Arabidopsis thimet oligopeptidases are redox-sensitive enzymes active in the local and systemic plant immune response.

Upon pathogen infection, receptors in plants will activate a localized immune response, the effector-triggered immunity (ETI), and a systemic immune response, the systemic acquired response (SAR). Infection also induces oscillations in the redox environment of plant cells, triggering response mechanisms involving sensitive cysteine residues that subsequently alter protein function. Arabidopsis thaliana thimet oligopeptidases TOP1 and TOP2 are required for plant defense against pathogens and the oxidative stress response. Herein, we evaluated the biochemical attributes of TOP isoforms to determine their redox sensitivity using ex vivo Escherichia coli cultures and recombinant proteins. Moreover, we explored the link between their redox regulation and plant immunity in wild-type and mutant Arabidopsis lines. These analyses revealed that redox regulation of TOPs occurs through two mechanisms: (1) oxidative dimerization of full-length TOP1 via intermolecular disulfides engaging cysteines in the N-terminal signal peptide, and (2) oxidative activation of all TOPs via cysteines that are unique and conserved. Further, we detected increased TOP activity in wild-type plants undergoing ETI or SAR following inoculation with Pseudomonas syringae strains. Mutants unable to express the chloroplast NADPH-dependent thioredoxin reductase C (NTRC) showed elevated TOP activity under unstressed conditions and were SAR-incompetent. A top1top2 knockout mutant challenged with P. syringae exhibited misregulation of ROS-induced gene expression in pathogen-inoculated and distal tissues. Furthermore, TOP1 and TOP2 could cleave a peptide derived from the immune component ROC1 with distinct efficiencies at common and specific sites. We propose that Arabidopsis TOPs are thiol-regulated peptidases active in redox-mediated signaling of local and systemic immunity.

Profiling thimet oligopeptidase-mediated proteolysis in Arabidopsis thaliana.

Protein homeostasis (proteostasis) is crucial for proper cellular function, including the production of peptides with biological functions through controlled proteolysis. Proteostasis has roles in maintenance of cellular functions and plant interactions with the environment under physiological conditions. Plant stress continues to reduce agricultural yields causing substantial economic losses; thus, it is critical to understand how plants perceive stress signals to elicit responses for survival. As previously shown in Arabidopsis thaliana, thimet oligopeptidases (TOPs) TOP1 (also referred to as organellar oligopeptidase) and TOP2 (also referred to as cytosolic oligopeptidase) are essential components in plant response to pathogens, but further characterization of TOPs and their peptide substrates is required to understand their contributions to stress perception and defense signaling. Herein, label-free peptidomics via liquid chromatography-tandem mass spectrometry was used to differentially quantify 1111 peptides, originating from 369 proteins, between the Arabidopsis Col-0 wild type and top1top2 knock-out mutant. This revealed 350 peptides as significantly more abundant in the mutant, representing accumulation of these potential TOP substrates. Ten direct substrates were validated using in vitro enzyme assays with recombinant TOPs and synthetic candidate peptides. These TOP substrates are derived from proteins involved in photosynthesis, glycolysis, protein folding, biogenesis, and antioxidant defense, implicating TOP involvement in processes aside from defense signaling. Sequence motif analysis revealed TOP cleavage preference for non-polar residues in the positions surrounding the cleavage site. Identification of these substrates provides a framework for TOP signaling networks, through which the interplay between proteolytic pathways and defense signaling can be further characterized.

Integrative network-centric approach reveals signaling pathways associated with plant resistance and susceptibility to Pseudomonas syringae.

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.

Proteome-Wide Analysis of Cysteine Reactivity during Effector-Triggered Immunity.

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 (Arabidopsis thaliana) wild-type Col-0 and top1top2 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 top1top2 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.

Multispecies genome-wide analysis defines the MAP3K gene family in Gossypium hirsutum and reveals conserved family expansions.

BACKGROUND: 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. RESULTS: 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. CONCLUSION: 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.

Evaluation of linear models and missing value imputation for the analysis of peptide-centric proteomics.

BACKGROUND: 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. RESULTS: 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. CONCLUSION: 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.

Proteomics and Proteogenomics Analysis of Sweetpotato ( Ipomoea batatas) Leaf and Root.

Two complementary protein extraction methodologies coupled with an automated proteomic platform were employed to analyze tissue-specific proteomes and characterize biological and metabolic processes in sweetpotato. A total of 74 255 peptides corresponding to 4321 nonredundant proteins were successfully identified. Data were compared to predicted protein accessions for Ipomoea species and mapped on the sweetpotato transcriptome and haplotype-resolved genome. The two methodologies exhibited differences in the number and class of the unique proteins extracted. Overall, 39 916 peptides mapped to 3143 unique proteins in leaves, and 34 339 peptides mapped to 2928 unique proteins in roots. Primary metabolism and protein translation processes were enriched in leaves, whereas genetic pathways associated with protein folding, transport, sorting, as well as pathways in the primary carbohydrate metabolism were enriched in storage roots. A proteogenomics analysis successfully mapped 90.4% of the total uniquely identified peptides against the sweetpotato transcriptome and genome, predicted 741 new protein-coding genes, and specified 2056 loci where gene annotations can be further improved. The proteogenomics results provide evidence for the translation of new open reading frames (ORFs), alternative ORFs, exon extensions, and intronic ORF sequences. Data are available via ProteomeXchange with identifier PXD012999.

The Raf-like Kinase ILK1 and the High Affinity K+ Transporter HAK5 Are Required for Innate Immunity and Abiotic Stress Response.

Plant perception of pathogen-associated molecular patterns (PAMPs) and other environmental stresses trigger transient ion fluxes at the plasma membrane. Apart from the role of Ca(2+) uptake in signaling, the regulation and significance of PAMP-induced ion fluxes in immunity remain unknown. We characterized the functions of INTEGRIN-LINKED KINASE1 (ILK1) that encodes a Raf-like MAP2K kinase with functions insufficiently understood in plants. Analysis of ILK1 mutants impaired in the expression or kinase activity revealed that ILK1 contributes to plant defense to bacterial pathogens, osmotic stress sensitivity, and cellular responses and total ion accumulation in the plant upon treatment with a bacterial-derived PAMP, flg22. The calmodulin-like protein CML9, a negative modulator of flg22-triggered immunity, interacted with, and suppressed ILK1 kinase activity. ILK1 interacted with and promoted the accumulation of HAK5, a putative (H(+))/K(+) symporter that mediates a high-affinity uptake during K(+) deficiency. ILK1 or HAK5 expression was required for several flg22 responses including gene induction, growth arrest, and plasma membrane depolarization. Furthermore, flg22 treatment induced a rapid K(+) efflux at both the plant and cellular levels in wild type, while mutants with impaired ILK1 or HAK5 expression exhibited a comparatively increased K(+) loss. Taken together, our results position ILK1 as a link between plant defense pathways and K(+) homeostasis.

MAPK target networks in Arabidopsis thaliana revealed using functional protein microarrays.

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.

ABC transporter PEN3/PDR8/ABCG36 interacts with calmodulin that, like PEN3, is required for Arabidopsis nonhost resistance.

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 Ca(2+) sensors, including calmodulin (CaM) 3, CaM7 and several CaM-like proteins, pointing to the importance of Ca(2+) 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 Ca(2+) sensing to plant immunity.

The tomato kinome and the tomato kinase library ORFeome: novel resources for the study of kinases and signal transduction in tomato and solanaceae species.

Protein kinase-driven phosphorylation constitutes the core of cellular signaling. Kinase components of signal transduction pathways are often targeted for inactivation by pathogens. The study of kinases and immune signal transduction in the model crop tomato (Solanum lycopersicum) would benefit from the availability of community-wide resources for large scale and systems-level experimentation. Here, we defined the tomato kinome and performed a comprehensive comparative analysis of the tomato kinome and 15 other plant species. We constructed a tomato kinase library (TOKN 1.0) of over 300 full-length open reading frames (ORF) cloned into a recombination-based vector. We developed a high-throughput pipeline to isolate and transform tomato protoplasts. A subset of the TOKN 1.0 library kinases were expressed in planta, were purified, and were used to generate a functional tomato protein microarray. All resources created were utilized to test known and novel associations between tomato kinases and Pseudomonas syringae DC3000 effectors in a large-scale format. Bsk7 was identified as a component of the plant immune response and a candidate effector target. These resources will enable comprehensive investigations of signaling pathways and host-pathogen interactions in tomato and other Solanaceae spp.

Methods to Analyze the Redox Reactivity of Plant Proteins.

Proteins can be covalently modified by a broad range of highly reactive chemicals and redox mechanisms. Reversible redox-mediated post-translational modifications of sensitive cysteine thiol groups in proteins impact protein characteristics such as interaction behavior and activity state. Evaluating the response of proteins to redox perturbation or reactive chemical species is critical for understanding the underlying mechanisms involved and their contribution to plant stress physiology. Here we provide a detailed workflow that includes procedures for (i) purification, processing, and analysis of protein samples with redox agents, (ii) determining redox-modulated monomer to oligomer transitions using size exclusion chromatography, and (iii) activity assays for monitoring the impact of redox agents on purified enzymes and in crude extracts from plants subjected to oxidative stress. We exemplified how to apply several of the methods discussed for analyzing redox-sensing metallopeptidases, such as thimet oligopeptidases. We anticipate that these protocols should find broad applications in monitoring biochemical properties of other classes of redox-sensitive plant proteins.

Metagenomic Analyses of the Soybean Root Mycobiome and Microbiome Reveal Signatures of the Healthy and Diseased Plants Affected by Taproot Decline.

Invading pathogens interact with plant-associated microbial communities, which can be altered under the pressure of pathogen infection. Limited information exists on plant-microbe interactions occurring during natural outbreaks in agricultural fields. Taproot decline (TRD) of soybean is an emerging disease caused by Xylaria necrophora. TRD disease occurrence and yield loss associated with TRD are outstanding issues in soybean production. We applied nuclear ribosomal DNA Internal Transcribed Spacers and 16S rRNA gene taxonomic marker sequencing to define the composition of the fungal and bacterial communities associated with healthy and diseased soybean roots collected from the Mississippi Delta. The plant compartment was a significant factor regulating taxonomic diversity, followed by the disease status of the plant. TRD impacted the root endophytes, causing imbalances; at the intermediate and advanced stages of TRD, X. necrophora decreased mycobiome diversity, whereas it increased microbiome richness. Networks of significant co-occurrence and co-exclusion relationships revealed direct and indirect associations among taxa and identified hubs with potential roles in assembling healthy and TRD-affected soybean biomes. These studies advance the understanding of host-microbe interactions in TRD and the part of biomes in plant health and disease.

Short waterlogging events differently affect morphology and photosynthesis of two cucumber (Cucumis sativus L.) cultivars.

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 CO2 assimilation rate (A) in leaves was significantly reduced and was caused by a corresponding decrease in stomatal conductance (gs). The downregulation of the maximum rate of Rubisco efficiency (Vcmax) and the maximum rate of photosynthetic electron transport (Jmax) 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 (ΦPSII). 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.

Back From the Dead: The Atypical Kinase Activity of a Pseudokinase Regulator of Cation Fluxes During Inducible Immunity.

Pseudokinases are thought to lack phosphotransfer activity due to altered canonical catalytic residues within their kinase domain. However, a subset of pseudokinases maintain activity through atypical phosphotransfer mechanisms. The Arabidopsis ILK1 is a pseudokinase from the Raf-like MAP3K family and is the only known plant pseudokinase with confirmed protein kinase activity. ILK1 activity promotes disease resistance and molecular pattern-induced root growth inhibition through its stabilization of the HAK5 potassium transporter with the calmodulin-like protein CML9. ILK1 also has a kinase-independent function in salt stress suggesting that it interacts with additional proteins. We determined that members of the ILK subfamily are the sole pseudokinases within the Raf-like MAP3K family and identified 179 novel putative ILK1 protein interactors. We also identified 70 novel peptide targets for ILK1, the majority of which were phosphorylated in the presence of Mn2+ instead of Mg2+ in line with modifications in ILK1's DFG cofactor binding domain. Overall, the ILK1-targeted or interacting proteins included diverse protein types including transporters (HAK5, STP1), protein kinases (MEKK1, MEKK3), and a cytokinin receptor (AHK2). The expression of 31 genes encoding putative ILK1-interacting or phosphorylated proteins, including AHK2, were altered in the root and shoot in response to molecular patterns suggesting a role for these genes in immunity. We describe a potential role for ILK1 interactors in the context of cation-dependent immune signaling, highlighting the importance of K+ in MAMP responses. This work further supports the notion that ILK1 is an atypical kinase with an unusual cofactor dependence that may interact with multiple proteins in the cell.

Waterlogging during the reproductive growth stage causes physiological and biochemical modifications in the leaves of cowpea (Vigna unguiculata L.) genotypes with contrasting tolerance.

Waterlogging causes various metabolic, physiological, and morphological changes in crops, resulting in yield loss of most legumes in rainfed and irrigated agriculture. However, research on cowpea genotypes using physiological and biochemical traits as a measure of tolerance to waterlogging stress is limited. We evaluated the impacts of 7 days of waterlogging (DOW) and 7 days of recovery (DOR) on the physiology and biochemistry of two cowpea (Vigna unguiculata (L.) Walp) genotypes (UCR 369 and EpicSelect.4) with contrasting waterlogging tolerance. Cowpea genotypes were grown in a controlled environment until the R2 stage and then subjected to 7 DOW. Later, the waterlogged plants were reoxygenated for an additional 7 DOR. Overall, cowpea genotypes had a contrasting response to waterlogging using different mechanisms. Compared to the control, the photosynthetic parameters of both cowpea genotypes were impaired under 7 DOW and could not recover at 7 DOR, with a larger decline in EpicSelect.4.7 DOW caused significant loss in the chlorophyll and carotenoid content of both genotypes. However, only waterlogged UCR 369 was not photo-inhibited and able to restore the levels of chlorophyll and carotenoids at 7 DOR. In addition, 7 DOW induced intense stress in UCR 369 with increased zeaxanthin, sucrose, and flavonoid content, while these metabolites were decreased in EpicSelect.4. On the other hand, glucose, fructose, and phenolic content were increased in EpicSelect.4 but decreased in UCR 369 at 7 DOR. In summary, compared to EpicSelect.4, UCR 369 restored their photosynthetic pigments and metabolites to the control levels at 7 DOR, indicating a likely tolerance to waterlogging stress.

Growth and Photosynthetic Responses of Cowpea Genotypes under Waterlogging at the Reproductive Stage.

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.

Methods for Optimization of Protein Extraction and Proteogenomic Mapping in Sweet Potato.

The complexity in chemical composition alongside the genomic complexity of crop plants poses significant challenges for the characterization of their proteomes. This chapter provides specific methods that can be used for the extraction and identification of proteins from sweet potato, and a proteogenomic method for the subsequent peptide mapping on the haplotype-derived sweet potato genome assembly. We outline two basic methods for extracting proteins expressed in root and leaf tissues for the label-free quantitative proteomics-one phenol-based procedure and one polyethylene glycol (PEG) 4000-based fractionation method-and discuss strategies for the organ-specific protein extraction and increased recovery of low-abundance proteins. Next, we describe computational methods for improved proteome annotation of sweet potato based on aggregated genomics and transcriptomics resources available in our and public databases. Lastly, we describe an easily customizable proteogenomics approach for mapping sweet potato peptides back to their genome location and exemplify its use in improving genome annotations using a mass spectrometry data set.

The Arabidopsis thaliana Knockout Mutant for Phytochelatin Synthase1 (cad1-3) Is Defective in Callose Deposition, Bacterial Pathogen Defense and Auxin Content, But Shows an Increased Stem Lignification.

The enzyme phytochelatin synthase (PCS) has long been studied with regard to its role in metal(loid) detoxification in several organisms, i.e., plants, yeasts, and nematodes. It is in fact widely recognized that PCS detoxifies a number of heavy metals by catalyzing the formation of thiol-rich oligomers, namely phytochelatins, from glutathione and related peptides. However, recent investigations have highlighted other possible roles played by the PCS enzyme in the plant cell, e.g., the control of pathogen-triggered callose deposition. In order to examine novel aspects of Arabidopsis thaliana PCS1 (AtPCS1) functions and to elucidate its possible roles in the secondary metabolism, metabolomic data of A. thaliana wild-type and cad1-3 mutant were compared, the latter lacking AtPCS1. HPLC-ESI-MS analysis showed differences in the relative levels of metabolites from the glucosinolate and phenylpropanoid pathways between cad1-3 and wild-type plants. Specifically, in control (Cd-untreated) plants, higher levels of 4-methoxy-indol-3-ylmethylglucosinolate were found in cad1-3 plants vs. wild-type. Moreover, the cad1-3 mutant showed to be impaired in the deposit of callose after Cd exposure, suggesting that AtPCS1 protects the plant against the toxicity of heavy metals not only by synthesizing PCs, but also by contributing to callose deposition. In line with the contribution of callose in counteracting Cd toxicity, we found that another callose-defective mutant, pen2-1, was more sensitive to high concentrations of Cd than wild-type plants. Moreover, cad1-3 plants were more susceptible than wild-type to the hemibiotrophic bacterial pathogen Pseudomonas syringae. The metabolome also revealed differences in the relative levels of hydroxycinnamic acids and flavonols, with consequences on cell wall properties and auxin content, respectively. First, increased lignification in the cad1-3 stems was found, probably aimed at counteracting the entry of Cd into the inner tissues. Second, in cad1-3 shoots, increased relative levels of kaempferol 3,7 dirhamnoside and quercetin hexoside rhamnoside were detected. These flavonols are endogenous inhibitors of auxin transport in planta; auxin levels in both roots and shoots of the cad1-3 mutant were in fact lower than those of the wild-type. Overall, our data highlight novel aspects of AtPCS1 functions in A. thaliana.

Insights into the Structure, Function, and Ion-Mediated Signaling Pathways Transduced by Plant Integrin-Linked Kinases.

Kinases facilitate detection of extracellular signals and set in motion cellular responses for plant adaptation and survival. Some of the energy utilized for kinase signal processing is produced through the activity of ion transporters. Additionally, the synergy between cellular ions and signal transduction influences plant response to pathogens, and their growth and development. In plants, the signaling elements that connect cell wall and membrane sensors with ion homeostasis and transport-mediated processes are largely unknown. Current research indicates that plant Integrin-Linked Kinases (ILKs), a subfamily Raf-like MAP2K Kinases, may have evolved to fulfill this role. In this review, we explore new findings on plant ILKs placing a particular focus on the connection between ILKs proteins unique structural features and ILKs functions. The ankyrin repeat motifs and the kinase domains of ILKs in Arabidopsis and land plants lineage, respectively, are analyzed and discussed as potential determinants of ILKs' metal ion cofactor specificity and their enzymatic and interaction activities. Further, ILKs regulation through gene expression, subcellular localization, and ions and ion transporters is reviewed in the context of recent studies. Finally, using evidence from literature and interactomics databanks, we infer ILKs-dependent cellular pathways and highlight their potential in transmitting multiple types of signals originating at the interface between the cell wall and plasma membrane.