In Arabidopsis, NHR requires membrane‐localized ATP‐binding cassette (ABC) transporter PENETRATION (PEN) 3. Upon perception of ...pathogen‐associated molecular patterns, PEN3 becomes phosphorylated, suggestive of PEN3 regulation by post‐translational modification. Here, we investigated the PEN3 protein interaction network. We probed the Arabidopsis protein microarray AtPMA‐5000 with the N‐terminal cytoplasmic domain of PEN3. Several of the proteins identified to interact with PEN3 in vitro represent cellular Ca²⁺ sensors, including calmodulin (CaM) 3, CaM7 and several CaM‐like proteins, pointing to the importance of Ca²⁺ sensing to PEN3‐mediated NHR. We demonstrated co‐localization of PEN3 and CaM7, and we confirmed PEN3–CaM interaction in vitro and in vivo by PEN3 pull‐down with CaM Sepharose, CaM overlay assay and bimolecular fluorescence complementation. We also show that just like in pen3, NHR to the nonadapted fungal pathogens Phakopsora pachyrhizi and Blumeria graminis f.sp. hordei is compromised in the Arabidopsis cam7 and pen3 cam7 mutants. Our study discloses CaM7 as a PEN3‐interacting protein crucial to Arabidopsis NHR and emphasizes the importance of Ca²⁺ sensing to plant immunity.
Gene families are sets of structurally and evolutionarily related genes - in one or multiple species - that typically share a conserved biological function. As such, the identification and subsequent ...analyses of entire gene families are widely employed in the fields of evolutionary and functional genomics of both well established and newly sequenced plant genomes. Currently, plant gene families are typically identified using one of two major ways: 1) HMM-profile based searches using models built on Arabidopsis thaliana genes or 2) coding sequence homology searches using curated databases. Integrated databases containing functionally annotated genes and gene families have been developed for model organisms and several important crops; however, a comprehensive methodology for gene family annotation is currently lacking, preventing automated annotation of newly sequenced genomes.
This paper proposes a combined measure of homology identification, motif conservation, phylogenomic and integrated gene expression analyses to define gene family structures in multiple plant species. The MAP3K gene families in seven plant species, including two currently unexamined species Gossypium hirsutum, and Zostera marina, were characterized to reveal new insights into their collective function and evolution and demonstrate the effectiveness of our novel methodology.
Compared with recent reports, this methodology performs significantly better for the identification and analysis of gene family members in several monocots/dicots, diploid as well as polyploid plant species.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Several methods to handle data generated from bottom-up proteomics via liquid chromatography-mass spectrometry, particularly for peptide-centric quantification dealing with post-translational ...modification (PTM) analysis like reversible cysteine oxidation are evaluated. The paper proposes a pipeline based on the R programming language to analyze PTMs from peptide-centric label-free quantitative proteomics data.
Our methodology includes variance stabilization, normalization, and missing data imputation to account for the large dynamic range of PTM measurements. It also corrects biases from an enrichment protocol and reduces the random and systematic errors associated with label-free quantification. The performance of the methodology is tested by performing proteome-wide differential PTM quantitation using linear models analysis (limma). We objectively compare two imputation methods along with significance testing when using multiple-imputation for missing data.
Identifying PTMs in large-scale datasets is a problem with distinct characteristics that require new methods for handling missing data imputation and differential proteome analysis. Linear models in combination with multiple-imputation could significantly outperform a t-test-based decision method.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Calmodulins (CaMs) are the most ubiquitous calcium sensors in eukaryotes. A number of CaM-binding proteins have been identified through classical methods, and many proteins have been predicted to ...bind CaMs based on their structural homology with known targets. However, multicellular organisms typically contain many CaM-like (CML) proteins, and a global identification of their targets and specificity of interaction is lacking. In an effort to develop a platform for large-scale analysis of proteins in plants we have developed a protein microarray and used it to study the global analysis of CaM/CML interactions. An Arabidopsis thaliana expression collection containing 1,133 ORFs was generated and used to produce proteins with an optimized medium-throughput plant-based expression system. Protein microarrays were prepared and screened with several CaMs/CMLs. A large number of previously known and novel CaM/CML targets were identified, including transcription factors, receptor and intracellular protein kinases, F-box proteins, RNA-binding proteins, and proteins of unknown function. Multiple CaM/CML proteins bound many binding partners, but the majority of targets were specific to one or a few CaMs/CMLs indicating that different CaM family members function through different targets. Based on our analyses, the emergent CaM/CML interactome is more extensive than previously predicted. Our results suggest that calcium functions through distinct CaM/CML proteins to regulate a wide range of targets and cellular activities.
The balance between accumulation of stress-induced polyamines and reactive oxygen species (ROS) is arguably a critical factor in plant tolerance to salt stress. Polyamines are compounds, which ...accumulate in plants under salt stress and help maintain cellular ROS homeostasis. In this review we first outline the role of polyamines in mediating salt stress responses through their modulation of redox homeostasis. The two proposed roles of polyamines in regulating ROS – as antioxidative molecules and source of ROS synthesis – are discussed and exemplified with recent studies. Second, the proposed function of polyamines as modulators of ion transport is discussed in the context of plant salt stress. Finally, we highlight the apparent connection between polyamine accumulation and programmed cell death induction during stress. Thus polyamines have a complex functional role in regulating cellular signaling and metabolism during stress. By focusing future efforts on how polyamine accumulation and turnover is regulated, research in this area may provide novel targets for developing stress tolerance.
Waterlogging induces growth and developmental changes in sensitive crops such as cucumber (
Cucumis sativus
L.) during early plant development. However, information on the physiological mechanisms ...underpinning the response of cucumber plants to waterlogging conditions is limited. Here, we investigated the effects of 10-day waterlogging stress on the morphology, photosynthesis, and chlorophyll fluorescence parameters in two cultivars of cucumber seedlings. Waterlogging stress hampered cultivars’ growth, biomass accumulation, and photosynthetic capacity. Both cultivars also developed adventitious roots (ARs) after 10 days of waterlogging (DOW). We observed differential responses in the light- and carbon-dependent reactions of photosynthesis, with an increase in light-dependent reactions. At the same time, carbon assimilation was considerably inhibited by waterlogging. Specifically, the CO
2
assimilation rate (
A
) in leaves was significantly reduced and was caused by a corresponding decrease in stomatal conductance (g
s
). The downregulation of the maximum rate of Rubisco efficiency (V
cmax
) and the maximum rate of photosynthetic electron transport (J
max
) were non-stomatal limiting factors contributing to
A
reduction. Exposure of cucumber to 10 DOW affected the PSII photochemistry by downregulating the PSII quantum yield (Φ
PSII
). The redox state of the primary quinone acceptor in the lake model (1-qL), a measure of the regulatory balance of the light reactions, became more oxidized after 10 DOW, indicating enhanced electron sink capacity despite a reduced
A
. Overall, the results suggest that waterlogging induces alterations in the photochemical apparatus efficiency of cucumber. Thus, developing cultivars that resist inhibition of PSII photochemistry while maintaining carbon metabolism is a potential approach for increasing crops’ tolerance to waterlogged environments.
A surge in the accumulation of oxidants generates shifts in the cellular redox potential during early stages of plant infection with pathogens and activation of effector-triggered immunity (ETI). The ...redoxome, defined as the proteome-wide oxidative modifications of proteins caused by oxidants, has a well-known impact on stress responses in metazoans. However, the identity of proteins and the residues sensitive to oxidation during the plant immune response remain largely unknown. Previous studies of the thimet oligopeptidases TOP1 and TOP2 placed them in the salicylic acid dependent branch of ETI, with a current model wherein TOPs sustain interconnected organellar and cytosolic pathways that modulate the oxidative burst and development of cell death. Herein, we characterized the ETI redoxomes in Arabidopsis (
) wild-type Col-0 and
mutant plants using a differential alkylation-based enrichment technique coupled with label-free mass spectrometry-based quantification. We identified cysteines sensitive to oxidation in a wide range of protein families at multiple time points after pathogen infection. Differences were detected between Col-0 and
redoxomes regarding the identity and number of oxidized cysteines, and the amplitude of time-dependent fluctuations in protein oxidation. Our results support a determining role for TOPs in maintaining the proper level and dynamics of proteome oxidation during ETI. This study significantly expands the repertoire of oxidation-sensitive plant proteins and can guide future mechanistic studies.
Waterlogging is an important environmental stress limiting the productivity of crops worldwide. Cowpea (Vigna unguiculata L.) is particularly sensitive to waterlogging stress during the reproductive ...stage, with a consequent decline in pod formation and yield. However, little is known about the critical processes underlying cowpea’s responses to waterlogging during the reproductive stage. Thus, we investigated the key parameters influencing carbon fixation, including stomatal conductance (gs), intercellular CO2 concentration, chlorophyll content, and chlorophyll fluorescence, of two cowpea genotypes with contrasting waterlogging tolerance. These closely related genotypes have starkly contrasting responses to waterlogging during and after 7 days of waterlogging stress (DOW). In the intolerant genotype (‘EpicSelect.4’), waterlogging resulted in a gradual loss of pigment and decreased photosynthetic capacity as a consequent decline in shoot biomass. On the other hand, the waterlogging-tolerant genotype (‘UCR 369’) maintained CO2 assimilation rate (A), stomatal conductance (gs), biomass, and chlorophyll content until 5 DOW. Moreover, there was a highly specific downregulation of the mesophyll conductance (gm), maximum rate of Rubisco (Vcmax), and photosynthetic electron transport rate (Jmax) as non-stomatal limiting factors decreasing A in EpicSelect.4. Exposure of EpicSelect.4 to 2 DOW resulted in the loss of PSII photochemistry by downregulating the PSII quantum yield (Fv/Fm), photochemical efficiency (ΦPSII), and photochemical quenching (qP). In contrast, we found no substantial change in the photosynthesis and chlorophyll fluorescence of UCR 369 in the first 5 DOW. Instead, UCR 369 maintained biomass accumulation, chlorophyll content, and Rubisco activity, enabling the genotype to maintain nutrient absorption and photosynthesis during the early period of waterlogging. However, compared to the control, both cowpea genotypes could not fully recover their photosynthetic capacity after 7 DOW, with a more significant decline in EpicSelect.4. Overall, our findings suggest that the tolerant UCR 369 genotype maintains higher photosynthesis under waterlogging stress attributable to higher photochemical efficiency, Rubisco activity, and less stomatal restriction. After recovery, the incomplete recovery of A can be attributed to the reduced gs caused by severe waterlogging damage in both genotypes. Thus, promoting the rapid recovery of stomata from waterlogging stress may be crucial for the complete restoration of carbon fixation in cowpeas during the reproductive stage.
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